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Published in final edited form as: Chem Res Toxicol. 2016 Oct 21;29(11):1828–1834. doi: 10.1021/acs.chemrestox.6b00203

Dihydromethysticin (DHM) Blocks Tobacco Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-Induced O6-Methylguanine in a Manner Independent of the Aryl Hydrocarbon Receptor (AhR) Pathway in C57BL/6 Female Mice

Sreekanth C Narayanapillai †, Shang-Hsuan Lin †, Pablo Leitzman †, Pramod Upadhyaya ‡, Carolyn J Baglole §, Chengguo Xing †,‡,‖,*
PMCID: PMC6532060  NIHMSID: NIHMS983501  PMID: 27728767

Abstract

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a key carcinogen responsible for tobacco smoke-induced lung carcinogenesis. Among the types of DNA damage caused by NNK and its metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), O6-methylguanine (O6-mG) is likely the most carcinogen in A/J mice. Results of our previous studies showed that levels of O6-mG and other types of NNAL-derived DNA damage were preferentially reduced in the lung of female A/J mice upon dietary treatment with dihydromethysticin (DHM), a promising lung cancer chemopreventive agent from kava. Such a differential blockage may be mediated via an increased level of NNAL glucuronidation, thereby leading to its detoxification. The potential of the aryl hydrocarbon receptor (AhR) as an upstream target of DHM mediating these events was evaluated herein using Ahr+/− and Ahr−/− C57BL/6 female mice because DHM was reported as an AhR agonist. DHM (0.05, 0.2, and 1.0 mg/g of diet) and dihydrokavain (DHK, an inactive analogue, 1.0 mg/g of diet) were given to mice for 7 days, followed by a single intraperitoneal dose of NNK at 100 mg/kg of body weight. The effects of DHM on the amount of O6-mG in the lung, on the urinary ratio of glucuronidated NNAL (NNAL-Gluc) and free NNAL, and on CYP1A½ activity in the liver microsomes were analyzed. As observed in A/J mice, DHM treatment significantly and dose-dependently reduced the level of O6-mG in the target lung tissue, but there were no significant differences in O6-mG reduction between mice from Ahr+/− and Ahr−/− backgrounds. Similarly, in both strains, DHM at 1 mg/g of diet significantly increased the urinary ratio of NNAL-Gluc to free NNAL and CYP1A½ enzymatic activity in liver with no changes detected at lower DHM dosages. Because none of these effects of DHM were dependent on Ahr status, AhR clearly is not the upstream target for DHM.

Graphical Abstract

graphic file with name nihms-983501-f0001.jpg

INTRODUCTION

As the leading cause of cancer-related death, lung cancer accounts for ~160000 deaths in the United States and more than 1.6 million deaths worldwide annually.1 Because of the lack of robust early diagnosis and effective treatment, the five-year survival rate of lung cancer patients has been around 15–18% for decades.2 Although developing effective therapy and early diagnosis are important in the clinical management of lung cancer, it is also essential to develop chemopreventive agents against this disease.

Tobacco use accounts for 85–90% of lung cancer incidence.3 Many chemicals detected in tobacco smoke have been classified as human carcinogens.4 Among these, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, a tobacco-specific nitrosamine) is a well-studied pulmonary carcinogen.5 Upon cytochrome P450 (CYP450) enzyme-mediated hydroxylation, NNK generates reactive intermediates that can form types of DNA damage. Alternatively, NNK can be metabolically reduced to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which will be bioactivated by CYP450 to form types of DNA damage (Scheme 1). These types of DNA damage, if not repaired, can lead to gene mutations and initiate lung tumorigenesis.6 Therefore, chemical entities that can block NNK- and NNAL-induced DNA damage have the potential to prevent lung carcinogenesis.

Scheme 1.

Scheme 1.

