Human Family 1-4 cytochrome P450 enzymes involved in the metabolic activation of xenobiotic and physiological chemicals: an update

Abstract

This is an overview of the metabolic activation of drugs, natural products, physiological compounds, and general chemicals by catalytic activity of cytochrome P450 enzymes belonging to Families 1–4. The data were collected from > 5,152 references. The total number of data entries of reactions catalyzed by P450s Families 1–4 was 7,696 of which 1,121 (~15%) were defined as bioactivation reactions of different degrees. The data were divided into groups of General Chemicals, Drugs, Natural Products, and Physiological Compounds, presented in tabular form. The metabolism and bioactivation of selected examples of each group are discussed. In most of the cases the metabolites are directly toxic chemicals reacting with cell macromolecules, but in some cases the metabolites formed are not direct toxicants but participate as substrates in succeeding metabolic reactions (e.g., conjugation reactions), the products of which are final toxicants. We identified a high level of activation for three groups of compounds (General Chemicals, Drugs, and Natural Products) yielding activated metabolites and the generally low participation of Physiological Compounds in bioactivation reactions. In the group of General Chemicals, P450 enzymes 1A1, 1A2, and 1B1 dominate in the formation of activated metabolites; Drugs are mostly activated by the enzyme P450 3A4, and Natural Products by P450s 1A2, 2E1, and 3A4. Physiological Compounds showed no clearly dominant enzyme, but the highest numbers of activations are attributed to P450 1A, 1B1, and 3A enzymes. The results thus show, perhaps not surprisingly, that Physiological Compounds are infrequent substrates in bioactivation reactions catalyzed by P450 enzyme Families 1–4, with the exception of estrogens and arachidonic acid. The results thus provide information on the enzymes that activate specific groups of chemicals to toxic metabolites.

Introduction

Human cytochrome P450 (P450, CYP) enzymes catalyze a great number of metabolic reactions that have important effects on the biological activities (physiologic, therapeutic, and/or toxic) of xenobiotics such as drugs, natural products, general chemicals (e.g., environmental chemicals such as pesticides, pro-carcinogens), and physiological compounds. Their general role and significance for metabolism in humans has been discussed and reviewed previously. In addition, in previous publications efforts were made to estimate the participation of the activity of different groups of enzymes, e.g. oxidoreductase enzymes (FMO (microsomal flavin-containing monooxygenase), AKR (aldo-keto reductase), MAO (monoamine oxidase), and P450 enzymes), in the metabolism of natural products and physiological chemicals and general chemicals in humans. When the groups of chemicals were analyzed, the results showed the highest values for participation of P450 enzymes in the metabolism of drugs and general chemicals as substrates. For P450 enzymes the calculations also showed that, regarding drug metabolism, more than three-fourths of the human P450 reactions can be accounted for by a set of five P450s: 1A2, 2C9, 2C19, 2D6, and 3A4, with the largest fraction of the P450 reactions being catalyzed by P450 3A enzymes. Compared to other oxidoreductase enzymes and taking into consideration chemicals that are classified as carcinogens, our calculations showed that metabolic activations of the compounds to toxic metabolites are dominantly catalyzed by P450 enzymes (66% of bioactivations) and that, within this group, six P450s (1A1, 1A2, 1B1, 2A6, 2E1, and 3A4) accounted for 77% of the P450 activation reactions. In the present review we have updated and extended our calculations to general activation reactions forming potentially toxic metabolites as a consequence of metabolic activation of drugs, natural products, physiological compounds, and general chemicals (Rendic 2002; Rendic and Di Carlo 1997; Rendic and Guengerich 2012; Rendic and Guengerich 2015). We recently reviewed the properties (mechanisms, induction, inhibition, toxic effects, and benefits) of human P450s belonging to the P450 Families 5–51 (i.e., 22 of the total 57 P450s) that are responsible for metabolism and biosynthesis of physiological compounds, including their substrate selectivity, information, and references (Rendic and Guengerich 2018). In the present paper we update and discuss important aspects of many of the P450s belonging to Families 1–4, including the reactions and the roles in metabolic activation of xenobiotics (drugs, natural products, general chemicals) and physiological compounds.

Results and discussion

A synopsis of of the data used for analysis of catalytic activity of P450 Families 1–4 is presented in Table 1. Data were collected from more than 5,152 references. The total number of data entries for enzymatic reactions catalyzed by P450s belonging to 1–4 Families was 7,686 of which 1,114 (~15%) were defined as bioactivation reactions of different degrees. When considering activation of all compounds the results show predominant participation of P450s 3A4, 1A2, and 1A1, followed by P450s 2E1 and 1B1. P450s 2C9, 2D6, 2A6, 2C19, and 2B6 also have significant participation in bioactivation reactions (Fig. 1).

Table 1.

Number of data entries related to metabolic activation of drugs, general chemicals, natural products, and physiological compounds catalyzed by human cytochrome P450 Families 1–4

Number of data entries
All compounds		General chemicals		Drugs		Natural products		Physiological compounds
Total	Activations	Total	Activations	Total	Activations	Total	Activations	Total	Activations
7686	1114	2165	618	4039	235	952	186	530	75

Fig 1.

Participation of human P450 Families 1–4 in activation of all compounds to potentially toxic metabolites (7686 reactions, 1114 activation reactions)

Data analyzed were divided into four groups of compounds: General Chemicals, Drugs, Natural Products, and Physiological Compounds. Of the 2,165 reactions for General Chemicals, 618 (29%) were classified as activations; for 4,032 Drugs entries, 237 (6%) were classified as activations; for the 952 reactions under Natural Products, 186 (20%) were classified as activations; for the 530 Physiological Compounds, 75 reactions (14%) were classified as activations (Table 1).

General chemicals

We reported previously that metabolism of General Chemicals catalyzed by human enzymes is predominately catalyzed by P450 enzymes in humans (~92%) (Rendic and Guengerich 2015). Other enzymes, besides P450s, that participate in a greater extent include those in the AKR, FMO, and MAO families (Rendic and Guengerich 2015). P450 enzymes dominate in bioactivation of carcinogens (66%) over other xenobiotic metabolizing enzymes (Rendic and Guengerich 2012). The present data show that among P450 enzymes, Family 1 enzymes (P450s 1A1, 1A2, B1) dominate in activations of General Chemicals, followed by P450s 2E1, 3A4, and 2A6 (Fig. 2).

Fig 2.

Participation of human P450 Families 1–4 in activation of general chemicals to potentially toxic metabolites (2165 reactions, 618 activation reactions)

The following examples illustrate the participation of P450 enzymes in the bioactivation of selected General Chemicals substrates.

Polycyclic aromatic hydrocarbons (PAHs)

Examples (213 data entries) of the metabolic activation of group of general chemicals (e.g., polycyclic aromatic hydrocarbons (PAHs), heterocyclic and aromatic amines, insecticides, organic solvents) are presented in Table 2. The majority of the data presented (75 data entries) involve PAHs and their metabolites. Of the 76 entries presented in Table 2, 24 are attributed as “high activity” or “high activation” and are catalyzed by P450 1A1, 1A2, 1B1, 2A13, and 2A6 enzymes. These data correlate well with experimental findings on the activation of PAHs by P450 enzymes (Shimada et al. 2013). The parent PAH compounds are not toxic per se but their products formed by hydroxylation and epoxidation reactions, catalyzed by P450 enzymes, are reactive and interact with cellular macromolecules. Consequently, the literature data on activation of PAHs are predominately focused on activation of the PAH metabolites (e.g., dihydrodiols possessing different stereochemical structures) to ultimate toxic dihydrodiol epoxides, as exemplified by the classic activation of benzo[a]pyrene (B[a]P) (Fig. 3).

Table 2.

Examples of the metabolic activation of groups of general chemicals (polycyclic aromatic hydrocarbons, heterocyclic and aromatic amines, insecticides, organic solvents) by human cytochrome P450 enzymes