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Putative Mechanism of Action of DHM via Activation of the AhR Pathway To Enhance NNAL Glucuronidation, Which Reduces the Level of NNK- and NNAL-Induced DNA Damage and Blocks Lung Carcinogenesis

Our earlier studies showed that dihydromethysticin (DHM), a natural product from Piper methysticum (kava), could block NNK-induced lung tumorigenesis in A/J mice, provided it was given during the tumor initiation phase.7‘8 DHM at a dose of 0.05 mg/g in the diet was able to reduce lung adenoma multiplicity by 97%, with a significant reduction in the level of DNA adduct O6-mG (likely the most carcinogenic type of DNA damage in A/J mice9). Further studies suggested that the reduction in the level of O6-mG by DHM may originate from the increased level of detoxification of NNAL.10 Specifically, dietary DHM increased the relative abundance of 4-(methylnitrosamino)-1-(3-pyridyl)-1-(O – β-d-glucopyranuronosyl)butane (NNAL-O-Gluc) in A/J mouse urine, which is likely mediated via the enhanced NNAL glucuronidating activity.10

NNAL glucuronidation is catalyzed by UDP-glucuronosyl-transferases (UGTs).11 UGTs are typically regulated by several transcriptional factors, including pregnane X receptor (PXR), constitutive androstane receptor (CAR),12,13 and aryl hydrocarbon receptor (AhR).14 Recently, Li et al. reported that DHM and methysticin, another structurally similar kavalactone in kava that also blocks NNK- and NNAL-induced O6-mG formation in A/J mice,8 could activate the AhR pathway in vitro.15 AhR is a ligand-dependent transcriptional factor strongly expressed in the lung.16 When its agonist binds, AhR translocates to the nucleus and dimerizes with ARNT, followed by the binding to the xenobiotic response element (XRE) of drug-metabolizing genes to activate their transcription.17 Besides UGTs and other phase II enzymes, phase I enzymes, particularly CYP1A1, are also dominantly regulated by AhR. While being essential for normal metabolism and other regular cellular functions,18 CYP1A½ enzymes may also be involved in the bioactivation of certain carcinogens, such as benzo[a]-pyrene (BaP)19 and 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP).20 Because of these complicated roles, the induction of CYP1A½ has been carefully evaluated in many herb-drug or drug-drug interactions.21 Indeed, Li et al. demonstrated that DHM and methysticin could induce CYP1A1 at mRNA, protein, and function levels in Hepa1c1c7 cells in an AhR-dependent manner.15 Several in vivo studies also reported that a large dose of kava extract led to increased levels of mRNA and protein of hepatic CYP1A1 in rodents.22,23 These results overall suggest that AhR might be one possible upstream target of DHM, which may activate UGTs and result in enhanced detoxification of NNAL that would account for its effect against NNK- and NNAL-induced DNA damage in A/J mice. At the same time, DHM may enhance the metabolic activation of other carcinogens and increase the risk of herb-drug interaction upon activation of AhR and induction of CYP1A½.24

Given DHM’s outstanding efficacy in blocking NNK- and NNAL-induced DNA damage and lung tumorigenesis, there is an urgent need to elucidate its mechanism of action. In this study, we investigated the role of AhR as a potential target of DHM using Ahr+/− and Ahr−/− C57BL/6 mice. We analyzed whether AhR deficiency has any effect on (1) DHM-mediated reduction in the level of O6-mG in target lung tissues, (2) the increased level of NNAL O-glucuronidation in urine, and (3) induction of CYP1A½ in the liver. Our results revealed that AhR status has no influence on any of these DHM-mediated effects, demonstrating that AhR is not the upstream target.

REAGENTS AND METHODS

Caution

NNK and NNAL are human carcinogens. Personnel handling them are expected to take appropriate safety measures.

Chemicals and Reagents

NNK, [13C6]NNK, [CD3]-O6-mG, and [4-CD2,CD3]NNAL-O-Gluc were purchased from Toronto Research Chemicals (Toronto, ON). NNAL and [13C6]NNAL were synthesized from NNK or [13C6]NNK via sodium borohydride reduction.10 (±)-DHM and (±)-DHK were synthesized in house with a slight modification of a reported procedure.25 AIN-G powdered diet was purchased from Harlan Teklad (Madison, WI). Recombinant β-glucuronidase was purchased from Sigma-Aldrich (St. Louis, MO). Ethoxyresorufin was purchased from Sigma-Aldrich. NADPH was purchased from RPI (Mount Prospect, IL). The qPCR primers were purchased from IDT (Coralville, IA), and the sequences are as follows: Gapdh, 5′-AACTTTGGCATTGTGGAAGG-3′ (sense) and 5′-ACA-CATTGGGGGTAGGAACA-3′ (antisense); Ahr, 5′-AGCCGGTGC-AGAAAACAGTAA-3′ (sense) and 5′-AGGCGGTCTAACTCTGT-GTTC-3′ (antisense); Cyplal, 5′-GGTTAACCATGACCGGGA-ACT-3′ (sense) and 5′-TGCCCAAACCAAAGAGAGTGA-3′ (antisense); Cyp1a2, 5′-TGGAGCTGGCTTTGACACAG-3′ (sense) and 5′-CGTTAGGCCATGTCACAAGTAGC-3′ (antisense). All other chemicals or solvents were purchased from either Fisher Scientific (Fairlawn, NJ) or Sigma-Aldrich, unless stated otherwise.