General chemical	P450	Category	Reaction	PMID numbers	References
N-Acetylaminofluorene (2-acetamidofluorene, 2-AAF)	1A2	Acyl arylamine	Hydroxylation, N- (major enzyme, activation)	8095200, 8313839, 1576936, 2813353, 9705755, 8200083, 10503887, 10517985, 11377247, 11013410, 11473383, 15279838, 11375903, 23432465	(Aryal et al. 2000; Butler et al. 1989; Edwards et al. 1994; Guengerich 1993; Guengerich et al. 1999; Ioannides and Parke 1993; Josephy et al. 2001; Juchau et al. 1992; Oda et al. 2001; Shimada et al. 2013; Turesky et al. 1999; Turesky et al. 1998; Yamazaki et al. 2004; Yueh et al. 2001)
2-AAF	1A1	Acetyl arylamine	Hydroxylation, N- (activation)	8095200, 8313839, 1576936, 11502724, 15279838, 7955101, 23432465	(Guengerich 1993; Ioannides and Parke 1993; Juchau et al. 1992; Shimada et al. 1994; Shimada et al. 2013; Shimada et al. 2001a; Yamazaki et al. 2004)
2-Aminoanthracene (2-AA)	2W1	Arylamine	Oxidation (activation)	16379042	(Brandon et al. 2006)
2-AA	1A2	Arylamine	Hydroxylation, N- (activation)	23432465	(Shimada et al. 2013)
2-AA	2A13	Arylamine	Hydroxylation, N- (activation)	23432465	(Shimada et al. 2013)
2-AA	2A6	Arylamine	Hydroxylation, N- (high activity, activation)	23432465	(Shimada et al. 2013)
2-AA	1A1	Arylamine	Hydroxylation, N- (high activity, activation), major enzyme	7955101, 11502724, 9705755, 11377247, 10964100, 23432465	(Oda et al. 2001; Shimada et al. 1994; Shimada et al. 2013; Shimada et al. 2001a; Turesky et al. 1998; Williams et al. 2000)
2-AA	1B1	Arylamine	Hydroxylation, N- (high activity, activation)	8674051, 10964100, 11377247, 11473383, 23432465, 27123158	(Chun and Kim 2016; Josephy et al. 2001; Oda et al. 2001; Shimada et al. 1996; Shimada et al. 2013; Williams et al. 2000)
6-Aminochrysene	3A4	Arylamine	N-Oxidation (high activity, activation)	2271712, 8330339	(Brian et al. 1990; Yamazaki et al. 1993)
6-Aminochrysene	1A1	Arylamine	Oxidation (high activity and activation)	7955101, 8961944, 11502724, 9685642	(Guengerich and Shimada 1998; Shimada et al. 1994; Shimada et al. 2001a; Shou et al. 1996a)
2-Amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ)	2A13	Heterocyclic amine	Activation	23432465	(Shimada et al. 2013)
MeIQ	2A6	Heterocyclic amine	Activation	23432465	(Shimada et al. 2013)
MeIQ	2W1	Heterocyclic amine	Activation	24278521	(Eun et al. 2010)
MeIQ	1A1	Heterocyclic amine	Hydroxylation, N- (activation)	7955101, 9152602, 11502724, 9705755, 8200083, 11377247, 11473383	(Edwards et al. 1994; Josephy et al. 2001; Oda et al. 2001; Shimada et al. 1994; Shimada et al. 1997b; Shimada et al. 2001a; Turesky et al. 1998)
MeIQ	1B1	Heterocyclic amine	Hydroxylation, N- (activation)	8674051, 10964100, 9152602, 10426814, 11377247, 11473383, 9721189, 11502724, 11719446, 23432465, 27123158	(Chun and Kim 2016; Chun et al. 2001; Josephy et al. 2001; Oda et al. 2001; Shimada et al. 1994; Shimada et al. 1997b; Shimada et al. 2013; Shimada et al. 2001a; Shimada et al. 1999; Shimada et al. 1998; Williams et al. 2000)
2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)	2A13	Heterocyclic amine	Activation	23432465	(Shimada et al. 2013)
MeIQx	2A6	Heterocyclic amine	Activation (weaker activation)	23432465	(Shimada et al. 2013)
MeIQx	1B1	Heterocyclic amine	Activation (weaker activation)	23432465, 27123158	(Shimada et al. 2013)
MeIQx	1A1	Heterocyclic amine	Hydroxylation, N- (activation)	7955101, 9705755, 8200083, 11377247, 11473383, 11502724, 17627018	(Bendaly et al. 2007; Edwards et al. 1994; Josephy et al. 2001; Oda et al. 2001; Shimada et al. 1994; Shimada et al. 2001a; Turesky et al. 1998)
MeIQx	1A2	Heterocyclic amine	Hydroxylation, N- (major enzyme, high activity and activation)	7955101, 9705755, 8200083, 11377247, 11473383, 9111224, 10220313, 11258970, 11453738, 12351158, 14744142, 14725854, 28879062	(Delannée et al. 2017; Edwards et al. 1994; Hammons et al. 1997; Josephy et al. 2001; Kim and Guengerich 2004; Langouët et al. 2001; Oda et al. 2001; Parikh et al. 1999; Shimada et al. 1994; Turesky et al. 1998; Turesky et al. 2002; Turesky et al. 2001; Zhou et al. 2004)
3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1)	2D6	Heterocyclic amine	Hydroxylation, N- (activation)	11377247	(Oda et al. 2001)
Trp-P-1	2E1	Heterocyclic amine	Hydroxylation, N- (activation)	11377247	(Oda et al. 2001)
Trp-P-1	3A4	Heterocyclic amine	Hydroxylation, N- (high activation)	11377247	(Oda et al. 2001)
Trp-P-1	2C9	Heterocyclic amine	Oxidation (activation)	11377247	(Oda et al. 2001)
Trp-P-1	2W1	Heterocyclic amine	Oxidation (activation)	16379042	(Brandon et al. 2006)
Trp-P-1	2A13	Heterocyclic amine	Activation (weaker activation)	23432465	(Shimada et al. 2013)
Trp-P-1	2A6	Heterocyclic amine	Activation (weaker activation)	23432465	(Shimada et al. 2013)
Trp-P-1	2W1	Heterocyclic amine	Oxidation (activation)	24278521	(Eun et al. 2010)
Trp-P-1	1A2	Heterocyclic amine	Hydroxylation, N- (major enzyme, activation)	11377247, 7955101, 9705755, 11473383, 8961944	(Josephy et al. 2001; Oda et al. 2001; Shimada et al. 1994; Shou et al. 1996a; Turesky et al. 1998)
Trp-P-1	1A1	Heterocyclic amine	Hydroxylation, N- (high activation)	7955101, 8200083, 11377247, 11502724, 11473383, 9705755	(Edwards et al. 1994; Josephy et al. 2001; Oda et al. 2001; Shimada et al. 1994; Shimada et al. 2001a; Turesky et al. 1998)
2-Aminofluorene (2-AF)	2W1	Arylamine	Oxidation (activation)	16379042	(Brandon et al. 2006)
2-AF	2A6	Arylamine	Hydroxylation, N- (activation)	23432465	(Shimada et al. 2013)
2-AF	2A13	Arylamine	Hydroxylation, N- (major enzyme, high activity, activation)	23432465	(Shimada et al. 2013)
2-AF	1A2	Arylamine	Hydroxylation, N- (major enzyme, high activity and activation)	2334931, 7955101, 8095200, 8313839, 1576936, 9705755, 8200083, 10503887, 10964100, 11377247, 11013410, 2803520, 10815771, 23432465	(Aoyama et al. 1989; Aryal et al. 2000; Edwards et al. 1994; Guengerich 1993; Ioannides and Parke 1993; Juchau et al. 1992; Lozano et al. 2000; McManus et al. 1990; Oda et al. 2001; Shimada et al. 1994; Shimada et al. 2013; Turesky et al. 1999; Turesky et al. 1998; Williams et al. 2000)
2-AF	1B1	Arylamine	Hydroxylation, N- (weaker activation)	23432465, 27123158	(Chun and Kim 2016; Shimada et al. 2013)
2-Amino-6-methyldipyrido[1,2-a,3,2’-d]-imidazole (Glu-P-1)	1A2	Heterocyclic amine	Hydroxylation, N- (high activity, major enzyme, activation)	7955101, 9705755, 10503887, 10517985, 11377247, 2803520, 14744142, 14725854	(Aoyama et al. 1989; Guengerich et al. 1999; Kim and Guengerich 2004; Oda et al. 2001; Shimada et al. 1994; Turesky et al. 1999; Turesky et al. 1998; Zhou et al. 2004)
2-Amino-3-methylimidazo[4,5-f]quinoline (IQ)	2A13	Heterocyclic amine	Activation	23432465	(Shimada et al. 2013)
IQ	2A6	Heterocyclic amine	Activation (weaker activation)	23432465	(Shimada et al. 2013)
IQ	2W1	Heterocyclic amine	Activation	24278521	(Eun et al. 2010)
IQ	1B1	Heterocyclic amine	Activation	23432465, 27123158	(Chun and Kim 2016; Shimada et al. 2013)
IQ	1A1	Heterocyclic amine	Hydroxylation, N- (activation)	7955101, 9705755, 8200083, 11377247, 11473383, 9918136, 8095200	(Edwards et al. 1994; Guengerich 1993; Josephy et al. 2001; Oda et al. 2001; Shimada et al. 1994; Turesky et al. 1998; Williams et al. 1998)
IQ	1A2	Heterocyclic amine	Hydroxylation, N- (high activity, major enzyme, activation)	9675256, 7955101, 8200083, 11377247, 9111224, 9918136, 14744142, 14725854, 11038156	(Barceló et al. 1998; Edwards et al. 1994; Hammons et al. 1997; Kim and Guengerich 2004; Miranda et al. 2000; Oda et al. 2001; Shimada et al. 1994; Williams et al. 1998; Zhou et al. 2004)
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, PhIP	2C9	Heterocyclic amine	Hydroxylation, N2- (activation)	11377247	(Oda et al. 2001)
PhIP	2D6	Heterocyclic amine	Hydroxylation, N2- (activation)	11377247	(Oda et al. 2001)
PhIP	2E1	Heterocyclic amine	Hydroxylation, N2- (low or no activation)	11377247	(Oda et al. 2001)
PhIP	1A1	Heterocyclic amine	Hydroxylation, N2- (activation)	11502724, 8961944, 9705755, 9855011, 8200083, 11377247, 11473383, 15279838, 9111224, 8095200	(Crofts et al. 1998; Edwards et al. 1994; Guengerich 1993; Hammons et al. 1997; Josephy et al. 2001; Oda et al. 2001; Shimada et al. 2001a; Shou et al. 1996a; Turesky et al. 1998; Yamazaki et al. 2004)
PhIP	1A2	Heterocyclic amine	Hydroxylation, N2- (high activity, major enzyme, major reaction, activation)	1913651, 7955101, 9705755, 8200083, 11377247, 11473383, 10220313, 11258970, 11453738, 12351158, 14725854, 15279838, 16167840	(Cheung et al. 2005a; Edwards et al. 1994; Josephy et al. 2001; Langouët et al. 2001; Oda et al. 2001; Parikh et al. 1999; Shimada et al. 1994; Shimada and Guengerich 1991; Turesky et al. 1998; Turesky et al. 2002; Turesky et al. 2001; Yamazaki et al. 2004; Zhou et al. 2004)
PhIP	1B1	Heterocyclic amine	Hydroxylation, N2- (activation)	8961944, 11502724, 9855011, 10964100, 11377247, 11473383, 9328177	(Crofts et al. 1997; Crofts et al. 1998; Josephy et al. 2001; Oda et al. 2001; Shimada et al. 2001a; Shou et al. 1996a; Williams et al. 2000)
PhIP	1B1	Heterocyclic amine	Hydroxylation, N2- and deamination (activation)	9855011, 9328177, 27123158	(Chun and Kim 2016; Crofts et al. 1997; Crofts et al. 1998)
Aniline reaction with norharman	1A1	Arylamine	Aminophenylnorharman formation (activation), high activity	15279827	(Nishigaki et al. 2004)
Aniline reaction with norharman	1A2	Arylamine	Aminophenylnorharman formation, activation, major enzyme	15279827	(Nishigaki et al. 2004)
Aniline reaction with norharman	1B1	Arylamine	Aminophenylnorharman formation, activation, very low activity	15279827	(Nishigaki et al. 2004)
Aniline reaction with norharman	2B6	Arylamine	Aminophenylnorharman formation, activation, very low activity	15279827	(Nishigaki et al. 2004)
Aniline reaction with norharman	2D6	Arylamine	Aminophenylnorharman formation, activation	15279827	(Nishigaki et al. 2004)
Aniline reaction with norharman	2E1	Arylamine	Aminophenylnorharman formation, activation, very low activity	15279827	(Nishigaki et al. 2004)
Aniline reaction with norharman	3A4	Arylamine	Aminophenylnorharman formation, activation, major enzyme	15279827	(Nishigaki et al. 2004)
Azinphos-methyl	2C19	Insecticide, organophosphate, benzotriazine organothiophosphate	Desulfuration (oxon formation), activation, medium Km, high activity	21782601, 12620367	(Buratti et al. 2002; Buratti et al. 2003)
Azinphos-methyl	1A1	Insecticide, organophosphate, benzotriazine organothiophosphate	Desulfuration (oxon formation), activation, medium Km, medium activity	21782601, 12620367	(Buratti et al. 2002; Buratti et al. 2003)
Azinphos-methyl	1A2	Insecticide, organophosphate, benzotriazine organothiophosphate	Desulfuration (oxon formation), activation, medium Km, medium activity	21782601, 12620367	(Buratti et al. 2002; Buratti et al. 2003)
Azinphos-methyl	2B6	Insecticide, organophosphate, benzotriazine organothiophosphate	Desulfuration (oxon formation), activation, medium Km, medium activity	21782601, 12620367	(Buratti et al. 2002; Buratti et al. 2003)
Azinphos-methyl	2C19	Insecticide, organophosphate, benzotriazine organothiophosphate	Desulfuration (oxon formation), activation, medium Km, high activity	21782601, 12620367	(Buratti et al. 2002; Buratti et al. 2003)
Azinphos-methyl	2C8	Insecticide, organophosphate, benzotriazine organothiophosphate	Desulfuration (oxon formation), activation, very low activity	21782601, 12620367	(Buratti et al. 2002; Buratti et al. 2003)
Azinphos-methyl	2C9	Insecticide, organophosphate, benzotriazine organothiophosphate	Desulfuration (oxon formation), activation, very low activity	21782601, 12620367	(Buratti et al. 2002; Buratti et al. 2003)
Azinphos-methyl	3A4	Insecticide, organophosphate, benzotriazine organothiophosphate	Desulfuration (oxon formation), activation, very low activity, at higher concentrations	21782601, 12620367	(Buratti et al. 2002; Buratti et al. 2003)
Benzo[a]anthracene-3,4-diol	1A1	Polycyclic aromatic hydrocarbon (PAH) metabolite	Activation (weaker)	14720319	(Shimada and Fujii-Kuriyama 2004)
Benzo[a]anthracene-3,4-diol	1A2	PAH metabolite	Activation (weaker)	14720319	(Shimada and Fujii-Kuriyama 2004)
trans-Benz[a]anthracene-3,4-diol	2W1	PAH metabolite	Oxidation (activation)	16379042	(Brandon et al. 2006)
Benzo[g]chrysene-11,12-diol	1A1	PAH metabolite	Activation	14720319	(Shimada and Fujii-Kuriyama 2004)
Benzo[g]chrysene-11,12-diol	1B1	PAH metabolite	Activation	14720319	(Shimada and Fujii-Kuriyama 2004)
Benzo[g]chrysene-11,12-diol	1A2	PAH metabolite	Activation (weaker)	14720319	(Shimada and Fujii-Kuriyama 2004)
Benzo[b]fluoranthene-9,10-diol	1A2	PAH metabolite	Oxidation (activation)	14720319	(Shimada and Fujii-Kuriyama 2004)
Benzo[b]fluoranthene-9,10-diol	2B6	PAH metabolite	Oxidation (activation)	14720319	(Shimada and Fujii-Kuriyama 2004)
Benzo[b]fluoranthene-9,10-diol	2C19	PAH metabolite	Oxidation (activation)	14720319	(Shimada and Fujii-Kuriyama 2004)
Benzo[c]phenanthrene (B[c]P)	2A13	PAH	Activation (weaker)	23432465	(Shimada et al. 2013)
B[c]P	2A6	PAH	Activation (weaker)	23432465	(Shimada et al. 2013)
B[c]P	1A1	PAH	3,4-Dihydrodiol-1,2-epoxide formation (weaker activation)	9168260, 21781864, 11409939	(Baum et al. 2001; Einolf et al. 1997; Seidel et al. 1998)
B[c]P	1A2	PAH	3,4-Dihydrodiol-1,2-epoxide formation (major enzyme, activation)	9168260, 21781864, 11409939	(Baum et al. 2001; Einolf et al. 1997; Seidel et al. 1998)
B[c]P	1B1	PAH	3,4-Dihydrodiol-1,2-epoxide formation (major enzyme, activation)	9168260, 21781864, 11409939, 23432465	(Baum et al. 2001; Einolf et al. 1997; Seidel et al. 1998; Shimada et al. 2013)
Benzo[c]phenanthrene 3,4-dihydrodiol (B[c]P-3,4-diol)	1A2	PAH metabolite	Activation (weak)	14720319	(Shimada and Fujii-Kuriyama 2004)
B[c]P-3,4-diol	1A1	PAH metabolite	Activation (weaker)	14720319	(Shimada and Fujii-Kuriyama 2004)
B[c]P-3,4-diol	1B1	PAH metabolite	Activation (weaker)	14720319, 23432465, 27123158	(Chun and Kim 2016; Shimada and Fujii-Kuriyama 2004; Shimada et al. 2013)
B[c]P-3,4-diol	2A13	PAH metabolite	Activation (weak)	23432465	(Shimada et al. 2013)
B[c]P-3,4-diol	2A6	PAH metabolite	Activation (weak)	23432465	(Shimada et al. 2013)
Benzo[a]pyrene (B[a]P)	1A1	PAH	trans-7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro formation (activation)	17640999, 14720319, 19330882	(Kabler et al. 2009; Kim et al. 2007; Shimada and Fujii-Kuriyama 2004)
B[a]P	1B1	PAH	trans-7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro formation. (medium Km, high activity, high efficiency, activation)	8674051, 9721189, 11502724, 12584184, 9806168, 12628515, 23432465, 14720319, 27123158	(Buters et al. 2003; Chun and Kim 2016; Guengerich et al. 2003; Kim et al. 1998; Shimada and Fujii-Kuriyama 2004; Shimada et al. 1996; Shimada et al. 2013; Shimada et al. 2001a; Shimada et al. 1998)
(±)-Benzo[a]pyrene (B[a]P)-7,8-dihydrodiol	2W1	PAH metabolite	Oxidation (activation)	16551781	(Wu et al. 2006)
(±)-B[a]P-7,8-dihydrodiol	1A1	PAH metabolite	Oxidation, diol-epoxide formation (high activation)	11952781, 21028851, 11502724, 14720319, 29219051, 18402469, 15720144, 16411658, 17295519	(Gelhaus et al. 2011; Jiang et al. 2005; Jiang et al. 2006; Kisselev et al. 2002; Quinn and Penning 2008; Rendic and Guengerich 2018; Ruan et al. 2007; Shimada and Fujii-Kuriyama 2004; Shimada et al. 2001a)
(±)-B[a]P-7,8-dihydrodiol	1B1	PAH metabolite	Oxidation, diol-epoxide formation (high activation)	16551781, 21028851, 11502724, 14720319, 29219051, 18402469, 15720144, 16411658, 17295519	(Gelhaus et al. 2011; Jiang et al. 2005; Jiang et al. 2006; Quinn and Penning 2008; Rendic and Guengerich 2018; Ruan et al. 2007; Shimada and Fujii-Kuriyama 2004; Shimada et al. 2001a; Wu et al. 2006)
cis-(−)-B[a]P-7,8-dihydrodiol	1A1	PAH metabolite	cis-(syn)-7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro formation (trans-diol epoxide formation), high activation	14720319, 29219051	(Rendic and Guengerich 2018; Shimada and Fujii-Kuriyama 2004)
cis-(−)-B[a]P-7,8-dihydrodiol	1B1	PAH metabolite	cis-(syn)-7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro formation (trans-diol epoxide formation), high activation	14720319, 29219051	(Rendic and Guengerich 2018; Shimada and Fujii-Kuriyama 2004)
trans-(−)-B[a]P-7,8-dihydrodiol	3A7	PAH metabolite	Oxidation (activation)	9328287	(Gillam et al. 1997)
trans-(−)-B[a]P-7,8-dihydrodiol	1A1	PAH metabolite	trans-(anti)-7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro formation (trans-diol epoxide formation), medium Km, high activity	7955101, 9152602, 8961944, 11502724, 15720144, 17295519, 9014198, 8043197, 1551116, 11238186, 7581497, 11952781, 12670496, 14633740, 19330882	(Doehmer et al. 1995; Gautier et al. 1996; Jiang et al. 2005; Kabler et al. 2009; Kisselev et al. 2002; Ruan et al. 2007; Schwarz et al. 2001; Schwarz et al. 2003; Schwarz and Roots 2003; Shimada et al. 1994; Shimada et al. 1997b; Shimada et al. 2001a; Shou et al. 1996a; Shou et al. 1994; Yun et al. 1992)
trans-(−)-B[a]P-7,8-dihydrodiol	1B1	PAH metabolite	trans-(anti)-7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro formation (trans-diol epoxide formation), low Km, high activity	9152602, 10426814, 12807732, 15720144, 15861043, 16411658, 17295519	(Jiang et al. 2005; Jiang et al. 2006; Mammen et al. 2005; Mammen et al. 2003; Ruan et al. 2007; Shimada et al. 1997b; Shimada et al. 1999)
trans-(+)-B[a]P-7,8-dihydrodiol	3A7	PAH metabolite	Oxidation (activation)	9328287	(Gillam et al. 1997)
trans-(+)-B[a]P-7,8-dihydrodiol	2A13	PAH metabolite	Oxidation (activation)	23432465	(Shimada et al. 2013)
trans-(+)-B[a]P-7,8-dihydrodiol	2A6	PAH metabolite	Oxidation (weak activation)	23432465	(Shimada et al. 2013)
trans-(+)-B[a]P-7,8-dihydrodiol	1B1	PAH metabolite	Oxidation, trans-(anti)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro formation (trans-diol epoxide formation), high efficiency activation	23432465, 14720319, 29219051	(Rendic and Guengerich 2018; Shimada and Fujii-Kuriyama 2004; Shimada et al. 2013)
trans-(+)-B[a]P-7,8-dihydrodiol	1A1	PAH metabolite	trans-(anti)-7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro formation (trans-diol epoxide formation), medium Km, high activity, high efficiency activation	7955101, 9152602, 8961944, 11502724, 15720144, 17295519, 9014198, 8043197, 1551116, 11238186, 7581497, 11952781, 12670496, 14633740, 14720319, 29219051, 19330882	(Doehmer et al. 1995; Gautier et al. 1996; Jiang et al. 2005; Kabler et al. 2009; Kisselev et al. 2002; Rendic and Guengerich 2018; Ruan et al. 2007; Schwarz et al. 2001; Schwarz et al. 2003; Schwarz and Roots 2003; Shimada and Fujii-Kuriyama 2004; Shimada et al. 1994; Shimada et al. 1997b; Shimada et al. 2001a; Shou et al. 1996a; Shou et al. 1994; Yun et al. 1992)
1,3-Butadiene	2A6	Olefin	Butadiene monoxide (epoxybutene) formation (high activity, activation)	8203896, 9016811, 8901879, 11397415,	(Bond and Medinsky 2001; Duescher and Elfarra 1994; Elfarra et al. 1996; Krause and Elfarra 1997)
1,3-Butadiene	1A2	Olefin	Butadiene monoxide (epoxybutene) formation (activation)	8203896, 9016811, 8901879, 11397415, 7586124	(Bond and Medinsky 2001; Duescher and Elfarra 1994; Elfarra et al. 1996; Krause and Elfarra 1997; Seaton et al. 1995)
1,3-Butadiene	2B6	Olefin	Butadiene monoxide (epoxybutene) formation (activation)	8203896, 9016811, 8901879, 11397415, 7586124	(Bond and Medinsky 2001; Duescher and Elfarra 1994; Elfarra et al. 1996; Krause and Elfarra 1997; Seaton et al. 1995)
1,3-Butadiene	2D6	Olefin	Butadiene monoxide (epoxybutene) formation (activation)	8203896, 9016811, 8901879, 11397415, 7586124	(Bond and Medinsky 2001; Duescher and Elfarra 1994; Elfarra et al. 1996; Krause and Elfarra 1997; Seaton et al. 1995)
1,3-Butadiene	2E1	Olefin	Butadiene monoxide (epoxybutene), (S)- and (R)- formation (high activity, activation, major enzyme)	8203896, 9635416, 17298833	(Boysen et al. 2007; Duescher and Elfarra 1994; Nieusma et al. 1998)
Butadiene monoxide (1,2-epoxy-3-butene)	3A4	Olefin, butadiene metabolite	Diepoxybutane (at high concentrations, activation)	8203896, 9016811, 8901879, 11397415, 7586124	(Bond and Medinsky 2001; Duescher and Elfarra 1994; Elfarra et al. 1996; Krause and Elfarra 1997; Seaton et al. 1995)
Butadiene monoxide (1,2-epoxy-3-butene)	2A6	Olefin, butadiene metabolite	Diepoxybutane, meso- (major) and (±)- formation (activation)	8203896, 9016811, 8901879, 11397415, 7586124	(Bond and Medinsky 2001; Duescher and Elfarra 1994; Elfarra et al. 1996; Krause and Elfarra 1997; Seaton et al. 1995)
Butadiene monoxide (1,2-epoxy-3-butene)	2C9	Olefin, butadiene metabolite	Diepoxybutane, meso- (major) and (±)- formation (activation)	8203896, 9016811, 8901879, 11397415, 7586124	(Bond and Medinsky 2001; Duescher and Elfarra 1994; Elfarra et al. 1996; Krause and Elfarra 1997; Seaton et al. 1995)
Butadiene monoxide (1,2-epoxy-3-butene)	2E1	Olefin, butadiene metabolite	Diepoxybutane, meso- (major) and (±)- formation (activation)	8203896, 9016811, 8901879, 11397415, 7586124, 17298833	(Bond and Medinsky 2001; Boysen et al. 2007; Duescher and Elfarra 1994; Elfarra et al. 1996; Krause and Elfarra 1997; Seaton et al. 1995)
Chloromethylindolines	2W1	Indoline	Oxidation (activation)	27257736, 23589180	(Guo et al. 2016; Travica et al. 2013)
Chlorpyrifos	2C9	Insecticide, organophosphate and acaricide, dicarboximide	Desulfuration (oxon formation), activation, low activity	11502728, 15764407	(Sams et al. 2004; Tang et al. 2001)
Chlorpyrifos	3A4	Insecticide, organophosphate and acaricide, dicarboximide	Desulfuration (oxon formation), activation, medium Km, major at high concentration (100 μM)	11502728, 21782601, 12620367, 11714865, 15764407, 16757081, 17079358, 17110060, 10996483, 20709133	(Buratti et al. 2006; Buratti et al. 2002; Buratti et al. 2003; Croom et al. 2010; Dai et al. 2001; Foxenberg et al. 2007; Mutch and Williams 2006; Sams et al. 2004; Sams et al. 2000; Tang et al. 2001)
Chlorpyrifos	1A2	Insecticide, organophosphate and acaricide, dicarboximide	Desulfuration (oxon formation, major enzyme), activation, low Km, medium activity	11502728, 21782601, 12620367, 16757081, 17079358	(Buratti et al. 2002; Buratti et al. 2003; Foxenberg et al. 2007; Mutch and Williams 2006; Tang et al. 2001)
Chlorpyrifos	2B6	Insecticide, organophosphate and acaricide, dicarboximide	Desulfuration (oxon formation), major enzyme, activation, medium activity, low Km, high activity and major reaction at low concentrations	11502728, 21782601, 12620367, 15764407, 16757081, 17079358, 29463407, 20709133, 22281205	(Buratti et al. 2002; Buratti et al. 2003; Foxenberg et al. 2007; Mutch and Williams 2006; Tang et al. 2001)
Chlorpyrifos	2C19	Insecticide, organophosphate and acaricide, dicarboximide	Desulfuration (oxon formation), activation, medium Km, medium to low activity	11502728, 21782601, 15764407, 16757081, 17079358, 20709133	(Buratti et al. 2002; Croom et al. 2010; Dai et al. 2001; Mutch and Williams 2006; Sams et al. 2004; Tang et al. 2001)
Chlorpyrifos	2D6	Insecticide, organophosphate and acaricide, dicarboximide	Desulfuration (oxon formation), activation, low activity	15764407, 16757081, 10996483	(Buratti et al. 2002; Croom et al. 2010; Dai et al. 2001; Mutch and Williams 2006; Sams et al. 2004; Tang et al. 2001)
Chlorpyrifos	3A5	Insecticide, organophosphate and acaricide, dicarboximide	Desulfuration (oxon formation), activation, medium Km, medium activity	16757081, 17079358, 17110060, 17079358	(Buratti et al. 2006; Foxenberg et al. 2007; Mutch and Williams 2006; Sams et al. 2004)
Chlorpyrifos	2C8	Insecticide, organophosphate and acaricide, dicarboximide	Desulfuration (oxon formation), activation	16757081, 21782601	(Buratti et al. 2002; Sams et al. 2004)
Chlorpyrifos	3A7	Insecticide, organophosphate and acaricide, dicarboximide	Desulfuration (oxon formation), activation, medium Km, low activity	17079358, 17110060	(Buratti et al. 2006; Mutch and Williams 2006)
trans-Chrysene-1,2-diol	1A2	PAH, chrysene metabolite	Oxidation (weaker activation)	14720319	(Shimada and Fujii-Kuriyama 2004)
trans-Chrysene-1,2-diol	2W1	PAH, chrysene metabolite	Oxidation (activation)	16379042	(Brandon et al. 2006)
trans-Chrysene-1,2-diol	1A1	PAH, chrysene metabolite	Oxidation (activation)	11502724, 8961944, 9152602, 14720319	(Shimada and Fujii-Kuriyama 2004; Shimada et al. 1997; Shimada et al. 2001a; Shou et al. 1996a)
trans-Chrysene-1,2-diol	1B1	PAH, chrysene metabolite	Oxidation (activation)	8674051, 9721189, 11502724, 12584184, 9152602, 10426814, 9685642, 14720319	(Buters et al. 2003; Guengerich and Shimada 1998; Shimada and Fujii-Kuriyama 2004; Shimada et al. 1997b; Shimada et al. 1996; Shimada et al. 2001a; Shimada et al. 1999; Shimada et al. 1998)
Diazinon	3A5	Insecticide, organophosphate, phosphorothioate	Desulfuration (oxon formation), activation, high activity	16757081	(Mutch and Williams 2006)
Diazinon	1A1	Insecticide, organophosphate, phosphorothioate	Desulfuration (oxon formation), activation, medium Km, medium activity	21969518	(Ellison et al. 2012)
Diazinon	2E1	Insecticide, organophosphate, phosphorothioate	Desulfuration (oxon formation), activation, low Km, medium activity, at lower concentration	21782601, 12620367	(Buratti et al. 2002; Buratti et al. 2003)
Diazinon	2B6	Insecticide, organophosphate, phosphorothioate	Desulfuration (oxon formation), activation, medium Km, medium to high activity	21782601, 12620367, 11708902, 15764407, 16757081, 21969518	(Buratti et al. 2002; Buratti et al. 2003; Dai et al. 2001a; Ellison et al. 2012; Kappers et al. 2001; Sams et al. 2004)
Diazinon	2C19	Insecticide, organophosphate, phosphorothioate	Desulfuration (oxon formation), activation, medium to high Km, medium to high activity, major enzyme	21782601, 12620367, 16757081, 11708902, 21969518	(Buratti et al. 2002; Buratti et al. 2003; Ellison et al. 2012; Kappers et al. 2001; Sams et al. 2004)
Diazinon	1A2	Insecticide, organophosphate, phosphorothioate	Desulfuration (oxon formation), activation, high Km, medium activity	21782601, 12620367, 21969518	(Buratti et al. 2002; Buratti et al. 2003; Ellison et al. 2012)
Diazinon	3A4	Insecticide, organophosphate, phosphorothioate	Desulfuration (oxon formation), activation, medium to high Km, high activity	21782601, 12620367, 21969518	(Buratti et al. 2002; Buratti et al. 2003; Ellison et al. 2012)
Dibenzo[b,k]fluoranthene	1A1	PAH	Oxidation (activation)	10613181	(Durant et al. 1999)
Dibenzo[a,l]pyrene (DB[a,l]P) (11R,12R)-dihydrodiol	1A2	PAH metabolite	Activation (weaker)	14720319	(Shimada and Fujii-Kuriyama 2004)
Dibenzo[a,l]pyrene (DB[a,l]P)	1A2	PAH	(−)-syn- and (−)-anti-11,12-dihydrodiol-13,14-epoxide form. (medium Km, high activity, high efficiency, activation)	14720319	(Shimada and Fujii-Kuriyama 2004)
DB[a,l]P	2W1	PAH	Oxidation (activation)	16379042	(Brandon et al. 2006)
DB[a,l]P	1B1	PAH	(−)-syn- and (−)-anti-11,12-dihydrodiol-13,14-epoxide formation (medium Km, high activity, high efficiency, activation)	8674051, 9721189, 11502724, 12584184, 9152602, 9625737, 10506751, 8968059, 9354437, 17623886, 12628515, 14720319, 27123158	(Buters et al. 2003; Chun and Kim 2016; Guengerich et al. 2003; King et al. 1999; Luch et al. 1998; Mahadevan et al. 2007; Shimada et al. 1997a; Shimada and Fujii-Kuriyama 2004; Shimada et al. 1997b; Shimada et al. 1996; Shimada et al. 2001a; Shimada et al. 1998; Shou et al. 1996c)
DB[a,l]P	1A1	PAH	(−)-syn- and (−)-anti-11,12-dihydrodiol-13,14-epoxide formation (medium Km, high activity, high efficiency, activation)	8961944, 9152602, 10207125, 10493514, 10506751, 8968059, 10613181, 14720319, 19330882	(Durant et al. 1999; Kabler et al. 2009; King et al. 1999; Luch et al. 1999a; Luch et al. 1999b; Shimada and Fujii-Kuriyama 2004; Shimada et al. 1997b; Shou et al. 1996a; Shou et al. 1996c)
trans-DB[a,l]P-11,12-diol	2W1	PAH metabolite	Oxidation (activation)	16379042	(Brandon et al. 2006)
trans-(−)-DB[a,l]P-(11R,12R)-diol	1A1	PAH metabolite	(−)-anti- and (+)-syn-11,12-dihydrodiol-13,14-epoxide formation (medium Km, high activity, activation)	7955101, 8961944, 11502724, 10207125, 10493514, 14720319	(Luch et al. 1999a; Luch et al. 1999b; Shimada and Fujii-Kuriyama 2004; Shimada et al. 1994; Shimada et al. 2001a; Shou et al. 1996a)
trans-(−)-DB[a,l]P-(11R,12R)-diol	1B1	PAH metabolite	(−)-anti-11,12-Dihydrodiol-13,14-epoxide formation (medium Km, high activity, high efficiency, activation)	8674051, 9721189, 11502724, 12584184, 9625737, 10207125, 10493514, 10506751, 10739169, 11465393, 9354437, 10426814, 12628515, 14720319	(Buters et al. 2003; Guengerich et al. 2003; King et al. 1999; Luch et al. 1998; Luch et al. 1999a; Luch et al. 1999b; Shimada et al. 1997a; Shimada and Fujii-Kuriyama 2004; Shimada et al. 1996; Shimada et al. 2001a; Shimada et al. 2001b; Shimada et al. 1999; Shimada et al. 1998; Watanabe et al. 2000)
trans-2,3-Dihydroxy-2,3-dihydrofluoranthene	1B1	PAH metabolite	Oxidation (activation)	8674051, 9721189, 11502724, 12584184, 9685642	(Buters et al. 2003; Guengerich and Shimada 1998; Shimada et al. 1996; Shimada et al. 2001a; Shimada et al. 1998)
7,12-Dimethylbenz[a]anthracene (7,12-DMBA)	2W1	PAH	Oxidation (activation)	16551781	(Wu et al. 2006)
7,12-DMBA	1B1	PAH	Oxidation (low Km, high activity and efficiency, activation)	11502724, 8674051, 9152602, 27123158	(Chun and Kim 2016; Shimada et al. 1997b; Shimada et al. 1996; Shimada et al. 2001a)
7,12-DMBA	1B1	PAH	Oxidation (activation)	23432465, 27123158	(Chun and Kim 2016; Shimada et al. 2013)
7,12-DMBA	1A1	PAH	Oxidation (low Km, high activity and efficiency, activation)	7955101, 8961944, 11502724, 10575002	(Ciolino and Yeh 1999; Shimada et al. 1994; Shimada et al. 2001a; Shou et al. 1996a)
7,12-DMBA	1A1	PAH	Oxidation (low Km, high activity and efficiency, activation)	7955101, 8961944, 11502724, 9152602, 23432465	(Shimada et al. 1994; Shimada et al. 1997b; Shimada et al. 2013; Shimada et al. 2001a; Shou et al. 1996a)
7,12-DMBA	1B1	PAH	Oxidation (activation)	8674051, 11502724	(Shimada et al. 1996; Shimada et al. 2001a)
trans-7,12-DMBA-3,4-diol	1A2	PAH metabolite	Oxidation (weaker activation)	14720319	(Shimada and Fujii-Kuriyama 2004)
trans-7,12-DMBA-3,4-diol	2W1	PAH metabolite	Oxidation (activation)	16379042	(Brandon et al. 2006)
trans-7,12-DMBA-3,4-diol	2A13	PAH metabolite	Activation	23432465	(Shimada et al. 2013)
trans-7,12-DMBA-3,4-diol	1A1	PAH metabolite	3,4-Dihydrodiol-1,2-epoxide formation (medium Km, high activity, high efficiency activation)	11502724, 9152602, 11360624, 8989918, 9685642, 14720319	(Guengerich and Shimada 1998; Shimada and Fujii-Kuriyama 2004; Shimada et al. 1997b; Shimada et al. 2001a; Shou et al. 1996b; Wu et al. 1997)
trans-7,12-DMBA-3,4-diol	1B1	PAH metabolite	3,4-Dihydrodiol-1,2-epoxide formation (medium Km, high activity, high efficiency activation)	8674051, 9721189, 11502724, 12584184, 9152602, 10426814, 23432465, 14720319	(Buters et al. 2003; Shimada and Fujii-Kuriyama 2004; Shimada et al. 1997b; Shimada et al. 1996; Shimada et al. 2013; Shimada et al. 2001a; Shimada et al. 1999; Shimada et al. 1998)
5,6-Dimethylchrysene-1,2-diol	1A1	PAH metabolite	Oxidation (activation)	7955101, 8961944, 11502724, 9152602, 16946553, 14720319	(Shimada 2006; Shimada and Fujii-Kuriyama 2004; Shimada et al. 1994; Shimada et al. 1997b; Shimada et al. 2001a; Shou et al. 1996a)
trans-5,6-Dimethylchrysene-1,2-diol	2W1	PAH metabolite	Oxidation (activation)	16379042	(Brandon et al. 2006)
trans-5,6-Dimethylchrysene-1,2-diol	1A2	PAH metabolite	Oxidation (activation)	7955101, 8961944, 16946553, 14720319	(Shimada 2006; Shimada and Fujii-Kuriyama 2004; Shimada et al. 1994; Shou et al. 1996a)
trans-5,6-Dimethylchrysene-1,2-diol	1B1	PAH metabolite	Oxidation (activation)	8674051, 9721189, 11502724, 12584184, 9152602, 10426814, 9685642, 14720319	(Buters et al. 2003; Guengerich and Shimada 1998; Shimada and Fujii-Kuriyama 2004; Shimada et al. 1997b; Shimada et al. 1996; Shimada et al. 2001a; Shimada et al. 1999; Shimada et al. 1998)
N,N-Dimethylformamide (DMF)	2E1	Organic solvent	N-Demethylation (high activity, activation)	1538706, 8477011, 11684354	(Amato et al. 2001; Hyland et al. 1992; Mráz et al. 1993)
N-Hydroxymethyl, N-methylformamide,	2E1	Organic solvent, dimethylformamide metabolite	Demethylation, N- (high activity, activation)	1538706, 8477011, 11684354	(Amato et al. 2001; Hyland et al. 1992; Mráz et al. 1993)
3-Methoxy-4-aminoazobenzene	2W1	Azoarylamine	Oxidation (activation)	16379042	(Turesky et al. 1998)
3-Methylcholanthrene	1A1	PAH	Oxidation (activation)	11360624, 14720319	(Shimada and Fujii-Kuriyama 2004; Wu et al. 1997)
5-Methylchrysene	1A1	PAH	Oxidation (medium Km, high activity, high efficiency, activation)	7955101, 8961944, 11502724, 27123158	(Chun and Kim 2016; Shimada et al. 1994; Shimada et al. 2001a; Shou et al. 1996a)
3-Methylchrysene-1,2-diol	1A1	PAH metabolite	Activation	14720319	(Shimada and Fujii-Kuriyama 2004)
3-Methylchrysene-1,2-diol	1B1	PAH metabolite	Activation	14720319	(Shimada and Fujii-Kuriyama 2004)
3-Methylchrysene-1,2-diol	1A2	PAH metabolite	Activation (weaker)	14720319	(Shimada and Fujii-Kuriyama 2004)
trans-5-Methylchrysene-1,2-diol	2W1	PAH metabolite	Oxidation (activation)	16379042	(Brandon et al. 2006)
trans-5-Methylchrysene-1,2-diol	2A6	PAH metabolite	Oxidation (medium Km, high activity, high efficiency activation)	23432465, 14720319	(Shimada and Fujii-Kuriyama 2004; Shimada et al. 2013)
trans-5-Methylchrysene-1,2-diol	2A13	PAH metabolite	Oxidation (medium Km, high activity, high efficiency activation)	23432465, 14720319	(Shimada and Fujii-Kuriyama 2004; Shimada et al. 2013)
trans-5-Methylchrysene-1,2-diol	1A1	PAH metabolite	Oxidation (medium Km, high activity, high efficiency activation)	7955101, 11502724, 9152602, 16485905, 16946553	(Shimada 2006; Shimada et al. 1994; Shimada et al. 1997b; Shimada and Guengerich 2006; Shimada et al. 2001a)
trans-5-Methylchrysene-1,2-diol	1B1	PAH metabolite	Oxidation (medium Km, high activity, high efficiency activation)	8674051, 9721189, 11502724, 12584184, 10426814, 9152602, 16485905, 9685642, 23432465, 14720319	(Buters et al. 2003; Guengerich and Shimada 1998; Shimada and Fujii-Kuriyama 2004; Shimada et al. 1997b; Shimada and Guengerich 2006; Shimada et al. 1996; Shimada et al. 2013; Shimada et al. 2001a; Shimada et al. 1999; Shimada et al. 1998)
N-Nitrosodi-n-propylamine	3A4	Nitrosamine	Depropylation, N- (medium Km, high activity, medium efficiency, activation)	10910959	(Teiber and Hollenberg 2000)
Parathion	2C9	Insecticide, organophosphate and acaricide, dicarboximide	Desulfuration (oxon formation), activation, medium Km, medium activity	17079358	(Foxenberg et al. 2007)
Parathion	1A1	Insecticide, organophosphate and acaricide, dicarboximide	Desulfuration (oxon formation), activation, medium Km, low activity	10414794, 21969518	(Ellison et al. 2012; Mutch et al. 1999)
Parathion	2A6	Insecticide, organophosphate and acaricide, dicarboximide	Desulfuration (oxon formation), activation	10794390, 12669189	(Eaton 2000; Mutch et al. 2003)
Parathion	3A5	Insecticide, organophosphate and acaricide, dicarboximide	Desulfuration (oxon formation), activation, medium Km, medium activity	10794390, 12669189, 10414794, 17079358, 17110060	(Buratti et al. 2006; Eaton 2000; Foxenberg et al. 2007; Mutch et al. 1999; Mutch et al. 2003)
Parathion	2B6	Insecticide, organophosphate and acaricide, dicarboximide	Desulfuration (oxon formation), activation, low Km, medium to high activity	10794390, 12669189, 10414794, 21782601, 12620367, 16757081, 17079358, 21969518	(Buratti et al. 2002; Buratti et al. 2003; Eaton 2000; Ellison et al. 2012; Foxenberg et al. 2007; Mutch et al. 1999; Mutch et al. 2003; Mutch and Williams 2006)
Parathion	1A2	Insecticide, organophosphate and acaricide, dicarboximide	Desulfuration (oxon formation), activation, low or medium Km, high activity	10794390, 12669189, 21782601, 12620367, 16757081, 17079358, 21969518	(Buratti et al. 2002; Buratti et al. 2003; Eaton 2000; Ellison et al. 2012; Foxenberg et al. 2007; Mutch et al. 2003; Mutch and Williams 2006)
Parathion	2C19	Insecticide, organophosphate and acaricide, dicarboximide	Desulfuration (oxon formation), activation, low Km, medium activity	10794390, 12669189, 21782601, 16757081, 17079358, 21969518	(Buratti et al. 2002; Eaton 2000; Ellison et al. 2012; Foxenberg et al. 2007; Mutch et al. 2003; Mutch and Williams 2006)
Parathion	2E1	Insecticide, organophosphate and acaricide, dicarboximide	Desulfuration (oxon formation), activation, very low activity	10794390, 12669189,16757081	(Eaton 2000; Mutch et al. 2003)
Parathion	2D6	Insecticide, organophosphate and acaricide, dicarboximide	Desulfuration (oxon formation), activation and 4-nitrophenol (p-nitrophenol) formation	10794390, 12669189, 16757081, 10996483	(Eaton 2000; Mutch et al. 2003; Mutch and Williams 2006; Sams et al. 2000)
Parathion	3A7	Insecticide, organophosphate and acaricide, dicarboximide	Desulfuration (oxon formation), activation	17110060, 17079358	(Buratti et al. 2006; Foxenberg et al. 2007)
Parathion	2C8	Insecticide, organophosphate and acaricide, dicarboximide	Desulfuration (oxon formation), activation	21782601, 10794390, 12669189, 10414794, 16757081	(Buratti et al. 2002; Eaton 2000; Mutch et al. 1999; Mutch et al. 2003; Mutch and Williams 2006)
Parathion	3A4	Insecticide, organophosphate and acaricide, dicarboximide	Desulfuration (oxon formation), activation, major enzyme, high Km, high activity	9023313, 10794390, 12669189, 10414794, 21782601, 12620367, 17079358, 17110060, 10996483	(Buratti et al. 2006; Buratti et al. 2002; Buratti et al. 2003; Butler and Murray 1997; Eaton 2000; Foxenberg et al. 2007; Mutch et al. 1999; Mutch et al. 2003; Sams et al. 2000)
3-(N-Phenylamino)propane-1,2-diol (PAP)	2C18	Toxic oil compound	Hydroxylation (aromatic), high Km, high activity, activation	17672514	(Martínez-Cabot et al. 2007)
PAP	2C8	Toxic oil compound	Hydroxylation (aromatic), high Km, high activity, activation	17672514	(Martínez-Cabot et al. 2007)
PAP	2C9	Toxic oil compound	Hydroxylation (aromatic), high Km, high activity, activation	17672514	(Martínez-Cabot et al. 2007)
PAP	2D6	Toxic oil compound	Hydroxylation (aromatic), high Km, high activity, activation	17672514	(Martínez-Cabot et al. 2007)
2,4,3´,5´-Tetramethoxystilbene	1B1	Cancer chemopreventive, trans-hydroxystilbene derivative	Oxidation (major enzyme, activation)	16120791, 11719446	(Chun et al. 2001; Chun et al. 2005)

Fig 3.

Metabolic activation of benzo[a]pyrene to toxic trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro product

The following examples taken from Table 2 illustrate PAH compounds for which metabolic activation is attributed as “high activity reaction” and/or “high activation” (for references see Table 2):

P450 1A1: 5-Methylchrysene, trans-5-methylchrysene-1,2-diol, 7,12-dimethylbenz[a]anthracene (7,12-DMBA), trans-7,12-DMBA-3,4-diol, (±)-benzo[a]pyrene (B[a]P) −7,8-dihydrodiol, cis-(−)-B[a]P-7,8-dihydrodiol, trans-(+)-B[a]P-7,8-dihydrodiol, trans-(−)-B[a]P-7,8-dihydrodiol, dibenzo[a,l]pyrene (DB[a,l]P), trans-(−)-DB[a,l]P-(11R,12R)-diol

P4501A2: Dibenzo[a,l]pyrene (DB[a,l]P)

P4501B1: B[a]P, (+,−)-B[a]P-7,8-dihydrodiol, cis-(−)-B[a]P-7,8-dihydrodiol, trans-(+)-B[a]P-7,8-dihydrodiol, trans-5-Methylchrysene-1,2-diol, trans-7,12-DMBA-3,4-diol), dibenzo[a,l]pyrene (DB[a,l]P), trans-(−)-DB[a,l]P-(11R,12R)-diol

P450 2A13: trans-5-Methylchrysene-1,2-diol

P450 2A6: trans-5-Methylchrysene-1,2-diol

Heterocyclic and aromatic amines

Activation of heterocyclic, aromatic, and azoaromatic amines is represented by 58 cadsentries (Table 2) of which 15 are attributed as “high activity” and/or “high activation” catalyzed by P450 1A1, 1A2, 1B1, 2A13, 2A6, and 3A4. The reactions of activation or aromatic and heterocyclic amines are presented in Fig. 4 and Fig. 5 as illustrated by activation of 2-aminofluorene and MeIQx, respectively.

Fig 4.

Activation of 2-amimnofluorene to toxic N-hydroxy product by P450 enzymes

Fig 5.

Activation of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQx) to toxic N-hydroxy product by P450 enzymes

The following examples illustrate metabolic activation of heterocyclic compounds by specific P450s (Table 2 and references therein):

P450 1A2: 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-6-methyldipyrido[1,2-a,3,2’-d]-imidazole (Glu-P-1)

P450 1A1: 2-Aminoanthracene (2-AA), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 6-aminochrysene

P450 1A2: 2-Aminofluorene (2-AF)

P450 1B1: 2-AA

P450 2A6: 2-AA

P450 2A13: 2-AF

P450 3A4: 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 6-aminochrysene

Insecticides

Activation of organophosphate insecticides is represented by chlorpyrifos, diazinon, parathion, and azinphos-methyl (Table 2 and references therein). The compounds are metabolically activated to neurotoxic metabolites (i.e. oxon derivatives) by desulfuration reactions catalyzed by P450 enzymes. Chlorpyrifos (Fig. 7) and parathion (Fig. 8) are activated by P450 1A2, 2B6, 2D6, 2C8, 2C19, 3A4, and 3A5 enzymes, of which P450 2B6 is the most prominent at lower concentrations (20 μM) and having the highest k_cat/K_m value. In addition to the oxon derivative, chloropyrifos is also metabolized to the less toxic 3,5,6-trichloro-2-pyridinol by P450 3A4 (Jan et al. 2016; Crane et al. 2012a; Crane et al. 2012b; Croom et al. 2010; Mutch and Williams 2006).

Fig 7.

Activation of parathion to a toxic oxon product by P450 enzymes

Fig 8.

Participation of human P450 Families 1–4 in activation of drugs to potentially toxic products (4039 reactions, 235 activation reactions)

Azinphos-methyl is activated primarily by P450 1A2 (at low concentrations), and 2B6 and 3A4 (at higher concentrations) (Table 2) (Buratti et al. 2002; Buratti et al. 2003). The entries in Table 2 show that at lower concentrations organophosphates are activated predominately by P450 1A1, 1A2, 2B6, and 2C19 and at high concentrations by P450 3A4.

Drugs

A present and historical view of the activation of drugs and their conversion to reactive metabolites as substrates of P450 enzymes has been reviewed recently by one of the authors of the present paper. It has been pointed out that P450 metabolic activity often prevents drug toxicity (for instance making drug elimination faster), but on the opposite side it can, in some cases, result in conversion of drugs to reactive metabolites that cause toxicity (Guengerich 2020). The final properties of the products of drug-P450 enzyme reactions can also be significantly affected by factors such as: a) variations in the activity caused by genetic polymorphism and thus primarily on the level of single nucleotide variations (SNVs), or b) by enzyme induction and/or inhibition of activity by environmental chemicals or by co-administered drug(s) (Guengerich and Rendic 2010; Guengerich 2020). Examples of drugs that are converted to toxic metabolites, due to activity of P450 enzymes, are listed in Table 3. It must be emphasized that most drugs, used in recommended doses, are not or are only slightly toxic per se due to extensive testing in preclinical and clinical testing of drugs. However, as mentioned before, toxic metabolites might be formed under circumstances of enhanced dose, when applied with other drugs/chemicals that might redirect metabolism pathway to formation of toxic metabolites, or when genetic polymorphism of the particular enzyme was not tested or observed in early drug testing. It is prudent to remember the words of Paracelsus, paraphrased, “the dose makes the poison (only the dose distinguishes a medicine from a poison)” (Borzelleca 2000). Selected examples of drugs taken from Table 3 are discussed, for which toxicity is related to metabolic conversion to toxic products and is known to occur during clinical use. In addition, therapeutic compounds are presented that are used as pro-drugs. Such pro-drugs are therapeutically inactive until activated by P450 enzymes but can became also cytotoxic in healthy cells/tissues when used in therapy (e.g., the anticancer drugs cyclophosphamide, ifosfamide, and AQ4N (banoxantrone; 1,4-bis{[2-(dimethylamino)ethyl]amino}−5,8-hydroxy-anthracene-9,10-dione bis-N-oxide)). Also included is the natural product drug ellipticine, which is used in cancer therapy and activated to a cytotoxic metabolite (Table 3). However, while the inherent toxicity of a drug might be lowered, the metabolites formed might be also less toxic and less therapeutically active. An example of such a drug is trabectedin (ecteinascidin 743), an anti-cancer drug of marine origin for which the side effects include myelosuppression, hepatotoxicity, and nausea and vomiting (Held-Warmkessel 2003). Trabectedin is metabolized by P450 3A4 (major enzyme) and in addition by P450s 2C9, 2C19, 2D6, and 2E1. Metabolic and inhibition studies revealed that the metabolites formed are less cytotoxic and less therapeutically active than the parent drug. Inhibitors of P450 enzymes significantly increased cytotoxicity of the drug in a human cell line model system (Reid et al. 2002; Brandon et al. 2005; Brandon et al. 2006).

Table 3.