Diet Preparation and Characterization

(±)-DHM- and (±)-DHK-supplemented AIN-G powdered diets were prepared by following our previously reported procedures.8 Briefly, (±)-DHM or (±)-DHK was reconstituted in absolute ethanol (50 mL) and then mixed with the AIN-93 G powdered diet (150 g). Absolute ethanol (50 mL) was mixed with the AIN-93 G powdered diet (150 g) for the control diet. The reconstituted diets were dried under vacuum to remove ethanol and then ground into fine powders. All diets were then mixed well with additional AIN-93 G powdered diet to the desired dose. The abundance of DHM and DHK in the diet was analyzed in triplicate by HPLC and confirmed to be within ±10% of the specified dose.

Animal Studies

The animal studies were approved by the University of Minnesota Institutional Animal Care and Use Committee and conducted following the National Institutes of Health guidelines. Five- to six-week-old C57BL/6 Ahr+/− and Ahr−/− female mice were procured from McGill University and housed in the core animal facilities of the Research Animal Resources of the University of Minnesota.26 Ahr+/− mice are phenotypically indistinguishable from wild-type (Ahr+/+) mice and are often used as controls when examining the physiological, pathophysiological, and toxicological parameters of the AhR, rendering AhR+/− mice as suitable controls for this study.27–29 After being acclimated for 1 week, 18 Ahr+/− and 18 Ahr−/− mice were randomized into six groups each (n = 3). From day 1 to day 7, they were fed with the corresponding diet. The body weights of the mice were measured every 2 or 3 days, and their food intake was monitored twice a week. On day 8, except for mice in the negative control groups, all other mice received a single dose of NNK in saline (100 μL) at 100 mg/kg of body weight via ip injection. Mice in the negative control groups were given saline. On the basis of the results from our previous studies, 4 h after NNK exposure, mice were euthanized with CO2 overdosing.10 Mouse sera, urine, lung, and liver tissues were collected and stored at −80 °C following our established procedures.10 For lung and liver tissues, small portions were stored in the RNA stabilization solution.

Quantification of the O6-mG DNA Adduct in the Lung Tissues

The isolation of DNA from the lung tissues and LC-MS/MS quantification were performed following the standard procedure.7 Briefly, DNA was isolated from half of the whole lung tissue of each individual mouse, following the Puregene DNA isolation protocol (Qiagen Corp.). O6-mG was quantified by liquid chromatography—electrospray ionization/tandem mass spectrometry (LC-ESI-MS/MS) with [CD3]-O6-mG as the internal standard.

Urinary NNAL-O-Gluc and Free NNAL Quantification

Urinary NNAL-O-Gluc and free NNAL were quantified via an established LC—MS/MS method.10 Briefly, urine samples were diluted 105 times with saline. The diluted samples (0.1 mL each) were mixed with [4-CD2,CD3]NNAL-O-Gluc and [13C6]NNAL at final concentrations of 5 and 10 ng/mL, respectively. LC—MS/MS analysis was performed using an Agilent 1100 series capillary high-pressure liquid chromatography system (Agilent Technologies, Palo Alto, CA) interfaced with a TSQ Quantum Discovery Max triple-quadrupole mass spectrometer (Thermo Electron, San Jose, CA). NNAL and NNAL-O-Gluc were analyzed simultaneously on a Phenomenex Luna C18 (150 mm × 0.5 mm, 3 μm) capillary column, under conditions reported previously.10