Examples of the metabolic activation of drugs by human cytochrome P450 enzymes

Drug	P450	Category	Reaction	PMIDs	References
Acetaminophen (paracetamol)	3A4	Analgesic, antipyretic	Oxidation to N-acetyl-p-benzoquinone imine (NABQI), activation, major at toxic concentrations	9152386, 7956731, 8374050, 2729995, 8380689, 10741631, 10872641, 9633991, 9246016, 15576447, 17894464, 19219744	(Cameron et al. 2007; Cheung et al. 2005b; Laine et al. 2009; Li et al. 1994; Manyike et al. 2000; Patten et al. 1993; Raucy et al. 1989; Roe et al. 1993; Sarich et al. 1997; Sinclair et al. 1998; Thummel et al. 2000; Zhou et al. 1997)
Acetaminophen (paracetamol)	2E1	Analgesic, antipyretic	Oxidation to NABQI, high Km, high activity, major in vivo enzyme, activation	9152386, 8374050, 2729995, 8380689, 10741631, 10872641, 9633991, 9246016, 15576447, 9056059, 8354023, 15532721, 31024054, 19219744, 9548799, 11095574, 11866476	(Bai and Cederbaum 2004; Chen et al. 1998; Cheung et al. 2005b; Dong et al. 2000; Hazai et al. 2002; Laine et al. 2009; Manyike et al. 2000; O’Shea et al. 1997; Patten et al. 1993; Rahman et al. 2019; Raucy et al. 1989; Roe et al. 1993; Sarich et al. 1997; Sinclair et al. 1998; Thummel et al. 2000; Zand et al. 1993; Zhou et al. 1997)
Acetaminophen (paracetamol)	2D6	Analgesic, antipyretic	Oxidation to NABQI, high Km, low to medium activity, activation	9152386, 9548799, 11095574, 11866476, 19219744	(Chen et al. 1998; Dong et al. 2000; Hazai et al. 2002; Laine et al. 2009; Zhou et al. 1997)
Acetaminophen (paracetamol)	1A2	Analgesic, antipyretic	Oxidation to NABQI, low to medium activity, activation	9152386, 7956731, 8374050, 2729995, 8380689, 10741631, 10872641, 9633991, 9246016, 15576447, 19219744	(Cheung et al. 2005b; Laine et al. 2009; Li et al. 1994; Manyike et al. 2000; Patten et al. 1993; Raucy et al. 1989; Roe et al. 1993; Sarich et al. 1997; Sinclair et al. 1998; Thummel et al. 2000; Zhou et al. 1997)
Acetaminophen (paracetamol)	2A6	Analgesic, antipyretic	Oxidation to NABQI, minor reaction, activation	9548799, 11095574, 11866476, 19219744	(Chen et al. 1998; Dong et al. 2000; Hazai et al. 2002; Laine et al. 2009)
Aminoflavone, NSC 68628	1A2	Anticancer, antiproliferative, flavone derivative	Hydroxylation, N4’-, N-5, activation	16775196, 12065765	(Chen et al. 2006a; Kuffel et al. 2002)
Aminoflavone, NSC 68628	1A1	Anticancer, antiproliferative, flavone derivative	Hydroxylation, N4’-, N5-, activation	12065765, 15210858, 16775196, 12065765	(Chen et al. 2006a; Kuffel et al. 2002; Loaiza-Pérez et al. 2004)
Aminoflavone, NSC 68628	2C9	Anticancer, antiproliferative, flavone derivative	Hydroxylation, N5-, activation,	16775196	(Chen et al. 2006a)
Aminoflavone, NSC 68628	2C19	Anticancer, antiproliferative, flavone derivative	Hydroxylation, N5- (activation, major enzyme at high conc.)	16775196	(Chen et al. 2006a)
Banoxantrone; 1,4-bis([2-(dimethylamino)ethyl]amino)-5,8-hydroxy-anthracene-9,10-dione bis-N-oxide (AQ4N)	1A1	Anticancer, anthraquinone prodrug	Reduction to AQ4, activation	12214668, 16410820	(Patterson 2002; Yakkundi et al. 2006)
AQ4N	1A2	Anticancer, anthraquinone prodrug	Reduction to AQ4, activation	12214668	(Patterson 2002)
AQ4N	1B1	Anticancer, anthraquinone prodrug	Reduction to AQ4, activation	12214668	(Patterson 2002)
AQ4N	2B6	Anticancer, anthraquinone prodrug	Reduction to AQ4, activation	15712360, 16410820	(McErlane et al. 2005; Yakkundi et al. 2006)
AQ4N	3A4	Anticancer, anthraquinone prodrug	Reduction to AQ4, activation	9845092, 10834269, 12214668, 12489027	(McCarthy et al. 2003; Patterson 2002; Patterson et al. 1999; Raleigh et al. 1998)
AQ4N	2S1	Anticancer, anthraquinone prodrug	Reduction to AQ4, activation	20566689, 21430234	(Nishida et al. 2010; Xiao et al. 2011)
AQ4N	2W1	Anticancer, anthraquinone prodrug	Reduction to AQ4, activation	20566689	(Nishida et al. 2010)
Carbamazepine	1A2	Antiepileptic	Activation	11760814, 9630846	(Masubuchi et al. 2001; Wolkenstein et al. 1998)
Carbamazepine	3A4	Antiepileptic	Activation	9630846	(Wolkenstein et al. 1998)
Carbamazepine, C2-hydroxy	3A4	Antiepileptic, carbamazepine metabolite	Iminostilbene formation (activation, major enzyme)	16135660	(Pearce et al. 2005)
Carbamazepine, C2-hydroxy	2C19	Antiepileptic, carbamazepine metabolite	Iminostilbene formation (activation, minor enzyme)	16135660, 18463198	(Pearce et al. 2008; Pearce et al. 2005)
Carbamazepine, C2-hydroxy	2D6	Antiepileptic, carbamazepine metabolite	Iminostilbene formation (activation, minor enzyme), at high concentration	16135660	(Pearce et al. 2005)
Carbamazepine, C3-hydroxy	3A5	Antiepileptic, carbamazepine metabolite	Hydroxylation, C2- (aromatic), activation	18463198	(Pearce et al. 2008)
Carbamazepine, C3-hydroxy	3A7	Antiepileptic, carbamazepine metabolite	Hydroxylation, C2- (aromatic), activation	18463198	(Pearce et al. 2008)
Carbamazepine, C3-hydroxy	2C19	Antiepileptic, carbamazepine metabolite	Hydroxylation, C2- (aromatic), activation, high activity	18463198	(Pearce et al. 2008)
Carbamazepine, C3-hydroxy	3A4	Antiepileptic, carbamazepine metabolite	Hydroxylation, C2- (aromatic), activation, major enzyme	18463198	(Pearce et al. 2008)
Carbamazepine, C3-hydroxy	1A1	Antiepileptic, carbamazepine metabolite	Hydroxylation, C2- (aromatic), activation, minor enzyme	18463198	(Pearce et al. 2008)
Carbamazepine, C3-hydroxy	1A2	Antiepileptic, carbamazepine metabolite	Hydroxylation, C2- (aromatic), activation, minor enzyme	18463198	(Pearce et al. 2008)
Carbamazepine, C3-hydroxy	2C18	Antiepileptic, carbamazepine metabolite	Hydroxylation, C2- (aromatic), activation, minor enzyme	18463198	(Pearce et al. 2008)
Clopidogrel	2C9	Antiplatelet, P2Y12 antagonist (thiophene)	Activation	19812348	(Kazui et al. 2010)
Clopidogrel	2B6	Antiplatelet, P2Y12 antagonist (thiophene)	Oxidation (2-oxo formation), low activity, or no activity, activation	12485953, 19812348, 26654298	(Clarke and Waskell 2003; Kazui et al. 2010; Zhai et al. 2016)
Clopidogrel	3A4	Antiplatelet, P2Y12 antagonist (thiophene)	Oxidation (2-oxo formation), major enzyme, activation	12485953, 10331074, 23770199, 19812348	(Clarke and Waskell 2003; Guengerich 1999; Kazui et al. 2010; Zahno et al. 2013)
Clopidogrel	2C19	Antiplatelet, P2Y12 antagonist (thiophene)	Oxidation (2-oxo formation), medium activity, activation	17682072, 19812348, 26654298	(Kazui et al. 2010; Walsky and Obach 2007; Zhai et al. 2016)
Clopidogrel	1A2	Antiplatelet, P2Y12 antagonist (thiophene)	Oxidation (2-oxo formation), very low to medium activity, activation	12485953, 19812348	(Clarke and Waskell 2003; Kazui et al. 2010)
Cyclophosphamide	3A7	Anticancer, alkylating, oxazaphosporine	N-Dechloroethylation (high Km, medium activity), activation to neuro- and nephrotoxic metabolite	15919850	(Chen et al. 2005)
Cyclophosphamide	3A4	Anticancer, alkylating, oxazaphosporine	N-Dechloroethylation (major enzyme, major reaction, high Km, high activity), activation to neuro- and nephrotoxic metabolite	8242617, 9010702, 9331082, 10692561, 15919850	(Bohnenstengel et al. 1996; Chang et al. 1993; Chen et al. 2005; Huang et al. 2000; Ren et al. 1997)
Cyclophosphamide	3A4	Anticancer, alkylating, oxazaphosporine	C4-Hydroxylation (high Km, high activity), activation to cytotoxic metabolite, major enzyme at high concentration	8242617, 9010702, 9331082, 9157990, 10348794, 10991840, 10692561, 9923542	(Bohnenstengel et al. 1996; Chang et al. 1993; Chang et al. 1997; Huang et al. 2000; Philip et al. 1999; Ren et al. 1997; Roy et al. 1999b; Zhou et al. 2000)
Cyclophosphamide	3A7	Anticancer, alkylating, oxazaphosporine	C4- Hydroxylation (high Km, low to high activity, major reaction), activation to cytotoxic metabolite	10348794, 15919850;9157990	(Chen et al. 2005; Roy et al. 1999b; Chang et al. 1997)
Cyclophosphamide	2C18	Anticancer, alkylating, oxazaphosporine	C4-Hydroxylation (low Km, activation to cytotoxic metabolite)	8242617, 9010702, 9331082, 10348794, , 10692561, 9157990	(Bohnenstengel et al. 1996; Chang et al. 1993; Chang et al. 1997; Huang et al. 2000; Ren et al. 1997; Roy 199b)
Cyclophosphamide	2B6	Anticancer, alkylating, oxazaphosporine	C4-Hydroxylation (major enzyme, major reaction, high Km, high activity, activation to cytotoxic metabolite), genetic polymorphism influence	8242617, 9010702, 9331082, 9157990, 10348794, 10991840, 10692561, 15919850, 10471061, 17502835, 11360624	(Bohnenstengel et al. 1996; Chang et al. 1993; Chang et al. 1997; Chen et al. 2005; Gervot et al. 1999; Huang et al. 2000; Nakajima et al. 2007; Ren et al. 1997; Roy et al. 1999b; Wu et al. 1997)
Dacarbazine	1A1	Anticancer, alkylating, imidazole carboxamide	N-Demethylation (major enzyme, activation)	10473105, 27428168	(Lewis et al. 2016; Reid et al. 1999)
Dacarbazine	1A2	Anticancer, alkylating, imidazole carboxamide	N-Demethylation (major enzyme, activation)	10473105, 27428168	(Lewis et al. 2016; Reid et al. 1999)
Dacarbazine	2E1	Anticancer, alkylating, imidazole carboxamide	N-Demethylation (activation, at higher concentration)	10473105	(Reid et al. 1999)
Dapsone	2B6	Antileprotic	Activation, minor contribution	19998329	(Ganesan et al. 2010)
Dapsone	2D6	Antileprotic	Activation, minor contribution	19998329	(Ganesan et al. 2010)
Dapsone	3A4	Antileprotic	N-Hydroxylation, (high Km, activation or no activity)	7586950, 8703658, 8181193, 8742227, 1588928, 19998329	(Fleming et al. 1992; Ganesan et al. 2010; Gill et al. 1995; Irshaid et al. 1996; May et al. 1994; Mitra et al. 1995)
Dapsone	2C8	Antileprotic	N-Hydroxylation, (high Km, minor enzyme), activation	10901692, 19998329	(Ganesan et al. 2010; Winter et al. 2000)
Dapsone	2C19	Antileprotic	N-Hydroxylation, (high Km, minor reaction), major enzyme for activation	10901692	(Winter et al. 2000)
Dapsone	2E1	Antileprotic	N-Hydroxylation, (low Km, activation), or no activity	7586950, 8703658, 8181193, 8742227, 1588928, 10901692	(Fleming et al. 1992; Gill et al. 1995; Irshaid et al. 1996; May et al. 1994; Mitra et al. 1995; Winter et al. 2000)
Dapsone	2C18	Antileprotic	N-Hydroxylation, (medium Km, minor reaction), activation	10901692	(Winter et al. 2000)
Dapsone	2C9	Antileprotic	N-Hydroxylation (medium Km, activation)	7586950, 8703658, 10901692, 9521735, 12920490	(Gill et al. 1995; Korzekwa et al. 1998; Li et al. 2003; Mitra et al. 1995; Winter et al. 2000)
Desogestrol	3A4	Estrogen, contraceptive	Oxygenation, mechanism-based inhibition	2133086, 2256525	{Guengerich, 1990, 12562; Guengerich, 1990, 13522}
Diclofenac	2C9	NSAID, cyclooxygenase-2 (COX-2) inhibitor	C4’-Hydroxylation, formation of 1´,4´-benzoquinoneimine (low or medium Km, medium or high activity, high efficiency, major enzyme, major reaction), activation	9698079, 8417277, 10027801, 10027798, 10950847, 10572000, 10449188, 12464247, 27130197, 19022234	(Bort et al. 1999; den Braver et al. 2016; Leemann et al. 1993; Mancy et al. 1999; Melet et al. 2003; Ngui et al. 2000; Shen et al. 1999; Tang et al. 1999; Yamazaki et al. 1998a)
Diclofenac	3A4	NSAID, COX-2 inhibitor	C5-Hydroxylation (major enzyme, high Km), reaction at high concentration (>100 μM), formation of proposed reactive intermediate	8417277, 10027801, 10027798, 10950847, 10572000, 12438516, 12871048, 17584015, 19022234	(Kalgutkar et al. 2007; Kumar et al. 2002; Lauer et al. 2009; Leemann et al. 1993; Mancy et al. 1999; Ngui et al. 2000; Shen et al. 1999; Tang 2003; Tang et al. 1999)
Diclofenac	3A4	NSAID, COX-2 inhibitor	C5-Hydroxylation, formation of 2,5-quinoneimine, activation	27130197	(den Braver et al. 2016)
Diclofenac 5-hydroxy	2C9	NSAID, COX-2 inhibitor	Activation, formation of 2,5-quinoneimine, activation	27130197	(den Braver et al. 2016)
Ellipticine	2B6	Anticancer, topoisomerase II inhibitor	C12-Hydroxylation, weak activation	11755121, 12123750, 22917556	(Frei et al. 2002; Stiborová et al. 2001a; Stiborová et al. 2012b)
Ellipticine	3A4	Anticancer, topoisomerase II inhibitor	C13- and C12-hydroxylation, activation (major enzyme)	11755121, 12123750, 15548707, 20027146, 22917556	(Frei et al. 2002; Martinkova et al. 2009:Stiborová, 2012b; Stiborová et al. 2001a; Stiborová et al. 2004)
Ellipticine	2C9	Anticancer, topoisomerase II inhibitor	C13- and C12-hydroxylation, activation, low activity	11755121, 12123750, 15548707, 22917556	(Frei et al. 2002; Stiborová et al. 2001a; Stiborová et al. 2012b; Stiborová et al. 2004)
Ellipticine	2D6	Anticancer, topoisomerase II inhibitor	C13- and C12-hydroxylation, activation, low activity	11755121, 12123750, 15548707, 22917556	(Frei et al. 2002; Stiborová et al. 2001a; Stiborová et al. 2012b; Stiborová et al. 2004)
Ellipticine	2E1	Anticancer, topoisomerase II inhibitor	C13- and C12-hydroxylation, activation, low activity	11755121, 12123750, 22917556	(Frei et al. 2002; Stiborová et al. 2001a; Stiborová et al. 2012b)
Ellipticine	2C19	Anticancer, topoisomerase II inhibitor	C13- and C12-hydroxylation, activation, low activity	11755121, 12123750, 15548707, 22917556	(Frei et al. 2002; Stiborová et al. 2001a; Stiborová et al. 2012b; Stiborová et al. 2004)
Ellipticine	1A1	Anticancer, topoisomerase II inhibitor	C13-Hydroxylation, activation	11755121, 12123750, 15548707, 22917556, 20027146	(Frei et al. 2002; Martinkova et al. 2009; Stiborová et al. 2001a; Stiborová et al. 2012b; Stiborová et al. 2004)
Ellipticine	1A2	Anticancer, topoisomerase II inhibitor	C13-Hydroxylation, activation, low activity	11755121, 12123750, 15548707, 22917556	(Frei et al. 2002; Stiborová et al. 2001a; Stiborová et al. 2012b; Stiborová et al. 2004)
Ellipticine	1B1	Anticancer, topoisomerase II inhibitor	C13-Hydroxylation, activation, low activity	11755121, 12123750, 22917556, 20027146	(Frei et al. 2002; Martinkova et al. 2009; Stiborová et al. 2001a; Stiborová et al. 2012b; Stiborová et al. 2004)
17α-Ethynylestradiol	3A4	Estrogen, contraceptive	Epoxidation (activation)	16251255	(Chen et al. 2006b)
17α-Ethynylestradiol	3A4	Estrogen, contraceptive	Oxygenation, mechanism-based inhibition	17251390, 17584015, 11907170, 3285175, 2133086, 2256525	(Guengerich 1988; Guengerich 1990a; Guengerich 1990b; Kalgutkar et al. 2007; Lin and Hollenberg 2007; Lin et al. 2002)
17α-Ethynylestradiol	3A5	Estrogen, Contraceptive	Oxygenation, mechanism-based inhibition	17251390, 17584015	(Kalgutkar et al. 2007; Lin and Hollenberg 2007)
Flutamide	2C9	Anticancer, antiandrogen, nonsteroidal, nitroaromatic	C2-Hydroxylation, activation	16507648	(Goda et al. 2006)
Flutamide	1B1	Anticancer, antiandrogen, nonsteroidal, nitroaromatic	C2-Hydroxylation (activation, major enzyme in cancer cells)	11160641	(Rochat et al. 2001)
Flutamide	1A2	Anticancer, antiandrogen, nonsteroidal, nitroaromatic	C2-Hydroxylation (activation, major enzyme)	8386241, 16507648, 9351907, 12052211, 18411402	(Berson et al. 1993; Goda et al. 2006; Kang et al. 2008; Patterson and Murray 2002; Shet et al. 1997)
Flutamide	1A2	Anticancer, antiandrogen, nonsteroidal, nitroaromatic	Amide oxidation, N-hydroxylation following amide cleavage	17403914, 18411402, 16507648	(Kang et al. 2007; Kang et al. 2008; Goda et al. 2006)
Flutamide	2C19	Anticancer, antiandrogen, nonsteroidal, nitroaromatic	Amide oxidation, N-hydroxylation following amide cleavage	17403914, 18411402, 16507648	(Kang et al. 2007; Kang et al. 2008; Goda et al. 2006)
Flutamide	3A4	Anticancer, antiandrogen, nonsteroidal, nitroaromatic	Amide oxidation, N-hydroxylation following amide cleavage	16507648	(Kang et al. 2007)
Gestodene	3A4	Estrogen, contraceptive	Oxygenation, mechanism-based inhibition, hydroxylation	2133086, 2256525, 8664172	(Guengerich 1990a; Guengerich 1990b; Ward and Back 1993)
Haloperidol	1A1	Antipsychotic, butyrophenone	Oxidation, pyridinium metabolite formation, activation	11717183, 16841959	(Avent et al. 2006; Fang et al. 2001)
Haloperidol	1B1	Antipsychotic, butyrophenone	Oxidation, pyridinium metabolite formation, activation	16841959	(Avent et al. 2006)
Haloperidol	2D6	Antipsychotic, butyrophenone	Oxidation, pyridinium metabolite formation, activation	9140699, 9844810	(Fang et al. 1997; Usuki et al. 1998)
Haloperidol	3A5	Antipsychotic, butyrophenone	Oxidation, pyridinium metabolite formation, activation	9140699, 9844810, 9431831, 10628896, 12584149, 11167668, 16841959	(Avent et al. 2006; Fang et al. 1997; Kalgutkar et al. 2003; Kudo and Ishizaki 1999; Pan et al. 1997; Shin et al. 2001; Usuki et al. 1998)
Haloperidol	3A7	Antipsychotic, butyrophenone	Oxidation, pyridinium metabolite formation, activation	16841959	(Avent et al. 2006)
Haloperidol	3A4	Antipsychotic, butyrophenone	Oxidation, pyridinium metabolite formation, activation	9140699, 9844810, 9431831, 10628896, 12584149, 11167668, 11717183, 24913773	(Fang et al. 1997; Fang et al. 2001; Kalgutkar et al. 2003; Kudo and Ishizaki 1999; Kurth et al. 2014; Pan et al. 1997; Shin et al. 2001; Usuki et al. 1998)
Haloperidol	3A4	Antipsychotic, butyrophenone	Oxidation, pyridinium metabolite formation, activation	9431831, 10628896, 12584149, 11167668, 11717183, 24913773	(Fang et al. 2001; Kalgutkar et al. 2003; Kudo and Ishizaki 1999; Kurth et al. 2014; Pan et al. 1997; Shin et al. 2001)
Halothane	2E1	General inhalation anesthetic, haloalkane	Dehalogenation, oxidative, activation (major reaction, major enzyme in vivo, low Km)	9616199, 9103523, 8886607, 10805064, 11506127, 11684364, 17584015, 24913773	(Kalgutkar et al. 2007; Kharasch et al. 2000; Kurth et al. 2014; Minoda and Kharasch 2001; Spracklin et al. 1997; Spracklin and Kharasch 1998; Spracklin et al. 1996; White and De Matteis 2001)
Halothane	2A6	General inhalation anesthetic, haloalkane	Dehalogenation, oxidative, activation (minor reaction, high Km)	9616199, 9103523, 8886607, 10805064, 11506127, 24913773	(Kalgutkar et al. 2007; Kharasch et al. 2000; Kurth et al. 2014; Spracklin et al. 1997; Spracklin and Kharasch 1998; Spracklin et al. 1996)
Halothane	3A4	General inhalation anesthetic, haloalkane	Dehalogenation, reductive, activation (high Km)	9616199, 9103523, 8886607, 10805064, 11506127, 24913773	(Kalgutkar et al. 2007; Kharasch et al. 2000; Kurth et al. 2014; Spracklin et al. 1997; Spracklin and Kharasch 1998; Spracklin et al. 1996)
Halothane	2A6	General inhalation anesthetic, haloalkane	Dehalogenation, reductive, activation (low Km)	9616199, 9103523, 8886607, 10805064, 11506127, 24913773	(Driscoll et al. 2007; Kharasch et al. 2000; Kurth et al. 2014; Spracklin et al. 1997; Spracklin and Kharasch 1998; Spracklin et al. 1996)
Halothane	2B6	General inhalation anesthetic, haloalkane	Oxidation, activation	11684364, 24913773	(Kalgutkar et al. 2007; White and De Matteis 2001)
Halothane	2E1	General inhalation anesthetic, haloalkane	Oxidation, activation	11684364, 24913773	(Kalgutkar et al. 2007; White and De Matteis 2001)
Ifosfamide, (R)-	3A5	Anticancer, alkylating, oxazaphosporine	Hydroxylation, C4, activation to cytotoxic product	10348794, 15821045, 10534317, 16854777	(Lu et al. 2006; McCune et al. 2005; Roy et al. 1999a)
Ifosfamide, (R)-, (S)-	3A4	Anticancer, alkylating, oxazaphosporine	Dechloroethylation, N2-, N3- (high Km, high activity, major enzyme, major reaction for (S)-), activation to neuro- and nephrotoxic metabolites	8242617, 10692561, 15919850, 15821045, 16854777, 10534317, 8161344, 8071856, 10101149, 9923542, 17464949	(Chang et al. 1993; Chen et al. 2005; Chugh et al. 2007; Granvil et al. 1999; Huang et al. 2000; Lu et al. 2006; McCune et al. 2005; Murray et al. 1994; Philip et al. 1999; Roy et al. 1999a; Walker et al. 1994)
Ifosfamide, (R)-, (S)-	2B6	Anticancer, alkylating, oxazaphosporine	Dechloroethylation, N2-, N3- (high Km, high activity, major enzyme, major reaction), activation to neuro- and nephrotoxic metabolites.	10692561, 15919850, 15821045, 16854777, 10534317	(Chen et al. 2005; Huang et al. 2000; Lu et al. 2006; McCune et al. 2005; Roy et al. 1999a)
Ifosfamide, (R)-, (S)-	2C9	Anticancer, alkylating, oxazaphosporine	Hydroxylation, C4- (low Km), activation to cytotoxic metabolites	8242617, 9157990	(Chang et al. 1993; Chang et al. 1997)
Ifosfamide, (S)-	2B6	Anticancer, alkylating, oxazaphosporine	Hydroxylation, C4- (high Km, medium (R-) and high (S-) activity, major enzyme), activation to cytotoxic metabolites	8242617, 10348794, 10692561, 9157990, 15919850, 25934575	(Calinski et al. 2015; Chang et al. 1993; Chang et al. 1997; Huang et al. 2000; Roy et al. 1999a)
Isoniazid	2E1	Antituberculotic, pyridine	Oxidation, activation, major enzyme	12668988, 18071298	(Huang et al. 2003; Shen et al. 2008)
3-Ketodesogestrol	3A4	Estrogen, contraceptive	Oxygenation, mechanism-based inhibition	2133086, 2256525	(Guengerich 1990a; Guengerich 1990b)
Levonorgestrol	3A4	Estrogen, contraceptive	Oxygenation, mechanism-based inhibition	2133086, 2256525	(Guengerich 1990a; Guengerich 1990b)
Norethisterone	3A4	Estrogen, contraceptive	Oxygenation, mechanism-based inhibition	2133086, 2256525	(Guengerich 1990a; Guengerich 1990b)
Phencyclidine	2B6	Hallucinogenic (angel dust, angel hair, angel mist), analgesic and anesthetic	C-Hydroxylation, (p-hydroxy and quinone methide formation), activation	17892269, 16326815, 16782764	(Driscoll et al. 2007; Jushchyshyn et al. 2006; Shebley et al. 2006)
Phencyclidine	2C19	Hallucinogenic (angel dust, angel hair, angel mist), analgesic and anesthetic	C-Hydroxylation (p-hydroxy and quinone methide formation), activation	17892269, 16326815, 16782764	(Driscoll et al. 2007; Jushchyshyn et al. 2006; Shebley et al. 2006)
Raloxifene	2D6	Anticancer and prevention of osteoporosis, antiestrogen, estrogen receptor modulator, benzothiophene	Oxidation, activation, minor enzyme	12119000	(Chen et al. 2002)
Raloxifene	3A4	Anticancer and prevention of osteoporosis, antiestrogen, estrogen receptor modulator, benzothiophene	3´-Hydroxylation, oxidation, glutathione (GSH) adduct formation, activation	12119000, 18052110, 17497897, 17584015, 20405834	(Baer et al. 2007; Chen et al. 2002; Hollenberg et al. 2008; Kalgutkar et al. 2007; Moore et al. 2010a)
Raloxifene	3A4	Anticancer and prevention of osteoporosis, antiestrogen, estrogen receptor modulator, benzothiophene	Dehydrogenation, activation, reactive diquinonemethide and 7-hydroxyraloxifene formation	20405834, 20812728, 17867646, 12119000	(Chen et al. 2002; Moore et al. 2010a; Moore et al. 2010b; Yukinaga et al. 2007)
Tamoxifen	1A1	Anticancer, antiestrogen, estrogen receptor modulator, triphenylethyleneamine	Hydroxylation, C3-, C4- (catechol formation, activation)	10348797, 8293548, 12018981, 12124303, 12419838, 12623757	(Boocock et al. 2002; Crewe et al. 2002; Dehal and Kupfer 1999; Hu et al. 2003; Notley et al. 2002; Styles et al. 1994)
Tamoxifen	3A5	Anticancer, antiestrogen, estrogen receptor modulator, triphenylethyleneamine	Hydroxylation, C3-, C4- (catechol formation, activation)	10348797, 8293548, 12018981, 12124303, 12419838, 12623757	(Boocock et al. 2002; Crewe et al. 2002; Dehal and Kupfer 1999; Hu et al. 2003; Notley et al. 2002; Styles et al. 1994)
Tamoxifen	3A5	Anticancer, antiestrogen, estrogen receptor modulator, triphenylethyleneamine	Cα-Hydroxylation, activation, at high concentration (250 μM)	15159443, 16533026	(Desta et al. 2004; Notley et al. 2005)
Tamoxifen	3A4	Anticancer, antiestrogen, estrogen receptor modulator, triphenylethyleneamine	Cα-Hydroxylation (major enzyme, activation), at low and high concentrations (18 and 250 μM)	10348797, 8293548, 12018981, 12124303, 12419838, 12623757, 16533026, 15159443, 14678348, 12971802	(Boocock et al. 2002; Coller et al. 2004; Crewe et al. 2002; Dehal and Kupfer 1999; Desta et al. 2004; Hu et al. 2003; Kim et al. 2003; Notley et al. 2005; Notley et al. 2002; Styles et al. 1994)
Tamoxifen	2B6	Anticancer, antiestrogen, estrogen receptor modulator, triphenylethyleneamine	Oxidation, activation	12061800, 7748182	(Stiborová et al. 2002; White et al. 1995)
Tamoxifen	3A4	Anticancer, antiestrogen, estrogen receptor modulator, triphenylethyleneamine	Oxidation, activation	7748182	(White et al. 1995)
Tamoxifen, 3-hydroxy (droloxifene)	2D6	Anticancer, antiestrogen, estrogen receptor modulator, triphenylethyleneamine	C4-Hydroxylation, (catechol form., low extent, activation)	10348797	(Dehal and Kupfer 1999)
Tamoxifen 3-hydroxy, (droloxifene)	3A4	Anticancer, antiestrogen, estrogen receptor modulator, triphenylethyleneamine	C4-Hydroxylation, (catechol formation, major reaction, activation)	10348797	(Dehal and Kupfer 1999)
Tamoxifen, 4-hydroxy	3A4	Anticancer, antiestrogen, estrogen receptor modulator, triphenylethyleneamine, tamoxifen metabolite	N-Demethylation, activation, formation of major antiestrogenic metabolite	15159443	(Desta et al. 2004)
Tamoxifen, 4-hydroxy	2D6	Anticancer, antiestrogen, estrogen receptor modulator, triphenylethyleneamine, tamoxifen metabolite	C3-Hydroxylation (catechol formation, low extent, activation)	10348797	(Dehal and Kupfer 1999)
Tamoxifen, 4-hydroxy	3A4	Anticancer, antiestrogen, estrogen receptor modulator, triphenylethyleneamine, tamoxifen metabolite	C3-Hydroxylation (catechol formation, major reaction, activation)	10348797, 15159443	(Dehal and Kupfer 1999; Desta et al. 2004)
Tamoxifen, N-desmethyl	2D6	Anticancer, antiestrogen, estrogen receptor modulator, triphenylethyleneamine, tamoxifen metabolite	C4-Hydroxylation (major enzyme, activation), formation of major antiestrogenic metabolite, genetic polymorphism influences clinical response, proposed clinically significant drug-drug interaction potential when inhibited	14652237, 15159443, 15111773, 16815318, 17518364, 23711794, 30536272, 29637493, 26423799	(Beverage et al. 2007; Bezerra et al. 2018; Borges et al. 2006; Desta et al. 2004; Hansten 2018; Johnson et al. 2004; Stearns et al. 2003; Watanabe et al. 2015; Yang et al. 2013)
Thiabendazole	1A1	Antifungal, benzimidazole	C5-Hydroxylation, activation,	12920490	(Li et al. 2003)
Thiabendazole	1A2	Antifungal, benzimidazole	C5-Hydroxylation (activation, major enzyme)	10936227, 9565779, 12920490	(Coulet et al. 1998; Coulet et al. 2000; Li et al. 2003)
Thiabendazole	1B1	Antifungal, benzimidazole	C5-Hydroxylation, activation,	12920490	(Li et al. 2003)
Tienilic acid	2C9	Diuretic and uricosuric, 2-aroylthiophene	Epoxidation (thiophene ring), activation, proposed reactive intermediate formation in mechanism-based inhibition process	8286335, 8075377, 17584015, 8477697	(Kalgutkar et al. 2007; Lecoeur et al. 1994; Lopez Garcia et al. 1993; López-Garcia et al. 1994)
Tienilic acid	2C9	Diuretic and uricosuric, 2-aroylthiophene	Oxidation, S-oxygenation (thiophene ring), activation, proposed reactive intermediate formation in mechanism-based inhibition process	17584015	(Kalgutkar et al. 2007)
Tolcapone, acetylamine	1A2	Dopaminergic, catechol-O-methyltransferase (COMT) inhibitor, tolcapone metabolite	Oxidation, activation, major enzyme	10806608, 12588182	(Jorga et al. 2000; Smith et al. 2003b)
Tolcapone, amine	1A2	Dopaminergic, COMT inhibitor, tolcapone metabolite	Oxidation, activation, major enzyme	10806608, 12588182	(Jorga et al. 2000; Smith et al. 2003b)
Tolmetin	3A4	Analgesic, anti-inflammatory	Epoxidation, activation	16251255	(Chen et al. 2006b)
Toremifene	3A4	Antiestrogen, estrogen receptor modulator, triphenylethylene	Cα-Hydroxylation, activation	12971802, 26423799	(Kim et al. 2003; Watanabe et al. 2015)
Toremifene, N-desmethyl	2C9	Antiestrogen, estrogen receptor modulator, triphenylethylene	4-Hydroxylation, endoxifen formation, activation	26423799	(Watanabe et al. 2015)
Toremifene, N-desmethyl	2D6	Antiestrogen, estrogen receptor modulator, triphenylethylene	4-Hydroxylation, activation	26423799	(Watanabe et al. 2015)
N,Ń,N´´-Triethylene thiophosphoramide (ThioTEPA)	3A4	Anticancer, alkylating agent, aziridine	Desulfuration, N,Ń,N´´-triethylene phosphoramide (TEPA) formation (major enzyme, activation)	12107550, 19076156	(Ekhart et al. 2009; Jacobson et al. 2002)
ThioTEPA	2B6	Anticancer, alkylating agent, aziridine	Desulfuration, TEPA formation (minor enzyme, activation)	12107550, 15121764, 17584015, 19076156	(Ekhart et al. 2009; Harleton et al. 2004; Jacobson et al. 2002; Kalgutkar et al. 2007)
ThioTEPA	1B1	Anticancer, alkylating agent, aziridine	Desulfuration, TEPA formation (minor enzyme, activation)	15121764	(Harleton et al. 2004)
Troglitazone	1A2	Peroxisome proliferation activation receptor (PPAR)-γ agonist, thiazolidinedione, glitazone	Oxidation of thiazolidinedione ring, activation	15155556	(He et al. 2004a)
Troglitazone	2C8	PPARγ agonist, thiazolidinedione, glitazone	Oxidation of thiazolidinedione ring, activation	10534310	(Yamazaki et al. 1999)
Troglitazone	2C19	PPARγ agonist, thiazolidinedione, glitazone	Oxidation of thiazolidinedione ring, activation	15155556	(He et al. 2004a)
Troglitazone	2D6	PPARγ agonist, thiazolidinedione, glitazone	Oxidation of thiazolidinedione ring, activation	15155556	(He et al. 2004a)
Troglitazone	2E1	PPARγ agonist, thiazolidinedione, glitazone	Oxidation of thiazolidinedione ring, activation	15155556	(He et al. 2004a)
Troglitazone	3A4	PPARγ agonist, thiazolidinedione, glitazone	Oxidation of thiazolidinedione ring, activation	15155556, 11511170, 11389877, 10534310, 11170509	(He et al. 2004a; He et al. 2001; Kassahun et al. 2001; Tettey et al. 2001; Yamazaki et al. 1999)
Troglitazone	3A5	PPARγ agonist, thiazolidinedione, glitazone	Oxidation of thiazolidinedione ring, activation	15155556	(He et al. 2004a)
Troglitazone	2C8	PPARγ agonist, thiazolidinedione, glitazone	Oxidation of thiazolidinedione ring (activation, major enzyme)	15155556	(He et al. 2004a)
Valproic acid	2A6	Antiepileptic, histone deacetylase (HDAC) inhibitor	Dehydrogenation (desaturation, 4-ene formation, activation), minor enzyme	16945988, 10461547, 9353388	(Ekins and Wrighton 1999; Kiang et al. 2006; Sadeque et al. 1997)
Valproic acid	2C9	Antiepileptic, HDAC inhibitor	Dehydrogenation (desaturation, 4-ene formation, activation), major enzyme	16945988, 10461547, 9353388, 14597963	(Ekins and Wrighton 1999; Ho et al. 2003; Kiang et al. 2006; Sadeque et al. 1997)
Valproic acid	2B6	Antiepileptic, HDAC inhibitor	Dehydrogenation (desaturation, 4-ene formation, activation), minor enzyme	16945988, 10461547	(Ekins and Wrighton 1999; Kiang et al. 2006)

The numbers of activation reactions of drugs as substrates of human P450 enzymes are presented in Fig. 8, calculated from our records. Of the total of 4,039 reactions, 235 (~ 6%) involve activation and formation of potentially toxic intermediates or metabolites. P450 3A4 clearly dominated in the formation of toxic metabolites compared with other P450s, catalyzing ~25% of the bioactivation reactions.

The following examples illustrate the participation of P450 enzymes in the bioactivation of selected drugs (Table 3).

AQ4N

AQ4N, an aliphatic amine di-N-oxide, is a potent topoisomerase II inhibitor and in clinical trials as a potential anticancer drug. It is inactive until enzymatically bioactivated to an active amine under the reductive conditions present in hypoxic tumor cells (Fig. 9) (Patterson 1993; Lalani et al. 2007; O’Rourke et al. 2008). Because the amine AQ4 is very toxic to normal cells, it is not inherently suitable for delivery as an anticancer drug. AQ4N is reported to be substrate of several P450s (i.e. 1A1, 1A2, 1B1, 2B6, 2W1, 2S1, and 3A4) (Table 3 and references therein), but most efficiently by P450s 1A1 and 2B6 (Yakkundi et al. 2006)). Under reducing oxygen conditions (hypoxia) AQ4N is reduced to the cytotoxic AQ4-mono-N-oxide (AQ4M) and amine (AQ4) (Patterson et al. 1999). Of the enzymes involved in the metabolism of AQN4, P450 2W1 is highly expressed in some human colon and adrenal tumors and was suggested as tumor-specific enzyme. In addition, strong expression of P450 2S1 has been reported in tumors of epithelial origin and hypoxic tumors and the gene was found to be overexpressed under hypoxic conditions (Karlgren et al. 2005; Saarikoski et al. 2005; Rivera et al. 2007; Nishida et al. 2010; Xiao et al. 2011).

Fig 9.

Activation of AQ4N by P450 enzymes

Cyclophosphamide and ifosfamide

Cyclophosphamide and ifosfamide are widely used anticancer agents that require metabolic activation by P450 enzymes (Figs. 10 and 11, respectively). While 4-hydroxylation yields DNA-alkylating and cytotoxic metabolites, N-dechloroethylation results in the generation of neuro- and nephrotoxic products. Cyclophosphamide and ifosfamide undergo extensive P450-catalyzed metabolism to yield both active (4-hydroxylated) and therapeutically inactive but neurotoxic N-dechloroethyl metabolites, and ovarian toxicity is a major concern with cyclophosphamide therapy. The human liver microsomal P450 metabolism of cyclophosphamide and ifosfamide 4-hydroxylation is well characterized (Table 3 and references therein). Of all P450 enzymes, P450 3A4 exhibited the highest N-dechloroethylation activity (bioactivation) toward both cyclophosphamide and ifosfamide, whereas P450 2B6 and P450 3A4 displayed high N-dechloroethylation activity toward ifosfamide but not cyclophosphamide. With cyclophosphamide as substrate, P450 3A4 was shown to catalyze ≥ 95% of liver microsomal N-dechloroethylation, whereas with ifosfamide as substrate, P450 3A4 catalyzed an average of ~70% of liver microsomal N-dechloroethylation (range 40–90%), with the balance of this activity catalyzed by P450 2B6 (range 10–70%, depending on the P450 2B6 content of the liver) (Huang and Waxman 2000). In the case of cyclophosphamide treatment, determination of selected P450 enzyme genotypes (such as frequencies of the variant alleles CYP2B6*5, CYP2C19*2, CYP2C9*2, and CYP3A5*3) has been proposed for predicting the risk of premature ovarian failure in lupus nephritis patients (Takada et al. 2004).

Fig 10.

Metabolic activation of CPA

Fig 11.

Metabolic activation of IFA

Acetaminophen

Acetaminophen (paracetamol, Tylenol®) is one of the most widely used drugs in the world. It is very safe when used at therapeutic doses. However, it is also involved in ~ ½ of the cases of drug-induced liver failure and well exemplifies the axiom of Paracelsus about the “the dose makes the poison” (Larson et al. 2005). When overdosed, the drug causes mitochondrial dysfunction and centrilobular necrosis in the liver in humans and experimental animals (Yoon et al. 2016). Normally most of the ingested drug is eliminated by glucuronidation and sulfation, catalyzed by UDP-glucuronosyltransferases (UGT1A1 and 1A6) and sulfotransferases (SULT1A1, 1A3/4, and 1E1), respectively, producing nontoxic conjugates (McGill and Jaeschke 2013).

The key mechanism in the hepatotoxicity is P450-catalyzed formation of the reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), which depletes hepatic glutathione and accumulates to cause hepatocellular liver damage, including oxidative stress (Fig. 13). Approximately 5% to 9% of orally administered acetaminophen is metabolized by P450 2E1, 1A2, and 3A4 catalyzed oxidation reactions (Table 3 and references therein). At a toxic concentration the formation of the NAPQI-glutathione conjugate was highest with P450 3A4, having the lowest K_m value of 130 μM (for comparison therapeutic concentrations of paracetamol are ~50 μM and toxic concentrations are ~1 mM) followed by P450 2E1 and P450 1A2 (Patten et al. 1993)). It has been proposed that human P450 3A4 is the major enzyme catalyzing acetaminophen oxidation to NAPQI (Laine et al. 2009). Other studies using inhibition studies with human recombinant enzymes indicated that P450 1A2 is the enzyme responsible for acetaminophen metabolic activation (Tan et al. 2008). In mice, the deletion of P450 2e1 or 1a2 blocks acetaminophen toxicity (Lee et al. 1996; Zaher et al. 1998).

Fig 13.

Metabolic activation of halothane

Halothane

Halothane was previously the most widely used anesthetic agent and in 1963 was reported to cause liver necrosis in humans. It was shown that halothane liver necrosis was induced following pretreatment of rats with polychlorinated biphenyls, known inducers of P450 enzymes. The liver necrosis caused by halothane anesthesia could be prevented by administration of metyrapone, a rather non-selective inhibitor of P450 enzymes, prior to anesthesia. These findings indicated that halothane toxicity resulted from metabolic activation of halothane by P450 enzymes (Nastainczyk et al. 1982). In addition, halothane hepatotoxicity could be potentiated in rats by chronic administration of ethanol, an inducer of P450 2E1 (Takagi et al. 1983). Human P450s activate halothane by both reductive and oxidative metabolism (Table 3, Fig. 14), and oxidative dehalogenation by P450 2E1 is a major in vivo reaction with low K_m values (and also results in lipid peroxidation). Limited participation of P450 2A6 activation has been indicated in vivo. Reductive activation of halothane is catalyzed by P450 2A6 and 3A4 enzymes (Table 3 and references therein). Halothane oxidation, the major metabolic pathway, leads to the production of the reactive electrophile trifluoroacetyl chloride, and subsequent acylation of liver proteins results in the formation of trifluoroacetylated protein neoantigens. Metabolic halothane reduction leads to the formation of the 2-chloro-1,1,1-trifluoroethyl radical by hemolytic cleavage of the C-Br bond (Fig. 13) (Kurth et al. 2014). Halothane has been replaced in most of countries by other, less toxic, inhalation anesthetics due to its induced hepatitis, but there is still the possibility that it is in use in some developing countries.

Fig 14.

Metabolic activation of 17α-ethynylestradiol

17α-Ethynylestradiol

17α-Ethynylestradiol is in use as the estrogenic component of oral contraceptives (Bolt 1979). Similar to natural estrogens (see the Physiological Compounds section, vide infra) (Lacassagne 1932), 17α-ethynylestradiol is a weak carcinogen in rats, and carcinogenic activity has been associated with the formation of catechol metabolites (Fig. 14) (Zhu et al. 1993). C2-hydroxylation catalyzed by P450 enzymes (mainly P450 3A4) was found to be the main metabolic pathway of 17α-ethynylestradiol in individuals using oral contraceptives (Guengerich 1988)). This metabolite is excreted as 2-methoxyethynylestradiol after O-methylation (Back et al. 1984). Induction of P450 3A4 by rifampicin, barbiturates, or herbal remedies such as St. John’s wort can lead to increased clearance and unplanned pregnancy (Bolt et al. 1977; Guengerich 1988).

Natural products

Natural products, including herbal supplements, can have multiple effects on activity of P450 enzymes, for instance inhibition or induction of activity and/or their expression (St. John’s wort, vide supra). By changing activity and/or expression of the enzymes and applied concomitantly with drugs, natural chemicals can provoke drug-phytochemical interactions. Such activity might result in altered therapeutic and or toxic properties of drugs (Guengerich and Rendic 2010; Rendic and Guengerich 2010). Examples of different classes of natural compounds that can be activated to toxic metabolites by cytochrome P450s (e.g., alkaloids, monoterpenes, mycotoxins, N-nitrosamines) are presented in Table 4. Natural products, as substrates of P450 enzymes, can be both activated to toxic and detoxicated to nontoxic products by P450 enzymes in different ways. For instance, aflatoxin B₁ (AFB₁) is activated to toxic and detoxicated to nontoxic metabolites by oxidative reactions, while aristolochic acid is activated by nitro reduction under (partially anaerobic conditions), and oxidative metabolism results in the formation of a nontoxic O-demethylated product. Estragole and safrole are examples in which metabolism by P450 enzymes to nontoxic metabolites can be followed by activation to toxic metabolites by conjugation to form a sulfate ester (Table 4 and references therein).

Table 4.

Examples of the metabolic activation of natural products by human cytochrome P450 enzymes