Mouse Liver Microsome Preparation and CYP1A½ Enzymatic Assay (EROD)

Mouse liver microsomes were prepared following the standard protocol.10 Briefly, liver tissue (~250 mg) was manually homogenized in ice cold microsome buffer [1 mL, 50 mM Tris-HCl, 1.15% KCl, and 1 mM EDTA (pH 7.0)]. The resulting homogenate was centrifuged at 10000 rpm and 4 °C for 30 min using a 70.1 Ti rotor (Beckman Coulter, Brea, CA). The supernatant was collected and further centrifuged at 40000 rpm and 4 °C for 90 min. The pellet was resuspended in ice-cold microsome buffer (100 μL). Upon protein quantification by the BCA method, the microsome was diluted to a total protein concentration of 5 mg/mL, aliquoted, and stored at −80 °C until use.

Aliquoted microsomes were thawed on ice right before the enzymatic assay. 7-Ethoxyresorufin O-deethylation (EROD) enzymatic assays were conducted following the standard protocols in 96-well plates.30 Briefly, the microsome was incubated with 7-ethoxyresorufin at 37 °C for 10 min followed by the addition of NADPH. With a final reaction volume of 100 uL, each well contained 0.2 mg/mL microsomal protein, 1 mM NADPH, and 1 μM 7-ethoxyresorufin in microsome buffer. The fluorescence intensity in each well was continuously monitored by a Tecan microplate reader (GENios Pro) with an excitation wavelength of 535 nm and an emission wavelength of 590 nm (20 measurements in each well over 15 min). During this period of time, the increase in fluorescence intensity was linear, suggesting minimal changes in enzymatic activity and substrate concentration (data not shown). The standard curve used for unit conversion was generated with resorufin purchased from Santa Cruz Biotechnology (Dallas, TX).

Western Blotting Analyses

Liver (~50 mg) and lung (~10 mg) tissue samples were first homogenized using TissueRuptor from Qiagen with T-PER Tissue Protein Extraction Reagent from Thermo Fisher, supplemented with protease inhibitors (Thermo Fisher) and phosphatase inhibitor cocktail (Cell Signaling, Danvers, MA). The protein concentration was determined by the BCA method. Western blotting was conducted with 50 μg of liver tissue lysates or 12.5 μg of lung tissue lysates per well. The primary antibody for AhR was purchased from Enzo Life Sciences (Farmingdale, NY) (catalog no. BML-SA210), at a 1:5000 dilution. The HRP-linked β-actin antibody was obtained from Sigma-Aldrich (catalog no. A3854), at a 1:25000 dilution. HRP-linked anti-rabbit IgG from Cell Signaling was used as secondary antibody for AhR with a dilution factor of 1:3000. β-Actin served as a loading control, and protein bands were visualized by the chemiluminescence method.

Quantitative Reverse Transcription PCR

Liver and lung tissue samples were saved in an RNA stabilization solution. Total RNA was isolated using the RNeasy Mini kit (Qiagen) following the manufacturer’s protocol. cDNA was synthesized from 2 μg of total RNA each using the SuperScript Vilo cDNA Synthesis kit (Invitrogen, Carlsbad, CA). qPCR was performed on a StepOnePlus Real-Time PCR System using SYBR Green PCR Master Mix (Invitrogen, Carlsbad, CA) with the following thermal cycling: 95 °C for 5 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min. Gapdh served as a reference gene.

Statistical Analyses

Data shown here represent three biological repeats (three mice) with their mean ± standard deviation. Statistical significance is denoted by asterisks with the following definition: not significant, p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. If not specified with any asterisks, there was no significant difference between compared groups. Except for the results of qRT-PCR, differences between treated and control groups were analyzed by one-way ANOVA, with Dunnett’s method used for multiple comparisons, at a 95% confidence interval. For qRT-PCR results, levels of mRNA expression between Ahr+/− and Ahr−/− groups were compared with a two-tailed Student’s t-test, at a 95% confidence interval.

RESULTS AND DISCUSSION

Characterization of Ahr Status and Health Monitoring

Before randomization and after euthanasia, all mice were genotyped for Ahr, following an established procedure (data not shown).26 In addition, Ahr mRNA and protein were analyzed via qRT-PCR and Western blotting, respectively, in the lung and liver tissues from representative animals (Figure S1). These results validated the Ahr status of mice used herein. During the study, there were no significant differences in body weight changes or food intake among mice with different treatments (data not shown).

Effect of Ahr Status on Reduction in O6-mG by DHM in Mouse Lung Tissues

O6-mG is likely the most carcinogenic DNA damage relative to other types of NNK-and NNAL-induced DNA damage in A/J mice,9 and it was effectively reduced by DHM in the target lung tissue.8 Therefore, pulmonary O6-mG was analyzed as the representative DNA damage in Ahr+/− and Ahr−/−mice. As shown in Figure 1, NNK treatment resulted in similar levels of O6-mG adduct formation irrespective of the Ahr status and DHM dose-dependently reduced O6-mG levels in both Ahr+/− and Ahr−/− mice. As expected, the extent of reduction in the level of O6-mG by DHK was much smaller, if any, relative to that of DHM in both genotypes. These results unambiguously demonstrated that the reduction in the level of O6-mG by DHM is AhR-independent.

Figure 1.

Figure 1.

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Effect of Ahr status on DHM and DHK on NNK- and NNAL-induced O6-mG in the target lung tissues. Data represent means ± the standard deviation of three mice per treatment group. Statistical comparison was made with the NNK-only treatment group in each strain of mice via one-way ANOVA.

Effect of Ahr Status on DHM-Induced NNAL Glucuronidation

Our earlier work revealed that dietary DHM increased the abundance of urinary NNAL-O-Gluc relative to that of free NNAL in A/J mice and enhanced the NNAL glucuronidating activity in the lung and liver tissues.10 Mechanistically, an increase in the level of UGT-mediated NNAL glucuronidation could lead to enhanced NNAL detoxification and therefore may contribute to the reduction in the level of O6-mG.10,31 Because AhR has been reported to transcriptionally regulate UGTs,14,32 the impact of Ahr status on NNAL glucuronidation was evaluated herein. The urinary ratio of NNAL-Gluc and free NNAL has been used as a convenient parameter to evaluate glucuronidation-mediated NNAL detoxification.33 The higher urinary ratio of NNAL-Gluc to free NNAL would indicate better detoxification, and such a ratio has been used to examine NNK detoxifying capability among smokers.34 Although both NNAL-N-Gluc and NNAL-O-Gluc have been detected in human smokers, only NNAL-O-Gluc has been detected in A/J mice.10 In this study, we therefore directly quantified NNAL-O-Gluc and free NNAL in the mouse urine,10 and the ratio of NNAL-O-Gluc to free NNAL was calculated. As shown in Figure 2, DHM at a dose of 1 mg/g of diet significantly increased the urinary ratio of NNAL-O-Gluc to NNAL while DHK had no effect, consistent with our results in A/J mice,10 supporting the possibility that DHM may exert its chemopreventive effects by increasing the level of NNAL detoxification. In agreement with the results of O6-mG reduction, the detoxification of NNAL by DHM also seemed to be independent of the AhR pathway. Although the extent of the increase by DHM in AhR+/− mice seemed to be larger than that in Ahr−/− mice, the basal level was slightly higher in AhR+/− mice, as well, indicating that further investigation is needed. While DHM was able to reduce the extent of formation of O6-mG at doses of 0.05 and 0.2 mg/g of diet, such treatments did not increase the NNAL-O-Gluc/NNAL ratio (data not shown). A simple explanation could be glucuronidation of NNAL is not the major mechanism responsible for the reduction of the NNK- and NNAL-induced level of O6-mG by DHM treatment. Alternatively, the urinary NNAL-O-Gluc/NNAL ratio may not accurately reflect the target lung tissue because liver is the major metabolizing tissue. Our earlier work showed that dietary DHM treatment only preferentially reduced the level of NNK- and NNAL-induced DNA damage in the target lung tissue but not in the liver tissue.8 It is possible that smaller doses of DHM may be sufficient to enhance NNAL glucuronidation in the target lung tissue while larger dosages of DHM may be needed to enhance NNAL glucuronidation in the liver, which requires further investigation.

Figure 2.

Figure 2.