Natural product	P450	Category	Reaction	Reference PMED IDs	Reference No.
Aflatoxin B1 (AFB1)	2A13	Mycotoxin, Aspergillus species	Hydroxylation (AFM1 formation) and epoxidation 8,9- (low activity, activation)	16385575, 30454686, 22743290	(Deng et al. 2018; He et al. 2006; Yang et al. 2012)
AFB1	2W1	Mycotoxin, Aspergillus species	Oxidation, activation	16379042	(Brandon et al. 2006)
AFB1	2B6	Mycotoxin, Aspergillus species	Epoxidation 8,9- (low activity, activation)	8597154, 9280407, 11360624	(Code et al. 1997; Neal 1995; Wu et al. 1997)
AFB1	2A6	Mycotoxin, Aspergillus species	Epoxidation, exo-8,9-, activation	8082563, 1944238, 16385575, 11189750	(Gonzalez and Gelboin 1994; He et al. 2006; Lewis et al. 1999; Yun et al. 1991)
AFB1	3A5	Mycotoxin, Aspergillus species	Epoxidation, exo-C8,9- (activation, a major enzyme at higher concentration), medium Km, medium activity	9385444, 9730826, 7893152, 16608170, 30454686, 11189750	(Deng et al. 2018; Gillam et al. 1995; Kamdem et al. 2006; Kim et al. 1997; Lewis et al. 1999; Wang et al. 1998)
AFB1	2E1	Mycotoxin, Aspergillus species	Oxidation (weak activation)	11189750	(Lewis et al. 1999)
AFB1	3A4	Mycotoxin, Aspergillus species	Epoxidation, exo-8,9- (activation), major enzyme, medium Km and activity	8261428, 7766804, 12079611, 1902334, 9385444, 11782366, 9730826, 11368545, 16608170, 9003190, 30454686, 8975785, 11497333, 1643250, 11189750	(Deng et al. 2018; Gallagher et al. 1996; Gallagher et al. 1994; Kamdem et al. 2006; Kim et al. 1997; Lewis et al. 1999; Ramsdell et al. 1991; Raney et al. 1992; Shimada and Guengerich 1989; Ueng et al. 1997; Ueng et al. 1995; Van Vleet et al. 2001; Van Vleet et al. 2002a; Van Vleet et al. 2002b; Wang et al. 1998; Xue et al. 2001)
AFB1	1A2	Mycotoxin, of Aspergillus species	Epoxidation, exo-8,9- (activation), and endo-8,9- (detoxication), medium Km and activity	8261428, 7766804, 12079611, 1902334, 11782366, 16385575, 16608170, 30454686, 8975785, 11497333, 11189750	(Deng et al. 2018; Gallagher et al. 1996; Gallagher et al. 1994; He et al. 2006; Kamdem et al. 2006; Lewis et al. 1999; Ramsdell et al. 1991; Shimada and Guengerich 1989; Ueng et al. 1995; Van Vleet et al. 2001; Van Vleet et al. 2002a; Van Vleet et al. 2002b)
AFB1	3A7	Mycotoxin, Aspergillus species	Epoxidation, exo-8,9- (activation, major enzyme at higher concentration), medium Km, medium activity	30454686	(Deng et al. 2018)
Aflatoxin G1 (AFG1)	2A13	Mycotoxin, Aspergillus species	Oxidation, activation	23907605	(Zhang et al. 2013)
AFG1	1A2	Mycotoxin, Aspergillus species	Oxidation, activation	11189750	(Lewis et al. 1999)
AFG1	3A4	Mycotoxin, Aspergillus species	Oxidation, activation	8082563, 8261428, 7766804, 12079611, 1902334, 352361, 12849689, 11189750	(Buening et al. 1978; Gallagher et al. 1994; Gonzalez and Gelboin 1994; Lewis et al. 1999; Ramsdell et al. 1991; Sabater Vilar et al. 2003; Ueng et al. 1995; Van Vleet et al. 2002a)
Aristolochic acid I	1A1	Nephrotoxin and carcinogen, from the plant Aristolochia fangchi and other species and several Asarum species	Nitroreduction, reductive activation, N-hydroxyaristolactam formation	11511187, 15386410, 23701164, 26593908, 24152141, 22086975, 23353840, 26861298	(Jerabek et al. 2012; Milichovský et al. 2016; Stiborová et al. 2015; Stiborová et al. 2014; Stiborová et al. 2005; Stiborová et al. 2001b; Stiborová et al. 2012a; Stiborová et al. 2013)
Aristolochic acid I	1A2	Nephrotoxin and carcinogen, from the plant Aristolochia fangchi and other species and several Asarum species	Nitroreduction, reductive activation, N-hydroxyaristolactam (high activity)	11511187, 15386410, 23701164, 26593908, 24152141, 22086975, 23353840, 26861298	(Jerabek et al. 2012; Milichovský et al. 2016; Stiborová et al. 2015; Stiborová et al. 2014; Stiborová et al. 2005; Stiborová et al. 2001b; Stiborová et al. 2012a; Stiborová et al. 2013)
Aristolochic acid I	1A1	Nephrotoxin and carcinogen, from the plant Aristolochia fangchi and other species and several Asarum species	O-Demethylation, detoxication	26593908, 22086975	(Stiborová et al. 2015; Stiborová et al. 2012a)
Aristolochic acid I	1A2	Nephrotoxin and carcinogen, from the plant Aristolochia fangchi and other species and several Asarum species	O-Demethylation, detoxication	26593908, 22086975	(Stiborová et al. 2015; Stiborová et al. 2012a)
Aristolochic acid I	2C9	Nephrotoxin and carcinogen, from the plant Aristolochia fangchi and other species and several Asarum species	O-Demethylation, detoxication	26593908	(Stiborová et al. 2015)
Aristolochic acid I	3A4	Nephrotoxin and carcinogen, from the plant Aristolochia fangchi and other species and several Asarum species	O-Demethylation, detoxication	26593908	(Stiborová et al. 2015)
Aristolochic acid II	1A1	Nephrotoxin and carcinogen, from the plant Aristolochia fangchi and other species and several Asarum species	Nitroreduction, reductive activation	11511187, 15386410	(Stiborová et al. 2005; Stiborová et al. 2001b)
Aristolochic acid II	1A2	Nephrotoxin and carcinogen, from the plant Aristolochia fangchi and other species and several Asarum species	Nitroreduction, reductive activation (high activity)	11511187, 15386410	(Stiborová et al. 2005; Stiborová et al. 2001b)
Benzophenone	1A1	Muscat grape and mango compound, flavoring substance, ultraviolet protection compound	Benzhydrol and 4-hydroxybezophenone formation, activation	12160905	(Takemoto et al. 2002)
Benzophenone	1A2	Muscat grape and mango compound, flavoring substance, ultraviolet protection compound	Benzhydrol and 4-hydroxybezophenone formation, activation	12160905	(Takemoto et al. 2002)
Benzophenone	1B1	Muscat grape and mango compound, flavoring substance, ultraviolet protection compound	Benzhydrol and 4-hydroxybezophenone formation, activation	12160905	(Takemoto et al. 2002)
Benzophenone	2A6	Muscat grape and mango compound, flavoring substance, ultraviolet protection compound	Benzhydrol and 4-hydroxybezophenone formation, activation	12160905	(Takemoto et al. 2002)
Curcumin	2D6	Coloring agent, yellow pigment from Curcuma longa, chemopreventive	O-Demethylation, activation	12220536	(Sakano and Kawanishi 2002)
Curcumin	1A1	Coloring agent, yellow pigment from Curcuma longa, chemopreventive	O-Demethylation, activation	12220536	(Sakano and Kawanishi 2002)
Curcumin	1A2	Coloring agent, yellow pigment from Curcuma longa, chemopreventive	O-Demethylation, activation	12220536	(Sakano and Kawanishi 2002)
Δ3-Carene	1A2	Bicyclic monoterpene	Epoxidation (high Km, medium activity, activation)	16379671	(Duisken et al. 2005)
Diallyl sulfone	2E1	Garlic oil compound, organosulfur	Oxidation (diallyl sulfoxide and diallyl sulfone formation, activation)	11062148, 16510538, 11238812	(Black et al. 2006; Forkert et al. 2000; Yang et al. 2001)
Ecteinascidin 743, trabectedin (ET-743)	3A4	Marine compound, tetrahydroisoquinoline	Oxidation, low Km, major enzyme, activation	12231541,16162970, 16379042	(Reid et al. 2002; Brandon et al. 2005; Brandon et al. 2006)
Ecteinascidin 743, trabectedin (ET-743)	2C9	Marine compound, tetrahydroisoquinoline	Oxidation, activation	12231541,16162970, 16379042	(Reid et al. 2002; Brandon et al. 2005; Brandon et al. 2006)
Ecteinascidin 743, trabectedin (ET-743)	2C19	Marine compound, tetrahydroisoquinoline	Oxidation, activation	16162970, 16379042	(Brandon et al. 2005; Brandon et al. 2006)
Ecteinascidin 743, trabectedin (ET-743)	2E1	Marine compound, tetrahydroisoquinoline	Oxidation, activation	12231541,16162970, 16379042	(Reid et al. 2002; Brandon et al. 2005; Brandon et al. 2006)
Ecteinascidin 743, trabectedin (ET-743)	2D6	Marine compound, tetrahydroisoquinoline	Oxidation, activation	12231541,16162970, 16379042	(Reid et al. 2002;Brandon et al. 2005; Brandon et al. 2006)
Estragole	2A6	Alkenylbenzene	C1´-Hydroxylation (major enzyme, medium Km, medium activity), activation after sulfation	17407329	(Jeurissen et al. 2007)
Estragole	2C19	Alkenylbenzene	C1´-Hydroxylation (minor enzyme, medium Km, medium activity), activation after sulfation	17407329	(Jeurissen et al. 2007)
Estragole	1A2	Alkenylbenzene	C1´-Hydroxylation, activation after sulfation, high Km, medium activity)	17407329	(Jeurissen et al. 2007)
Estragole	2D6	Alkenylbenzene	C1´-Hydroxylation, activation after sulfation, at high concentrations	17407329	(Jeurissen et al. 2007)
Estragole	2E1	Alkenylbenzene	C1´-Hydroxylation, activation after sulfation, at high concentrations	17407329	(Jeurissen et al. 2007)
Ethanol	2C19	Organic solvent	Oxidation (acetaldehyde formation), activation, high Km, high activity	17084997	(Hamitouche et al. 2006)
Ethanol	1A1	Organic solvent	Oxidation (acetaldehyde formation), activation, high Km (~10 mM), high activity	17084997	(Hamitouche et al. 2006)
Ethanol	1B1	Organic solvent	Oxidation (acetaldehyde formation), activation, high Km (~10 mM), high activity	17084997	(Hamitouche et al. 2006)
Ethanol	2B6	Organic solvent	Oxidation (acetaldehyde formation), activation, high Km (~10 mM), high activity; in vivo after long-term treatment or at high doses	17084997	(Hamitouche et al. 2006)
Ethanol	2D6	Organic solvent	Oxidation (acetaldehyde formation), activation, high Km (~10 mM), high activity; in vivo after long-term treatment or at high doses	17084997	(Hamitouche et al. 2006)
Ethanol	2C8	Organic solvent	Oxidation (acetaldehyde formation), activation, high Km (~10 mM), high activity; in vivo after long-term treatment or at high doses	17084997	(Hamitouche et al. 2006)
Ethanol	2C9	Organic solvent	Oxidation (acetaldehyde formation), activation, high Km (~10 mM), high activity; in vivo after long-term treatment or at high doses	17084997	(Hamitouche et al. 2006)
Ethanol	2J2	Organic solvent	Oxidation (acetaldehyde formation), activation, high Km (~10 mM), high activity; in vivo after long-term treatment or at high doses	17084997	(Hamitouche et al. 2006)
Ethanol	4A11	Organic solvent	Oxidation (acetaldehyde formation), activation, high Km (~10 mM), high activity; in vivo after long-term treatment or at high doses	17084997	(Hamitouche et al. 2006)
Ethanol	1A2	Organic solvent	Oxidation (acetaldehyde formation), activation, high Km (~10 mM), high activity; in vivo after long-term treatment or at high doses	17084997, 10446146, 9368031, 8627510, 9884161	(Asai et al. 1996; Bell and Guengerich 1997; Bell-Parikh and Guengerich 1999; Hamitouche et al. 2006)
Ethanol	2E1	Organic solvent	Oxidation (acetaldehyde formation), activation, high Km (~10 mM), high activity; in vivo after long-term treatment or at high doses	17084997, 8313838, 10446146, 9368031, 8627510, 7687464, 17084997, 9143349, 30362088	(Asai et al. 1996; Dai et al. 1993; Hamitouche et al. 2006; Guengerich and Avadhani 2018; Yang and Cederbaum 1997)
Ethanol	3A4	Organic solvent	Oxidation (acetaldehyde formation), activation, high Km (~10 mM), high activity; in vivo after long-term treatment or at high doses	17084997, 8571359, 10976571, 10446146, 9368031, 8627510, 9884161	(Asai et al. 1996; Bell and Guengerich 1997; Bell-Parikh and Guengerich 1999; Hamitouche et al. 2006; Novak and Woodcroft 2000; Raucy 1995; Salmela et al. 1998)
Ethyl carbamate (urethane)	2E1	Carbamic acid derivative, fermentation byproduct	Oxidation to vinyl carbamate epoxide, activation	1664256, 1912327	(Guengerich and Kim 1991; Guengerich et al. 1991)
4-Ipomeanol	1A2	Pulmonary toxin, alkylating, from Fusarium solani	Oxidation, activation, major enzyme	1651809, 15892579	(Baer et al. 2005; Czerwinski et al. 1991)
4-Ipomeanol	2C19	Pulmonary toxin, alkylating, from F. solani	Oxidation, activation, major enzyme	15892579	(Baer et al. 2005)
4-Ipomeanol	2D6	Pulmonary toxin, alkylating, from F. solani	Oxidation, activation	15892579	(Baer et al. 2005)
4-Ipomeanol	2E1	Pulmonary toxin, alkylating, from F. solani	Oxidation, activation	15892579	(Baer et al. 2005)
4-Ipomeanol	2F1	Pulmonary toxin, alkylating, from F. solani	Oxidation, activation	1651809	(Czerwinski et al. 1991)
4-Ipomeanol	3A4	Pulmonary toxin, alkylating, from F. solani	Epoxidation, activation	14967002, 17584015	(Alvarez-Diez and Zheng 2004; Kalgutkar et al. 2007)
4-Ipomeanol	3A4	Pulmonary toxin, alkylating, from F. solani	Oxidation, activation, minor enzyme	1651809, 15892579	(Baer et al. 2005; Czerwinski et al. 1991)
4-Ipomeanol	4B1	Pulmonary toxin, alkylating, from F. solani	Oxidation, activation	1651809, 23748241, 27092941, 30409834	(Czerwinski et al. 1991; Roellecke et al. 2016; Teitelbaum et al. 2019)
3-Methylindole (skatole)	1A2	Pulmonary toxin	Dehydrogenation (3-methyleneindolenine formation, low Km, high activity, high efficiency, activation)	8558432, 11408359	(Lanza and Yost 2001; Thornton-Manning et al. 1996)
3-Methylindole (skatole)	2A6	Pulmonary toxin	Epoxidation (3-methyloxindole formation, at high concentration, activation)	8558432, 11408359	(Lanza and Yost 2001; Thornton-Manning et al. 1996)
3-Methylindole (skatole)	1A2	Pulmonary toxin	Epoxidation (3-methyloxindole formation, low Km, high activity, high efficiency, activation)	8558432, 11408359	(Lanza and Yost 2001; Thornton-Manning et al. 1996)
3-Methylindole (skatole)	1B1	Pulmonary toxin	Epoxidation (3-methyloxindole form., low Km, high activity, high efficiency, activation)	8558432, 11408359	(Lanza and Yost 2001; Thornton-Manning et al. 1996)
3-Methylindole (skatole)	1A1	Pulmonary toxin	Epoxidation (3-methyloxindole form., low Km, medium activity, high efficiency, activation)	8558432, 11408359	(Lanza and Yost 2001; Thornton-Manning et al. 1996)
3-Methylindole (skatole)	2E1	Pulmonary toxin	Epoxidation (3-methyloxindole form., low Km, medium activity, high efficiency, activation)	8558432, 11408359	(Lanza and Yost 2001; Thornton-Manning et al. 1996)
3-Methylindole (skatole)	1A2	Pulmonary toxin	Hydroxylation, C- (indole-3-carbinol formation, low Km, high activity, high efficiency, activation)	8558432, 11408359	(Lanza and Yost 2001; Thornton-Manning et al. 1996)
3-Methylindole (skatole)	1A1	Pulmonary toxin	Hydroxylation, C- (indole-3-carbinol formation, low Km, medium activity, high efficiency, activation)	8558432, 11408359	(Lanza and Yost 2001; Thornton-Manning et al. 1996)
3-Methylindole (skatole)	1B1	Pulmonary toxin	Hydroxylation, C- (indole-3-carbinol formation, low Km, medium activity, high efficiency, activation)	8558432, 11408359	(Lanza and Yost 2001; Thornton-Manning et al. 1996)
3-Methylindole (skatole)	2F1	Pulmonary toxin	Dehydrogenation (desaturation, 3-methyleneindolenine formation, low Km, medium activity, high efficiency and activation)	8558432, 11408359, 10383923, 17962375	(Kartha and Yost 2008; Lanza et al. 1999; Lanza and Yost 2001; Thornton-Manning et al. 1996)
3-Methylindole (skatole)	2F1	Pulmonary toxin	Dehydrogenation (desaturation, 3-methyleneindolenine formation, high activation)	8558432, 11408359, 10383923, 17962375, 20795680, 20187624	(Kartha and Yost 2008; Lanza et al. 1999; Lanza and Yost 2001; Thornton-Manning et al. 1996; Weems et al. 2010; Weems and Yost 2010)
3-Methylindole (skatole)	1A1	Pulmonary toxin	Dehydrogenation (desaturation, 3-methyleneindolenine form., low Km, medium activity, high efficiency, activation)	8558432, 11408359, 20795680, 20187624	(Lanza and Yost 2001; Thornton-Manning et al. 1996; Weems et al. 2010; Weems and Yost 2010)
3-Methylindole (skatole)	2A13	Pulmonary toxin	Dehydrogenation (desaturation, 3-methyleneindolenine formation, activation)	8558432, 11408359, 20795680, 20187624	(Lanza and Yost 2001; Thornton-Manning et al. 1996; Weems et al. 2010; Weems and Yost 2010)
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)	2E1	Tobacco-specific nitrosamine	Activation	19156262	(Krishnan et al. 2009)
NNK	1B1	Tobacco-specific nitrosamine	Activation (low activation)	19156262	(Krishnan et al. 2009)
NNK	2A13	Tobacco-specific nitrosamine	Hydroxylation, Cα-methylene (keto aldehyde formation), activation, medium Km, medium activity, or high activity	11016631, 12975327, 15333516, 15528319, 15962925, 12130698	(Bao et al. 2005; He et al. 2004b; Jalas et al. 2003; Su et al. 2000; Wong et al. 2005b; Zhang et al. 2002)
NNK	2A13	Tobacco-specific nitrosamine	Hydroxylation, Cα-methyl (keto alcohol), major enzyme for activation, medium Km, medium activity, or high activity	11016631, 12975327, 15333516, 15528319, 15962925, 12130698, 17671098, 21473878, 23917075	(Bao et al. 2005; Chiang et al. 2011; He et al. 2004b; Jalas et al. 2003; Megaraj et al. 2014; Su et al. 2000; Wong et al. 2005b; Zhang et al. 2007; Zhang et al. 2002)
NNK	2B6	Tobacco-specific nitrosamine	Hydroxylation, α-methyl (keto alcohol formation), activation, major reaction	11360624, 12920169, 16174803, 9280407, 1312898, 8806763, 9106248, 8485585	(Code et al. 1997; Crespi et al. 1997; Dicke et al. 2005; Patten et al. 1996; Penman et al. 1993; Smith et al. 2003a; Smith et al. 1992; Wu et al. 1997)
NNK	2F1	Tobacco-specific nitrosamine	Hydroxylation, α-methyl (keto alcohol formation), activation	1312898, 8806763	(Patten et al. 1996; Smith et al. 1992)
NNK	2A6	Tobacco-specific nitrosamine	Hydroxylation, Cα-methyl (keto alcohol and ketoaldehyde formation), high Km, low activity, minor reaction, weak activation	1312898, 8806763, 1423839, 9106248, 9280407, 8485585, 10837014, 11600130, 12920169, 11016631, 11080669, 14668073, 15333516, 16364922, 21473878	(Chiang et al. 2011; Code et al. 1997; Crespi et al. 1997; Fujita and Kamataki 2001b; He et al. 2004b; Kushida et al. 2000; Patten et al. 1996; Penman et al. 1993; Sellers et al. 2003; Smith et al. 2003a; Smith et al. 1992; Su et al. 2000; von Weymarn et al. 2006; Yamazaki et al. 1992)
NNK	2E1	Tobacco-specific nitrosamine	Hydroxylation, Cα-methylene (keto aldehyde formation), high Km, low activity, minor reaction, activation	1312898, 8806763, 1423839, 9106248, 9280407, 8485585, 10837014, 11600130, 12920169	(Code et al. 1997; Crespi et al. 1997; Fujita and Kamataki 2001b; Kushida et al. 2000; Patten et al. 1996; Penman et al. 1993; Smith et al. 2003a; Smith et al. 1992; Yamazaki et al. 1992)
NNK	2E1	Tobacco-specific nitrosamine	Hydroxylation, Cα-methyl (keto alcohol), high Km, medium activity, major reaction, activation	1312898, 8806763, 1423839, 9106248, 9280407, 8485585, 10837014, 11600130	(Code et al. 1997; Crespi et al. 1997; Fujita and Kamataki 2001b; Kushida et al. 2000; Patten et al. 1996; Penman et al. 1993; Smith et al. 1992; Yamazaki et al. 1992)
NNK	2D6	Tobacco-specific nitrosamine	Hydroxylation, Cα-methyl (keto alcohol), high Km, medium activity, or high activity, major reaction, activation	1312898, 8806763, 9106248, 9280407, 8485585	(Code et al. 1997; Crespi et al. 1997; Patten et al. 1996; Penman et al. 1993; Smith et al. 1992)
NNK	1A2	Tobacco-specific nitrosamine	Hydroxylation, α-methyl (keto alcohol formation), high Km, medium activity, activation	1312898, 8806763, 9106248, 9280407, 8485585, 11774366, 12214673, 16174803, 21473878, 19156262	(Chiang et al. 2011; Code et al. 1997; Crespi et al. 1997; Dicke et al. 2005; Fujita and Kamataki 2001a; Kamataki et al. 2002; Krishnan et al. 2009; Patten et al. 1996; Penman et al. 1993; Smith et al. 1992)
4-Methylphenol (p-cresol)	1A1	Antiseptic, disinfectant	Hydroxylation, C-methyl, 4-hydroxybenzaldehyde formation (activation, low activity)	16174805	(Yan et al. 2005)
4-Methylphenol (p-cresol)	1A2	Antiseptic, disinfectant	Hydroxylation, C-methyl, 4-hydroxybenzaldehyde formation (activation, high activity)	16174805	(Yan et al. 2005)
4-Methylphenol (p-cresol)	2C19	Antiseptic, disinfectant	Hydroxylation, C-methyl, 4-hydroxybenzaldehyde formation (activation, low activity)	16174805	(Yan et al. 2005)
4-Methylphenol (p-cresol)	2D6	Antiseptic, disinfectant	Hydroxylation, C-methyl, 4-hydroxybenzaldehyde formation (activation, low activity)	16174805	(Yan et al. 2005)
4-Methylphenol (p-cresol)	2C9	Antiseptic, disinfectant	Hydroxylation, C-methyl, 4-hydroxybenzaldehyde formation (activation, low activity)	16174805	(Yan et al. 2005)
4-Methylphenol (p-cresol)	3A4	Antiseptic, disinfectant	Hydroxylation, C-methyl, 4-hydroxybenzaldehyde formation (activation, low activity)	16174805	(Yan et al. 2005)
4-Methylphenol (p-cresol)	2E1	Antiseptic, disinfectant	Hydroxylation, C-aromatic, formation of 4-methyl-o-hydroquinone (activation, high activity)	16174805	(Yan et al. 2005)
4-Methylphenol (p-cresol)	2C19	Antiseptic, disinfectant	Hydroxylation, C-aromatic, formation of 4-methyl-o-hydroquinone (activation, low activity)	16174805	(Yan et al. 2005)
4-Methylphenol (p-cresol)	2C9	Antiseptic, disinfectant	Hydroxylation, C-aromatic, formation of 4-methyl-o-hydroquinone (activation, low activity)	16174805	(Yan et al. 2005)
4-Methylphenol (p-cresol)	1A1	Antiseptic, disinfectant	Hydroxylation, C-aromatic, formation of 4-methyl-o-hydroquinone (activation, low activity)	16174805	(Yan et al. 2005)
4-Methylphenol (p-cresol)	1A2	Antiseptic, disinfectant	Hydroxylation, C-aromatic, formation of 4-methyl-o-hydroquinone (activation, low activity)	16174805	(Yan et al. 2005)
4-Methylphenol (p-cresol)	2D6	Antiseptic, disinfectant	Hydroxylation, C-aromatic, formation of 4-methyl-o-hydroquinone (activation, low activity)	16174805	(Yan et al. 2005)
4-Methylphenol (p-cresol)	3A4	Antiseptic, disinfectant	Hydroxylation, C-aromatic (activation, low activity)	16174805	(Yan et al. 2005)
4-Methylphenol (p-cresol)	2E1	Antiseptic, disinfectant	Hydroxylation, C-methyl, 4-hydroxybenzaldehyde formation (low activity)	16174805	(Yan et al. 2005)
3-N-Nitrosoguvacine (NGC)	1A1	Nitrosamine, betel quid, Areca nut compound	Oxidation, activation	23983642	(Lin et al. 2013)
NGC	2A6	Nitrosamine, betel quid, Areca nut compound	Oxidation, activation	23983642	(Lin et al. 2013)
NGC	2E1	Nitrosamine, betel quid, Areca nut compound	Oxidation, activation	23983642	(Lin et al. 2013)
3-N-Nitrosoguvacoline (NGL)	2A13	Nitrosamine, betel quid, Areca nut compound	Oxidation, major enzyme, activation	15725615	(Miyazaki et al. 2005)
NGL	2A6	Nitrosamine, betel quid, Areca nut compound	Oxidation, major enzyme, activation	15725615, 23983642	(Lin et al. 2013; Miyazaki et al. 2005)
NGL	2E1	Nitrosamine, betel quid, Areca nut compound	Oxidation, activation	23983642	(Lin et al. 2013)
3-(N-Nitrosomethylamino)propionaldehyde (NMPA)	1A1	Nitrosamine, betel quid, Areca nut compound	Oxidation, activation	15725615, 23983642	(Lin et al. 2013; Miyazaki et al. 2005)
NMPA	1B1	Nitrosamine, betel quid, Areca nut compound	Oxidation, activation	15725615	(Miyazaki et al. 2005)
NMPA	2A13	Nitrosamine, betel quid, Areca nut compound	Oxidation, major enzyme, activation	15725615	(Miyazaki et al. 2005)
NMPA	2A6	Nitrosamine, betel quid, Areca nut compound	Oxidation, activation	15725615, 23983642	(Lin et al. 2013; Miyazaki et al. 2005)
NMPA	2E1	Nitrosamine, betel quid, Areca nut compound	Oxidation, activation	15725615, 23983642	(Lin et al. 2013; Miyazaki et al. 2005)
3-(N-Nitrosomethylamino)propionitrile (NMPN)	1B1	Nitrosamine, betel quid, Areca nut compound	Oxidation, activation	15725615	(Miyazaki et al. 2005)
NMPN	2A13	Nitrosamine, betel quid, Areca nut compound	Oxidation, activation	15725615	(Miyazaki et al. 2005)
NMPN	2A6	Nitrosamine, betel quid, Areca nut compound	Oxidation, major enzyme, activation	15725615, 23983642	(Lin et al. 2013; Miyazaki et al. 2005)
NMPN	2E1	Nitrosamine, Betel quid, Areca nut compound	Oxidation, major enzyme, activation	15725615, 23983642	(Lin et al. 2013; Miyazaki et al. 2005)
NMPN	1A1	Nitrosamine, betel quid, Areca nut compound	Oxidation, major enzyme, activation	15725615, 23983642	(Lin et al. 2013; Miyazaki et al. 2005)
Menthofuran	1A2	Monoterpene, pennyroyal herb and oil compound, pulegone metabolite	Activation	26969934	(Lassila et al. 2016)
Menthofuran	3A4	Monoterpene, pennyroyal herb and oil compound, pulegone metabolite	Activation	26969934	(Lassila et al. 2016)
Menthofuran	2B6	Monoterpene, pennyroyal herb and oil compound, pulegone metabolite	Activation	26969934	(Lassila et al. 2016)
Menthofuran (R)-(+)-	2A6	Monoterpene, pennyroyal herb and oil compound, pulegone metabolite	C2-Hydroxylation, (2-hydroxymethofuran formation), activation	10220485	(Khojasteh-Bakht et al. 1999)
Menthofuran (R)-(+)-	1A2	Monoterpene, pennyroyal herb and oil compound, pulegone metabolite	C2-Hydroxylation, (2-hydroxymethofuran formation), high Km, low activity, activation	10220485, 26969934	(Khojasteh-Bakht et al. 1999; Lassila et al. 2016)
Menthofuran (R)-(+)-	2C19	Monoterpene, pennyroyal herb and oil compound, pulegone metabolite	C2-Hydroxylation, (2-hydroxymethofuran formation), high Km, low activity, activation	10220485	(Khojasteh-Bakht et al. 1999)
Menthofuran (R)-(+)-	2E1	Monoterpene, pennyroyal herb and oil compound, pulegone metabolite	C2-Hydroxylation, (2-hydroxymethofuran formation), medium Km, low activity, activation	10220485	(Khojasteh-Bakht et al. 1999)
Methyleugenol	2C19	Phenylpropene, from Rhizoma acorigraminei	C1´-Hydroxylation, medium activity, high Km, activation, followed by formation of 1´-sulfooxymethyleugenol	16411663	(Jeurissen et al. 2006)
Methyleugenol	2D6	Phenylpropene, from Rhizoma acorigraminei	C1´-Hydroxylation, medium activity, high Km, activation, followed by formation of 1´-sulfooxymethyleugenol	16411663	(Jeurissen et al. 2006)
Methyleugenol	2E1	Phenylpropene, from Rhizoma acorigraminei	C1´-Hydroxylation, medium activity, high Km, activation, followed by formation of 1´-sulfooxymethyleugenol	16411663, 9328175	(Gardner et al. 1997; Jeurissen et al. 2006)
Methyleugenol	1A2	Phenylpropene, from Rhizoma acorigraminei	C1´-Hydroxylation, major enzyme, activation, followed by formation of 1´-sulfooxymethyleugenol	16411663, 25549870	(Al-Subeihi et al. 2015; Jeurissen et al. 2006)
Methyleugenol	2C9	Phenylpropene, from Rhizoma acorigraminei	C1´-Hydroxylation, major enzyme, activation, followed by formation of 1´-sulfooxymethyleugenol	16411663	(Jeurissen et al. 2006)
Methyleugenol	2B6	Phenylpropene, from Rhizoma acorigraminei	Epoxidation, major enzyme, activation, followed by formation of 1-sulfooxymethyleugenol	25549870	(Al-Subeihi et al. 2015)
Monocrotaline	3A4	Pyrrolizidine alkaloid, genotoxic	Pyrrole formation, dehydrogenation, activation	15649625	(Wang et al. 2005)
N´-Nitrosonornicotine (NNN)	1A1	Tobacco-specific nitrosamine	Activation	11774366	(Fujita and Kamataki 2001a)
NNN	1A2	Tobacco-specific nitrosamine	Activation	11774366	(Fujita and Kamataki 2001a)
NNN	1B1	Tobacco-specific nitrosamine	Activation	11774366	(Fujita and Kamataki 2001a)
NNN	2A6	Tobacco-specific nitrosamine	5´-Hydroxylation (activation, major enzyme)	11774366, 12214673, 9029045, 9276639, 15651850	(Fujita and Kamataki 2001a; Kamataki et al. 2002; Patten et al. 1997; Staretz et al. 1997; Wong et al. 2005b)
NNN	2C19	Tobacco-specific nitrosamine	Activation	11774366	(Fujita and Kamataki 2001a)
NNN	3A4	Tobacco-specific nitrosamine	2´-Hydroxylation (activation)	11774366, 9029045, 9276639	(Fujita and Kamataki 2001a; Patten et al. 1997; Staretz et al. 1997)
NNN	3A5	Tobacco-specific nitrosamine	Activation	11774366	(Fujita and Kamataki 2001a)
NNN	2A13	Tobacco-specific nitrosamine	2´-Hydroxylation (activation, major enzyme)	15651850	(Wong et al. 2005b)
NNN	2E1	Tobacco-specific nitrosamine	5´-Hydroxylation (activation)	9276639	(Patten et al. 1997)
NNN	2D6	Tobacco-specific nitrosamine	5´-Hydroxylation (activation, major enzyme)	9276639	(Patten et al. 1997)
Ochratoxin A	2C9	Mycotoxin, from Aspergillus ochraceus and Penicillium verrucosum	Oxidation, activation	10712746	(El Adlouni et al. 2000)
Ochratoxin A	1A1	Mycotoxin, from Aspergillus ochraceus and Penicillium verrucosum	Oxidation, activation	8542584	(de Groene et al. 1996)
Ochratoxin A	1A2	Mycotoxin, from Aspergillus ochraceus and Penicillium verrucosum	Oxidation, activation	8542584, 11189750	(de Groene et al. 1996; Lewis et al. 1999)
Ochratoxin A	3A4	Mycotoxin, from Aspergillus ochraceus and Penicillium verrucosum	Oxidation, activation	8542584, 16139406	(de Groene et al. 1996; Simarro Doorten et al. 2006)
Pulegone (R)-(+)-	1A2	Monoterpene, pennyroyal herb and oil compound	Oxidation, menthofuran formation (high Km, medium activity, medium efficiency, activation)	10220485	(Khojasteh-Bakht et al. 1999)
Pulegone (R)-(+)-	2C19	Monoterpene, pennyroyal herb and oil compound	Oxidation, menthofuran formation (medium Km, medium activity, medium efficiency, activation)	10220485	(Khojasteh-Bakht et al. 1999)
Pulegone (R)-(+)-	2E1	Monoterpene, pennyroyal herb and oil compound	Oxidation, menthofuran formation (major enzyme, medium Km, high activity, medium efficiency, activation)	10220485	(Khojasteh-Bakht et al. 1999)
Retrorsine	3A4	Pyrrolizidine alkaloid, genotoxic	Pyrrole formation, activation	15649625, 25651456, 24799337, 32469285, 19818743	(Dai et al. 2010; Fashe et al. 2015; Lu et al. 2020; Tu et al. 2014; Wang et al. 2005)
Retrorsine	2C19	Pyrrolizidine alkaloid, genotoxic	Pyrrole formation, activation	19818743	(Dai et al. 2010)
Riddelliine	3A4	Pyrrolizidine alkaloid, genotoxic	Pyrrole formation, activation dehydrogenation	15649625, 32798647	(Li et al. 2020; Wang et al. 2005)
Riddelliine	3A5	Pyrrolizidine alkaloid, genotoxic	Pyrrolic metabolite formation, dehydrogenation, activation	32798647	(Li et al. 2020)
Riddelliine	3A7	Pyrrolizidine alkaloid, genotoxic	Pyrrolic metabolite formation, dehydrogenation, activation, low activity	32798647	(Li et al. 2020)
Safrole	1B1	Methylenedioxyphenyl, sassafras oil and betel quid component	C1´-Hydroxylation, activation	15310247	(Ueng et al. 2004)
Safrole	2A6	Methylenedioxyphenyl, sassafras oil and betel quid component	C1´-Hydroxylation, major enzyme at low concentration, medium Km, medium activity, activation	15377158, 15310247, 17407329, 23112005	(Jeurissen et al. 2004; Jeurissen et al. 2007; Ueng et al. 2004; Uno et al. 2013)
Safrole	2C19	Methylenedioxyphenyl, sassafras oil and betel quid component	C1´-Hydroxylation, high Km, medium activity, activation	15377158, 17407329	(Jeurissen et al. 2004; Jeurissen et al. 2007)
Safrole	2C9	Methylenedioxyphenyl, sassafras oil and betel quid component	C1´-Hydroxylation, major enzyme at higher conc., high Km, medium activity, activation	15377158, 15310247, 17407329	(Jeurissen et al. 2004; Jeurissen et al. 2007; Ueng et al. 2004)
Safrole	3A4	Methylenedioxyphenyl, sassafras oil and betel quid component	C1´-Hydroxylation, activation	15310247	(Ueng et al. 2004)
Safrole	2E1	Methylenedioxyphenyl, sassafras oil and betel quid component	C1´-Hydroxylation, major enzyme, activation	15310247, 15377158	(Jeurissen et al. 2004; Ueng et al. 2004)
Safrole	2D6	Methylenedioxyphenyl, sassafras oil and betel quid component	C1´-Hydroxylation, activation, high Km, low activity	15377158	(Jeurissen et al. 2004)
Safrole	1A2	Methylenedioxyphenyl, sassafras oil and betel quid component	o-Quinone formation, activation	30865484, 29082813	(Hu et al. 2019; Yang et al. 2018)
Safrole	1A1	Methylenedioxyphenyl, sassafras oil and betel quid component	C1´-Hydroxylation, activation	15310247	(Ueng et al. 2004)
Senecionine	3A4	Pyrrolizidine alkaloid, genotoxic	Pyrrole formation, N-oxygenation, dehydrogenation, activation	2009596, 8095200	(Guengerich 1993; Miranda et al. 1991)
Sterigmatocystin	1A1	Mycotoxin, fungal product	Epoxidation, activation (minor reaction)	7955101, 20929267, 21913247	(Cabaret et al. 2011; Cabaret et al. 2010; Shimada et al. 1994)
Sterigmatocystin	1A2	Mycotoxin, fungal product, xanthon	Epoxidation, activation (minor reaction)	7955101, 20929267, 21913247	(Cabaret et al. 2011; Cabaret et al. 2010; Shimada et al. 1994)
Sterigmatocystin	2W1	Mycotoxin, fungal product	Oxidation, activation	16551781	(Wu et al. 2006)
Sterigmatocystin	3A4	Mycotoxin, fungal product	Epoxidation, activation (minor reaction)	9328287, 20929267, 21913247, 7554070	(Cabaret et al. 2011; Cabaret et al. 2010; Gillam et al. 1997; Yamazaki et al. 1995)
Sterigmatocystin	3A7	Mycotoxin, fungal product	Oxidation, activation	9328287	(Gillam et al. 1997)
Sterigmatocystin	1B1	Mycotoxin, fungal product	Oxidation (weak activation)	16551781	(Wu et al. 2006)
Sterigmatocystin	2W1	Fungal product	Oxidation (activation)	16379042	(Brandon et al. 2006)

3-Methylindole (skatole) is formed in nature by microbial degradation of tryptophan and tyrosine (Carlson and Breeze 1984), but is also present humans where it is formed by the decarboxylation of tryptophan in the large intestine. 3-Methylindole is selective pulmonary toxicant and, in addition to intestinal formation and absorption, cigarette smoke is additional source of 3-methylindole in smokers. 3-Methylindole may provoke pneumotoxicity and lung cancer by activity of P450 1A1 and P450 2F1 (Weems et al. 2010). Toxicity of 3-methylindole depends on bioactivation by several reactions: epoxidation (3-methyloxindole formation, P450 1A1, 1A2, 1B1, 2E1, 2A6), C-hydroxylation (indole-3-carbinol formation, P450 1A1, 1A2, 1B1), and dehydrogenation (3-methyleneindolenine formation, P450 1A1, 1A2, 2A13, 2F1 (Table 4 and references therein).

The numbers of activation reactions catalyzed by human P450 enzymes reacting with natural products as substrates are presented in Fig. 15. Of the total of 952 reactions identified in our records, 152 (~16%) involve bioactivation and the formation of potentially toxic products. The major P450s involved in the activations are P450s 1A2 (~12%), P450s 2E1 and P450 3A4 (~11% each), followed by P450 1A1 and 2A6 (~10%).

Fig 15.

Participation of human P450 Families 1–4 in activation of natural products to potentially toxic products (952 reactions, 186 activation reactions)

The following examples illustrate the participation of P450 enzymes in the bioactivation of selected natural compounds (Table 4).

Aflatoxins

AFB₁ is a potent hepatocarcinogen in animal models and also classified as a hepatocarcinogen in humans. AFB₁ is metabolically activated by P450 enzymes to form cytotoxic and DNA-reactive intermediates (Fig. 16). AFB₁ is activated to the toxic exo-8,9 epoxide most prominently by P450 Subfamily 3A enzymes in liver and P450 2A13 in lung (Shimada and Guengerich 1989; Deng et al. 2018). In addition to its hepatotoxicity, AFB₁ can be toxic in lungs (at least in animal models) due to the activity of P450 2A enzymes. P450 3A enzymes (3A4 and 3A5) oxidize AFB₁ to the highly mutagenic exo-8,9-epoxide (Fig. 16), while P450 1A2 oxidizes it to a roughly equimolar mixture of toxic exo- plus the endo-epoxide, the latter of which is essentially non-mutagenic (Iyer et al. 1994). Both P450 3A4 and 1A2 enzymes also catalyze AFB₁ detoxication reactions, i.e. 3α-hydroxylation in the case of P450 3A4 (aflatoxin Q₁ formation) and 9a-hydroxylation in the case of P450 1A2 (aflatoxin M₁ formation) (Rendic and Guengerich 2012)). This example illustrates that P450 enzymes can catalyze both activation and detoxication reactions acting on the same substrate. The toxic AFB₁-exo-8,9-epoxide is detoxicated by glutathione (GSH) transferases by conjugation of GSH to the epoxide (Johnson et al. 1997; Deng et al. 2018; Yang et al. 2012). In addition to a being substrate of P450 enzymes, AFB₁ is an inducer of P450 1A1, 1B1, and 3A4 in monocytes (Bahari et al. 2014), and the compound might enhance its own metabolism or metabolism of another substrate of the enzyme.

Fig 16.

Activation (formation of 8,9-exo-epoxide) and detoxication (formation of AFQ₁) of AFB₁ by P450 3A4

Artistocholic acid

Aristolochic acids constitute a group of compounds found naturally in many types of plants known as Aristolochiaceae, including Aristolochia and Asarum (wild ginger) grown worldwide. Aristolochic acid I and II are the predominant chemical toxins in the plants. Aristolochic acid compounds were shown to be the cause of a kidney disease called Chinese herb nephropathy, now renamed aristolochic acid nephropathy (Arlt et al. 2002; Schmeiser et al. 2009; Gökmen et al. 2013). Aristocholic acid is classified by the International Agency for Research on Cancer as a Group I carcinogen. This natural product has also been implicated in the development of another kidney disease, Balkan endemic nephropathy, and its associated urothelial malignancy. The disease is endemic in certain rural areas of Balkan countries located closed to the tributaries of the Danube river basin (Arlt et al. 2007; Grollman et al. 2007; Han et al. 2019). As already mentioned, aristolochic acid I is activated by reduction of the nitro group (under partially anaerobic conditions), and oxidative metabolism results in formation of nontoxic O-demethylated metabolites. Nitro reduction of aristolochic acid I, considered as the major factor causing its toxicity, is required to exert its carcinogenic properties. The reaction catalyzed by P450s 1A1 and 1A2 results in the generation of N-hydroxyaristolactam I, which leads to the formation of a cyclic acyl nitrenium ion, the intermediate that either forms DNA adducts or rearranges to 7-hydroxyaristolactam I (Fig. 17). Aristolochic acid I oxidation to a nontoxic metabolite by O-demethylation of the methoxy group is catalyzed by the same enzymes, i.e. P450 1A1 and 1A2, with contribution from P450 2C9 and 3A4 (Table 4). The product of the reactions is 8-hydroxyaristolochic acid I, a detoxication product. The O-demethylated metabolite is excreted either in its free or conjugated form (Chan et al. 2006; Shibutani et al. 2010; Arlt et al. 2011; Stiborová et al. 2012a).

Fig 17.

Activation of aristolochic acid I and II by P450 enzymes

Estragole

Estragole is a common component of herbs and spices and is a natural constituent of basil oil. It is also a genotoxic hepatocarcinogen in rodents, and its potential toxic effect in humans is still under consideration. One of the major sources of human exposure to this phytochemical is Foeniculum vulgare Mill. (fennel) (Levorato et al. 2018). Toxicity is ascribed to its hydroxylation in position C1´, catalyzed by P450s 1A2, 2A6, 2C19, 2D6, and 2E1, of which P450s 1A2 and 2A6 are the major enzymes (Table 4 and references therein). Other enzymes can also contribute but at relatively high concentrations of estragole. The metabolite of P450 oxidation is not inherently toxic; however, C1´-hydroxylation of estragole is the first step in activation followed by sulfate conjugation by a sulfotransferase to produce genotoxic 3´-sulfoxyestragole (Fig. 18) (Monien et al. 2019).

Fig 18.

Activation of estragole by P450 enzymes and sulfotransferases

Ethanol

Ethanol is widely consumed and is metabolically activated to toxic acetaldehyde (Fig. 19). The metabolism and activation of ethanol is primarily catalyzed by alcohol dehydrogenase and, to a lesser extent, catalase but under certain circumstances (e.g., high doses) P450 enzymes can also be involved. Many P450 enzymes in Families 1–4 oxidize ethanol to acetaldehyde at high concentrations, namely 1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 (Table 3), but P450 2E1 has the highest catalytic activity (i.e., specificity constant, k_cat/K_m).

Fig 19.

Activation of ethanol to toxic acetaldehyde by P450 enzymes

The role of P450 2E1 in ethanol metabolism has been reviewed recently. P450 2E1, 3A4, and 1A2 were reported as P450s that are significantly involved in oxidation of ethanol under conditions of high concentration (K_m ~10 mM) and chronic use (Hamitouche et al. 2006; Guengerich and Avadhani 2018; Guengerich 2020).

Safrole

Safrole is a natural compound categorized as an IARC Group 2B carcinogen. It is extracted from sassafras oil or certain other essential oils and also from betel quid. Safrole was reported to be a rodent hepatocarcinogen, and DNA adducts were identified in liver samples of patients having a history of betel quid chewing (Bolton et al. 1994; Chung et al. 2008). In addition, betel quid chewing is associated with oral and hypopharynx cancers (Shield et al. 2017; Chen et al. 2017). The metabolism of safrole was reported to be predominantly catalyzed by P450 1A2, with minor contributions by P450 2E1. It was suggested that the ortho-quinone metabolite may mediate safrole hepatotoxicity (Fig. 20, Table 4 and references therein). Safrole can also, as in the case of estragole, undergo bioactivation by sequential 1´-hydroxylation and sulfation, resulting in reactive intermediates capable of forming DNA adducts (Jeurissen et al. 2004). In addition, it has been reported that safrole is a mechanism-based inhibitor of P450 1A2 (Hu et al. 2019; Yang et al. 2018). It has been also reported that safrole induced P450 2A6 activity and tobacco specific 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolic activation, resulting in higher NNK-induced genotoxicity (Tsou et al. 2019).

Fig 20.

Activation of safrole by P450 enzymes and sulfotransferases

N´-Nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

The tobacco specific nitrosamines NNN and NNK are potent carcinogens in animal models and are believed to be causative agents for esophageal cancer in smokers and those using chewing tobacco and snuff. Metabolic activation of NNN is required to exert carcinogenic potential (Fig. 21) and occurs through P450 catalyzed 2´- and 5´-hydroxylation, which generates unstable metabolites that decompose to 4-hydroxy-1-(3-pyridyl)-1-butanone (‘keto alcohol’) and 4-hydroxy-4-(3-pyridyl)butanal, respectively. The latter cyclizes to 5-(3-pyridyl)-2-hydroxytetrahydrofuran (‘lactol’). P450s 2E1, 2A6, and 3A4 were identified as major catalysts for NNN 5´-hydroxylation in human liver microsomes (Yamazaki et al. 1992; Hecht 1998; Wong et al. 2005a; Patten et al. 1996; Patten et al. 1997; Carlson et al. 2016; Fan et al. 2019; Staretz et al. 1997; Fujita and Kamataki 2001a).

Fig 21.

Activation of NNN by P450 enzymes

NNK, a potent tobacco-specific carcinogen, has been demonstrated to induce lung tumors in animals and is suspected to be a human carcinogen. P450s are the major enzymes responsible for the activation of NNK in lung and liver microsomes of rats and mice, as well as in human liver. Human P450s 2A6 and 3A4 are involved in the activation of NNK (Smith et al. 1995; Staretz et al. 1997). In addition, it was demonstrated that P450s 1A2, 2A6, and 3A4 may be important for the activation of NNK to a DNA-methylating agent (‘keto aldehyde’) via the α-methylene hydroxylation pathway (Fig. 22). P450s 1A2, 2E1, and 2D6 are selective for α-methyl hydroxylation of NNK, leading to keto alcohol and a DNA-pyridyloxobutylating agent. P450 1A2 exhibits at least twice the specificity toward NNK bioactivation compared to P450 2E1 and catalyzed the formation of both, keto alcohol and 4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde) with the keto alcohol being the major product (Patten et al. 1996; Patten et al. 1997; Krishnan et al. 2009; Smith et al. 1996) (Table 4 and references therein).

Fig 22.

Activation of NNK by P450 enzymes

Physiological compounds

Physiological substrates of P450 Family 1–4 enzymes include eicosanoids, estrogens (e.g., estradiol), fatty acids (e.g., arachidonic acid), cholesterol, fat-soluble vitamins (e.g., vitamins A, D₃, E, and K), neurotransmitters (serotonin, tryptamine), leukotrienes, prostaglandins, fatty acids (e.g. arachidonic acid), bile acids (e.g. lithocholic, deoxycholic, cholic acid), corticosteroids, androgens (e.g., androstenedione, testosterone, dihydrotestosterone), and progesterone. In addition to being substrates of P450s Families 1–4, these compounds are predominately substrates of the enzymes belonging to Families 5–51 (with 22 enzymes) (Rendic and Di Carlo 1997; Rendic and Guengerich 2018). The data presented in Fig. 23 show the participation of Family 1–4 P450s in the activation of physiological compounds to some potentially toxic products. Of the total 530 metabolic reactions (data from our records), 75 (14 %) involve bioactivation. The highest involvement is with P450s 1A1, 1A2, 1B1, 3A4, and 3A5 (~9% each), followed closely by P450 2C9 (8%). Physiological substrates in activation reactions include estrogenic hormones (17β-estradiol and estrone) and fatty acids (Table 5). In addition to being activated to toxic products, fatty acids (exemplified by arachidonic acid) can both down-regulate (Palacharla et al. 2017) or induce P450 activity by changing their expression (Finn et al. 2009). In some cases, the reaction products are not inherently reactive but may have deleterious signaling properties (e.g., 20-HETE, EETs (Sausville et al. 2018)).

Fig 23.

Participation of human P450 Families 1–4 in metabolic activation of physiological compounds to potentially toxic metabolites (530 reactions, 75 activation reactions)

Table 5.

Examples of the metabolic activation of physiological compounds by human cytochrome P450 enzymes.

Compound	Enzyme	Subcategory	Reaction	PMIDs	References
Arachidonic acid	2E1	Fatty acid, polyunsaturated	Oxidation	9169410, 14744237	(Caro and Cederbaum 2004; Chen et al. 1997)
Arachidonic acid	2W1	Fatty acid, polyunsaturated	Epoxidation, epoxyeicosatrienoic acid (EET) formation, very low activity, if any, under physiological conditions	26936974	(Zhao et al. 2016)
Arachidonic acid	2C8	Fatty acid, polyunsaturated	Epoxidation, EET formation	28701518, 7996455, 7625847, 15652503, 8651708, 7574697, 15766564, 11668219	(Barbosa-Sicard et al. 2005; Dai et al. 2001b; Daikh et al. 1994; Fan and Roman 2017; Lundblad et al. 2005; Rifkind et al. 1995; Zeldin et al. 1995; Zeldin et al. 1996)
Arachidonic acid	2C9	Fatty acid, polyunsaturated	Epoxidation, EET formation, 14,15-, 11,12- and 8,9-EETs	28701518, 7996455, 7625847, 8651708, 7574697, 15766564, 15652503	(Barbosa-Sicard et al. 2005; Daikh et al. 1994; Fan and Roman 2017; Lundblad et al. 2005; Rifkind et al. 1995; Zeldin et al. 1995)
Arachidonic acid	2J2	Fatty acid, polyunsaturated	Epoxidation, EET formation	28701518	(Fan and Roman 2017)
Arachidonic acid	4F3	Fatty acid, polyunsaturated	Hydroxylation, 20-hydroxy eicosatetraenoic acid (HETE) formation, activation to pro-hypertensive activity	28701518, 12709424	(Christmas et al. 2003; Fan and Roman 2017)
Arachidonic acid	4F2	Fatty acid, polyunsaturated	Hydroxylation, 20-HETE formation, low activity, activation to pro-hypertensive activity	9618440, 10660572, 28701518, 18662666	(Fan and Roman 2017; Hirani et al. 2008; Lasker et al. 2000; Powell et al. 1998)
Arachidonic acid	4A11	Fatty acid, polyunsaturated	Hydroxylation, 20-HETE formation, activation to pro-hypertensive activity	9618440, 10660572, 28701518	(Fan and Roman 2017; Lasker et al. 2000; Powell et al. 1998)
Arachidonic acid	1A2	Fatty acid, polyunsaturated	Epoxidation, EET formation	7625847, 15258110, 8651708, 7574697	(Choudhary et al. 2004; Rifkind et al. 1995; Zeldin et al. 1995; Zeldin et al. 1996)
Arachidonic acid	2E1	Fatty acid, polyunsaturated	Hydroxylation, 20-HETE formation, activation to pro-hypertensive activity	7625847	(Rifkind, 1995)
Arachidonic acid	2B6	Fatty acid, polyunsaturated	Epoxidation, EET formation, activation, low activity	7625847, 8651708, 7574697	(Rifkind, 1995;Zeldin et al. 1996; Zeldin et al. 1995)
Arachidonic acid	1A1	Fatty acid, polyunsaturated	Epoxidation, EET formation	15041462	(Schwarz et al. 2004)
Arachidonic acid	1B1	Fatty acid, polyunsaturated	Epoxidation, EET formation	15258110	(Choudhary et al. 2004)
Arachidonic acid	2C19	Fatty acid, polyunsaturated	Epoxidation, EET formation	9866708, 9435160	(Bylund et al. 1998a; Bylund et al. 1998b)
17β-Estradiol	2D6	Estrogen	C16α-Hydroxylation, minor reaction	9667077, 9625734	(Niwa et al. 1998; Yamazaki et al. 1998b)
17β-Estradiol	3A5	Estrogen	C4-Hydroxylation, activation	11454902, 15784278, 12865317, 12124305	(Lee et al. 2003a; Lee et al. 2001; Williams et al. 2002; Zhu and Lee 2005)
17β-Estradiol	1B1	Estrogen	C4-Hydroxylation, major extrahepatic enzyme, medium to low Km, medium activity, medium to low efficiency, activation	8790407, 9498279, 9152602, 15784278, 12865317, 12902195, 10426814, 14703066, 10739169, 11465393, 10403516, 16112414, 12423652, 10862525, 10963622, 15142886, 11555828, 10910054, 11854143 11854439, 11719446, 25678418	(Aklillu et al. 2002; Badawi et al. 2001; Chen et al. 2004; Chun et al. 2001; Hanna et al. 2000; Hayes et al. 1996; Jefcoate et al. 2000; Lee et al. 2003a; Li et al. 2000; Modugno et al. 2003; Niwa et al. 2015; Pang et al. 1999; Shimada et al. 1997b; Shimada et al. 2001b; Shimada et al. 1999; Spink et al. 2002a; Spink et al. 2002b; Spink et al. 1998; Tsuchiya et al. 2005; van Duursen et al. 2003; Watanabe et al. 2000; Zhu and Lee 2005)
17β-Estradiol	1A2	Estrogen	C4-Hydroxylation, minor reaction, activation	1449532, 15784278, 9625734, 9054608, 9667077, 9152602, 11555828, 12865317	(Badawi et al. 2001; Kerlan et al. 1992; Lee et al. 2003a; Niwa et al. 1998; Shimada et al. 1997b; Shou et al. 1997; Yamazaki et al. 1998b; Zhu and Lee 2005)
17β-Estradiol	3A4	Estrogen	C4-Hydroxylation, minor reaction, medium Km, medium activity, activation	1449532, 11454902, 15784278, 9625734, 9054608, 9667077, 12865317, 12124305, 11555828, 9003190	(Badawi et al. 2001; Kerlan et al. 1992; Lee et al. 2003a; Lee et al. 2001; Niwa et al. 1998; Shou et al. 1997; Ueng et al. 1997; Williams et al. 2002; Yamazaki et al. 1998b; Zhu and Lee 2005)
17β-Estradiol	1A2	Estrogen	C16α-Hydroxylation (major enzyme, high Km, low activity)	9625734, 9054608, 12865317, 9667077, 11555828	(Badawi et al. 2001; Lee et al. 2003a; Niwa et al. 1998; Shou et al. 1997; Yamazaki et al. 1998b)
17β-Estradiol	1B1	Estrogen	C16α-Hydroxylation,(minor enzyme, medium and high Km, low activity)	9667077, 11555828, 11854439, 10910054	(Aklillu et al. 2002; Badawi et al. 2001; Hanna et al. 2000; Niwa et al. 1998)
17β-Estradiol	1A1	Estrogen	C16α-Hydroxylation (high Km, low activity)	9667077, 11555828, 12865317, 15784278, 10403516	(Badawi et al. 2001; Lee et al. 2003a; Niwa et al. 1998; Pang et al. 1999; Zhu and Lee 2005)
17β-Estradiol	1A1	Estrogen	C4-Hydroxylation (minor reaction, medium Km, medium efficiency, low activity, activation)	8790407, 11555828, 12865317, 15784278, 10403516, 25678418	(Badawi et al. 2001; Hayes et al. 1996; Lee et al. 2003a; Niwa et al. 2015; Pang et al. 1999; Zhu and Lee 2005)
17β-Estradiol	2A6	Estrogen	C4-Hydroxylation (low activity, activation)	12865317, 15784278	(Lee et al. 2003a; Zhu and Lee 2005)
17β-Estradiol	2C19	Estrogen	C4-Hydroxylation (low activity, activation)	11067738	(Satoh et al. 2000)
17β-Estradiol	2C19	Estrogen	C16α-Hydroxylation (low activity)	9667077, 9625734, 15784278	(Niwa et al. 1998; Yamazaki et al. 1998b; Zhu and Lee 2005)
17β-Estradiol	2C8	Estrogen	C4-Hydroxylation (low activity, activation)	12865317, 11067738	(Lee et al. 2003a; Satoh et al. 2000)
17β-Estradiol	2C9	Estrogen	C4-Hydroxylation (very low activity, activation)	9625734, 12865317, 15784278	(Lee et al. 2003a; Yamazaki et al. 1998b; Zhu and Lee 2005)
17β-Estradiol	2C9	Estrogen	C16α-Hydroxylation (minor reaction, low activity)	9667077, 9625734, 15784278	(Niwa et al. 1998; Yamazaki et al. 1998b; Zhu and Lee 2005)
17β-Estradiol	2D6	Estrogen	C4-Hydroxylation (very low activity, activation)	11067738	(Satoh et al. 2000)
17β-Estradiol	3A4	Estrogen	C16α-Hydroxylation, high Km, low activity	9625734, 11454902, 12865317, 14559847, 9667077, 14703066, 11555828	(Badawi et al. 2001; Lee et al. 2003a; Lee et al. 2003b; Lee et al. 2001; Modugno et al. 2003; Niwa et al. 1998; Yamazaki et al. 1998b)
17β-Estradiol	3A5	Estrogen	C16α-Hydroxylation, high Km, low activity	12865317, 14559847	(Lee et al. 2003a; Lee et al. 2003b)
17β-Estradiol	3A7	Estrogen	C4-Hydroxylation, high Km, low activity, activation	14559847	(Lee et al. 2003b)
17β-Estradiol	3A4	Estrogen	C2-Hydroxylation, activation, major enzyme in liver	11454902, 11067738, 14703066, 12865317, 11555828, 16112414	(Badawi et al. 2001; Lee et al. 2003a; Lee et al. 2001; Modugno et al. 2003; Satoh et al. 2000; Tsuchiya et al. 2005)
17β-Estradiol	3A5	Estrogen	C2-Hydroxylation, activation	11454902, 12865317	(Lee et al. 2003a; Lee et al. 2001)
17β-Estradiol	2D6	Estrogen	C2-Hydroxylation, activation	12865317	(Lee et al. 2003a)
17β-Estradiol	1A1	Estrogen	C15α-Hydroxylation	12865317	(Lee et al. 2003a)
17β-Estradiol	1A1	Estrogen	C6α-Hydroxylation,	12865317	(Lee et al. 2003a)
17β-Estradiol	1A1	Estrogen	C7α-Hydroxylation	12865317	(Lee et al. 2003a)
17β-Estradiol	1B1	Estrogen	C2-Hydroxylation, low Km and low activity, activation	8790407, 12865317, 11555828, 25678418	(Badawi et al. 2001; Hayes et al. 1996; Lee et al. 2003a; Niwa et al. 2015)
17β-Estradiol	3A4	Estrogen	C16β-Hydroxylation, low activity	12865317, 14703066	(Lee et al. 2003a; Modugno et al. 2003)
17β-Estradiol	1A2	Estrogen	C2-Hydroxylation, high activity, activation, major enzyme in liver	12865317, 11555828, 16112414	(Badawi et al. 2001; Lee et al. 2003a; Tsuchiya et al. 2005)
17β-Estradiol	1A1	Estrogen	C2-Hydroxylation, high activity, major extrahepatic enzyme, activation	8790407, 9498279, 11854143, 12902195, 14703066, 16112414, 15142886, 25678418	(Chen et al. 2004; Hayes et al. 1996; Modugno et al. 2003; Niwa et al. 2015; Spink et al. 2002b; Spink et al. 1998; Tsuchiya et al. 2005; van Duursen et al. 2003)
17β-Estradiol	2W1	Estrogen	C2-Hydroxylation, very low activity under physiological conditions, activation	26936974	(Zhao et al. 2016)
17β-Estradiol	2A6	Estrogen	C2-Hydroxylation, activation	12865317	(Lee et al. 2003a)
17β-Estradiol	2B6	Estrogen	C2-Hydroxylation, activation	12865317	(Lee et al. 2003a)
17β-Estradiol	2C8	Estrogen	C2-Hydroxylation, activation	12865317	(Lee et al. 2003a)
17β-Estradiol	2C9	Estrogen	C2-Hydroxylation, activation	12865317	(Lee et al. 2003a)
17β-Estradiol	2C19	Estrogen	C2-Hydroxylation, activation	12865317	(Lee et al. 2003a)
17β-Estradiol	P450 2E1	Estrogen	C4-Hydroxylation (low activity, activation)	11067738, 15784278	(Satoh et al. 2000; Zhu and Lee 2005)
17β-Estradiol	3A7	Estrogen	C16α-Hydroxylation, very low activity	12865317, 14559847	(Lee et al. 2003a; Lee et al. 2003b)
Estrone	1A1	Estrogen	C15α-Hydroxylation, high activity	12865317, 15784278	(Lee et al. 2003a; Zhu and Lee 2005)
Estrone	2A6	Estrogen	C16α-Hydroxylation, at higher concentration	9635876	(Huang et al. 1998)
Estrone	3A7	Estrogen	C16α-Hydroxylation, minor reaction	12865317, 14559847	(Lee et al. 2003a; Lee et al. 2003b)
Estrone	1A1	Estrogen	C16α-Hydroxylation, minor reaction	9635876, 15784278, 16537715	(Cribb et al. 2006; Huang et al. 1998; Zhu and Lee 2005)
Estrone	1B1	Estrogen	C4-Hydroxylation, low Km, major reaction, activation	9152602, 15784278, 12865317, 16537715, 10426814, 10739169, 11465393, 16207128, 25678418	(Cribb et al. 2006; Lee et al. 2003a; Niwa et al. 2015; Paracchini et al. 2005; Shimada et al. 1997b; Shimada et al. 2001b; Shimada et al. 1999; Watanabe et al. 2000; Zhu and Lee 2005)
Estrone	1A1	Estrogen	C4-Hydroxylation, medium activity, activation	12865317, 15784278, 16537715, 25678418	(Cribb et al. 2006; Lee et al. 2003a; Niwa et al. 2015; Zhu and Lee 2005)
Estrone	1A1	Estrogen	C7α-Hydroxylation, minor reaction, activation	12865317, 15784278	(Lee et al. 2003a; Zhu and Lee 2005)
Estrone	1A2	Estrogen	C4-Hydroxylation (medium Km, very low to medium activity, activation)	9625734, 9054608, 12865317, 15784278, 16537715	(Cribb et al. 2006; Lee et al. 2003a; Shou et al. 1997; Yamazaki et al. 1998b; Zhu and Lee 2005)
Estrone	1A2	Estrogen	C16α-Hydroxylation (minor reaction, very low activity, activation)	9625734, 9054608, 12865317, 25678418	(Lee et al. 2003a; Niwa et al. 2015; Shou et al. 1997; Yamazaki et al. 1998b)
Estrone	3A7	Estrogen	C4-Hydroxylation (low activity, medium Km, activation)	12865317, 14559847	(Lee et al. 2003a; Lee et al. 2003b)
Estrone	3A7	Estrogen	C2-Hydroxylation (low activity, activation)	14559847	(Lee et al. 2003b)
Estrone	3A5	Estrogen	C16α-Hydroxylation (high Km, low activity)	9635876, 9625734, 9054608, 12865317, 9667077, 15784278, 16537715, 14559847	(Cribb et al. 2006; Huang et al. 1998; Lee et al. 2003a; Lee et al. 2003b; Niwa et al. 1998; Shou et al. 1997; Yamazaki et al. 1998b; Zhu and Lee 2005)
Estrone	2C9	Estrogen	C16α-Hydroxylation (low activity, activation)	9635876, 9625734, 9054608, 14703066, 9667077, 15784278	(Huang et al. 1998; Modugno et al. 2003; Niwa et al. 1998; Shou et al. 1997; Yamazaki et al. 1998b; Zhu and Lee 2005)
Estrone	3A4	Estrogen	C16α-Hydroxylation (high Km, low activity, major enzyme)	9625734, 9054608, 9667077, 9635876, 12865317, 15784278, 14559847, 25678418	(Huang et al. 1998; Lee et al. 2003a; Lee et al. 2003b; Niwa et al. 2015; Niwa et al. 1998; Shou et al. 1997; Yamazaki et al. 1998b; Zhu and Lee 2005)
Estrone	3A4	Estrogen	C4-Hydroxylation (high Km, low activity, major enzyme, activation)	9635876, 9625734, 9054608, 9667077, 12865317, 15784278	(Huang et al. 1998; Lee et al. 2003a; Niwa et al. 1998; Shou et al. 1997; Yamazaki et al. 1998b; Zhu and Lee 2005)
Estrone	2C9	Estrogen	C4-Hydroxylation, (very low activity, activation)	9625734, 12865317, 15784278	(Lee et al. 2003a; Yamazaki et al. 1998b; Zhu and Lee 2005)
Estrone	2C19	Estrogen	C16α-Hydroxylation (low activity)	9054608, 9625734, 15784278, 16537715	(Cribb et al. 2006; Shou et al. 1997; Yamazaki et al. 1998b; Zhu and Lee 2005)
Estrone	2E1	Estrogen	C16α-Hydroxylation (very low activity)	9054608, 9625734, 15784278	(Shou et al. 1997; Yamazaki et al. 1998b; Zhu and Lee 2005)
Estrone	2B6	Estrogen	C4-Hydroxylation (very low activity, activation)	9054608, 9625734, 15784278	(Shou et al. 1997; Yamazaki et al. 1998b; Zhu and Lee 2005)
Estrone	2E1	Estrogen	C4-Hydroxylation (very low activity, activation)	9054608, 9625734, 15784278	(Shou et al. 1997; Yamazaki et al. 1998b; Zhu and Lee 2005)
Estrone	2C8	Estrogen	C-4 Hydroxylation (very low activity, activation)	9054608, 9625734	(Shou et al. 1997; Yamazaki et al. 1998b)
Estrone	1A1	Estrogen	C6α-Hydroxylation, low activity	12865317	(Lee et al. 2003a)
Estrone	1A1	Estrogen	C2-Hydroxylation, major metabolite, activation	12865317, 25678418	(Lee et al. 2003a; Niwa et al. 2015)
Estrone	1A2	Estrogen	C2-Hydroxylation, high activity, activation	12865317	(Lee et al. 2003a)
Estrone	1B1	Estrogen	C2-Hydroxylation, low activity, activation	12865317, 25678418	(Lee et al. 2003a; Niwa et al. 2015)
Estrone	3A4	Estrogen	C2-Hydroxylation, activation	12865317, 25678418	(Lee et al. 2003a; Niwa et al. 2015)
Estrone	3A5	Estrogen	C2-Hydroxylation, activation	12865317	(Lee et al. 2003a)
Estrone	3A5	Estrogen	C4-Hydroxylation, activation	12865317	(Lee et al. 2003a)

Although a relatively low number of activations are ascribed to P450 enzymes interacting with physiological compounds, some of them are important because they can possibly cause either cancer (e.g., estrogenic hormones) or have important impact on physiological processes related to high blood pressure (arachidonic acid).

17β-Estradiol and estrone

Estrogenic hormones (e.g., 17β-estradiol and estrone) can induce tumors in various organs of experimental animals (Lacassagne 1932). In humans, elevated circulating estrogen levels increase the risk of breast and uterine cancer. Estrogens can act as hormone stimulating cell proliferators and also as procarcinogens, inducing genetic damage (Yager 2000; Liehr 2000). 17β-Estradiol and estrone are eliminated from the body by metabolic conversion to inactive metabolites that are excreted in the urine and/or feces following oxidations and conjugation reactions. The first step in the metabolism of estrogens is hydroxylation catalyzed by P450 enzymes (Fishman et al. 1970; Zhu and Lee 2005). A large number of hydroxylated metabolites are formed and catalyzed by P450 Family 1–4 (Table 5); however, we focus here on reactions leading to formation of activated and toxic metabolites. Activations of 17β-estradiol and estrone by hydroxylation at positions C2 and C4 have been suggested to be major reactions involved in mammary carcinogenesis and other cancers (Cavalieri and Rogan 2006; Cavalieri et al. 2006). The data (Table 5 and references therein) also show that formation of the major metabolite of 17β-estradiol, 2-hydroxyestradiol, is mainly catalyzed by P450s 1A2 and 3A4, and by P450 1A1 in extrahepatic tissues. P450 1B1, which is highly expressed in estrogen target tissues including mammary, ovary, and uterus, selectively catalyzes the 4-hydroxylation of 17β-estradiol (Guengerich et al. 2003; Chun and Kim 2016; Wen et al. 2007) Formation of catechols of estrone and estradiol is considered as a part of the carcinogenic process, in that these compounds can readily be further oxidized to reactive quinones, semiquinones, and reactive oxygen species are formed (Bolton and Thatcher 2008). 4-Hydroxyestradiol can generate free radicals from redox cycling, with formation of corresponding semiquinone and quinone forms causing cellular damage. Local formation of 4-hydroxyestradiol in breast and endometrial cancers has been reported (Tsuchiya et al. 2005; Hayes et al. 1996; Spink et al. 1997; Liehr 2000; Shimada et al. 1999; Bolton 2002; Bolton and Thatcher 2008; Fussell et al. 2011). Estradiol-3,4-quinone is more reactive with DNA than estradiol-2,3-quinone, and the relative reactivities of estradiol-3,4-quinone and estradiol-2,3-quinone to form depurinating adducts have been correlated with the carcinogenicity, mutagenicity, and cell-transforming activity of their precursors, the catechol estrogens 4-hydroxyestradiol and 2-hydroxyestradiol (Zahid et al. 2006).

Numerous P450s have been detected in breast tumor or adjacent tissue, including P450s 1A1, 1B1, 2A5, 2B6, 2C9, 2D6, 2E1, 2J2, 2S1, 2U1, 3A4, 3A5, 3A43, 4A11, 4V2, 4X1, 4Z1, 26A1, and of course 19A1 (Hellmold et al. 1998; Huang et al. 1996; Iscan et al. 2001; Schmidt et al. 2004). Of these, three enzymes are involved to major extent in estradiol hydroxylation (i.e. P450s 1A1, 1B1, and 3A4) (Fig. 24). P450 2C9 is also involved in the conversion of both estradiol and estrone, with low activity in forming C4- and C16-hydroxylated products (Table 5). P450 enzymes involved in estrogen metabolism are expressed in both tumor and non-tumor breast tissue; however, higher levels of P450 1B1 and 3A4 were found more often in non-tumor tissue than in tumor tissue. It has been suggested that local activation of estrogen to potentially reactive metabolites by the P450s in breast tissue may play a role in initiating and promoting the carcinogenic process (Modugno et al. 2003).

Fig 24.

Metabolism and activation of 17β-estradiol and estrone by P450 enzymes

In breast tumor cells, P450 1A1 and 1B1 mRNA levels and rates of both estradiol 2- and 4-hydroxylation were elevated following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Spink et al. 1998). In addition, the inhibitory effects of ketoconazole, cyclosporin A, and cimetidine (inhibitors of P450 enzymes) toward P450 3A4-catalyzed estradiol 2-hydroxylation were reported, and the IC₅₀ values were 7 nM, 64 nM, and 290 μM, respectively (Satoh et al. 2000). It was also reported that non-ortho-substituted polychlorinated biphenyl congeners can, depending on the structure, induce or inhibit P450 1B1 and 1A1 activity and consequently that they might affect the formation of 2- and 4- hydroxylated metabolites of estradiol and the potential for mammary tumorigenesis (Spink et al. 2002a; Pang et al. 1999). Resveratrol was reported to strongly inhibit the TCDD-induced aryl hydrocarbon receptor DNA binding activity, the expression of P450 1A1 and 1B1, and P450 1A1 and 1B1 catalytic activities in MCF-10A breast cancer cells. Resveratrol also reduced the formation of 2- and 4-hydroxyestradiol from 17β-estradiol by recombinant human P450s 1A1 and 1B1, respectively. Furthermore, resveratrol significantly attenuated intracellular reactive oxygen species formation and oxidative DNA damage, and the cytotoxicity induced by the catechol estrogens (Chen et al. 2004). In addition to chemicals that induce or inhibit activity of P450 enzymes, genetic variation of the enzymes (e.g., P450 1B1) can also affect the metabolic activation and carcinogenesis of 17β-estradiol and estrone, although the effects have not been shown to be large (Shimada et al. 1999; Watanabe et al. 2000). Changes in the expression levels of estrogen-metabolizing P450s not only alter the activity of substrates but may also have physiological effects in liver and target tissues (Chun and Kim 2016).

Arachidonic acid

Arachidonic acid metabolites are key mediators involved in the pathogenesis of numerous cardiovascular, pulmonary, inflammatory, and thromboembolic diseases. Thromboxane A₂ is produced by the action of thromboxane synthase (P450 5A1) on the prostaglandin endoperoxide H₂ (PGH₂), a product of the enzymatic transformation of arachidonic acid by the cyclooxygenases (Rendic and Guengerich 2018). Arachidonic acid is metabolized in a number of tissues (liver, kidney, lung, brain, and the vasculature) by P450 enzymes that form hydroxyeicosatetraenoic acids (HETEs) or epoxides (epoxyeicosatrienoic acids, EETs) (Fig. 25). The reactions occur in different organs (brain, kidney, lung, vasculature, liver). EETs and HETEs have different biological properties, based on sites of production, and can be stored in tissue lipids and released in response to hormonal stimuli.

Fig 25.

Metabolism and activation of arachidonic acid to 20-HETE and EETs by P450 enzymes

20-HETE has both pro- and anti-hypertensive actions that result from modulation of vascular and kidney function. 20-HETE is a potent vasoconstrictor, and upregulation of the production of this compound can contributes to elevation of endothelial dysfunction and the increase in peripheral vascular resistance associated with some forms of hypertension. In kidney, 20-HETE exerts anti-hypertensive action by inhibiting sodium reabsorption by the kidney in both the proximal tubule and thick ascending limb of Henle (Williams et al. 2010; Garcia et al. 2017; Zhang et al. 2018; Roman 2002). Formation of 20-HETE is catalyzed by human P450s 4A11, 4F2, and F3B and the epoxygenation of arachidonic acid to EETs is catalyzed by P450s 2C8, 2C9, 2C19, and 2J2 and (to a much lesser extent) by P450 2W1 (Table 5 and references therein). The arachidonic acid products 20-HETE and EETs compose a group of compounds that participate in the regulation of liver metabolic activity and hemodynamics, may be involved in abnormalities related to liver diseases (e.g., cirrhosis), and play a key role in the pathophysiology of portal hypertension and renal failure (Sacerdoti et al. 2003). Arachidonic acid, as a model for metabolic activation of polyunsaturated fatty acids, produced a concentration- and time-dependent toxicity to Hep G2-MV2E1–9 cells, which express P450 2E1, proposed to be related to reactive oxygen intermediates and lipid peroxidation (Chen et al. 1997).

Concluding remarks

The data on activation of xenobiotics and endobiotics catalyzed by P450 enzymes in Families 1–4 are divided into groups of General Chemicals, Drugs, Natural Products, and Physiological Compounds. The metabolites formed are direct toxicants reacting with cell macromolecules in many cases. However, in selected cases the metabolites are not direct toxicants but participate as substrates in additional metabolic reactions (e.g., conjugation reactions) and the resulting products are final toxicants (e.g., estragole). In other cases, the product elicits physiological responses through indirect biological activities (e.g., 20-HETE, EETs). We have emphasized the observed higher number of activations of three groups of compounds (General Chemicals, Drugs, and Natural Products) yielding activated metabolites and the lower fraction of Physiological Compounds involved as substrates in activation reactions catalyzed by P450 enzymes belonging to Families 1–4, exemplified by estrogen hormones and arachidonic acid. In the group of General Chemicals, P450s 1A1, 1A2, and 1B1 are dominant in the formation of activated metabolites, followed by P450s 3A4 and 2E1 (Fig. 2); in the group Drugs (Fig. 9) P450 3A4 dominates in the formation of activated metabolites. In the group of Natural Products, P450s 1A2, 3A4, and 2E1 dominate in the formation of activated metabolites, followed by P450s 1A1 and 2A6 (Fig. 16); in the group of Physiological Compounds there was no clearly dominant P450 but the highest number of activations is attributed to P450s 1A, 1B1 and 3A (Fig. 23). The results show that Physiological Compounds are substrates infrequently in bioactivation reactions catalyzed by P450 enzymes belonging to Families 1–4, with the gexception of estrogens and arachidonic acid.

The results presented give information on the enzymes that dominate in bioactivation of specific group of chemicals and might be used as guide on which enzymes to direct research when testing their bioactivation to toxic metabolites.

Fig 6.

Activation of chlorpyrifos to a toxic oxon product by P450 enzymes

Fig 12.

Metabolic activation of acetaminophen (paracetamol)