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Effect of Ahr status on DHM-induced NNAL glucuronidation in urine. The NNAL-O-Gluc/free NNAL ratio was used as a measurement of NNAL glucuronidation. Both DHM and DHK were present at a dose of 1 mg/g of diet. Data represent means ± the standard deviation of three mice per treatment group. Statistical comparison was made with the NNK-only treatment group in each strain of mice via one-way ANOVA.

Effect of Ahr Status on CYP1A1/2 Activity by DHM in Mouse Liver Microsomes

In addition to its chemopreventive potential, kava usage has been linked to hepatotoxicity in humans.35 Here we characterized the impact of dietary DHM on CYP1A1/2 to further explore its safety. We focused on CYP1A1/2 in this study mainly because these two CYP isozymes are dominantly regulated by AhR36 and Li et al. has demonstrated CYP1A1 activation by DHM in an AhR-dependent manner in vitro.15 In addition, CYP1A1 has been reported to be upregulated in vivo with large doses of kava.22 Moreover, as discussed before, CYP1A1/2 may activate other carcinogens to potentially promote carcinogenesis.20,37

Because DHM was given to mice in their diet and its impact on Cyplal and Cyp1a2 mRNA was expected to be temporal, we attempted to quantify CYP1A1 and CYP1A2 proteins in the liver tissues via Western blotting. Because of the lack of well-validated isoform-specific antibodies for CYP1A1 and CYP1A2, Western blotting results were inconclusive (data not shown). In the next step, enzymatic activity of CYP1A1/2 was evaluated through a 7-ethoxyresorufin O-deethylation (EROD) assay (Figure 3).30 In spite of previous in vitro evidence showing CYP1A1 induction by DHM via the AhR pathway,15 DHM’s effect on the liver microsomal CYP1A1/2 activity (EROD) was independent of Ahr status in this study. Such a discrepancy can be due to many reasons, including in vitro and in vivo models, tissue differences, dosage, metabolism, and the temporal and spatial distribution of DHM. In comparison to the NNK control groups, DHM at the largest dose (1 mg/g) significantly increased EROD activity, while the extent of the increase induced by DHK was much smaller and statistically non-significant. With smaller doses of DHM (0.05 or 0.2 mg/g), liver microsomal CYP1A1/2 activities did not show a significant difference when compared to NNK control groups in both Ahr+/− and Ahr−/− mice.

Figure 3.

Figure 3.

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Effect of Ahr status on CYP1A1/2 activity (EROD) in the liver microsome. Data represent means ± the standard deviation of three mice per treatment group. Statistical comparison was made with the NNK-only treatment group in each strain of mice via one-way ANOVA.

It appeared that the basal level of liver microsomal EROD activity was lower in the Ahr−/− mice than in the Ahr+/− mice, although the differences were not statistically significant, suggesting that even in the liver, AhR may contribute to the basal levels of CYP1A1/2.

CONCLUSION

Because the effect of DHM on pulmonary NNK- and NNAL-induced O6-mG, urinary ratio of NNAL-O-Gluc/NNAL, and liver microsomal EROD were similar in both Ahr+/− and Ahr−/− C57BL/6 mice, AhR clearly is not the upstream target of DHM. Because of the low sensitivity of C57BL/6 mice to NNK- and NNAL-induced lung tumorigenesis in comparison to the A/J mice and the lack of Ahr−/− A/J mice, we were unable to evaluate the effect of Ahr status on DHM’s tumor reduction potential. It is clear that O6-mG formation is unlikely the only molecular basis responsible for NNK-induced lung tumorigenesis, particularly in C57BL/6 mice.38 Nevertheless, a strong positive correlation between O6-mG levels and lung tumor multiplicity in A/J mice has been observed.9 It is reasonable to speculate that DHM may block NNK- and NNAL-induced lung tumorigenesis in A/J mice in a manner independent of the AhR pathway, which needs further validaton.

It was also observed that DHM at 0.2 or 0.05 mg/g of diet, while retaining the complete chemopreventive effect, did not cause any induction of CYP1A1/2 activity in the liver microsome. DHM therefore has a decent therapeutic window to effectively block lung tumorigenesis without enhancing CYP1A1/2 activity. In addition, flavokawains A and B, not DHM, have been recently identified by us and others as the compounds potentially responsible for kava’s hepatotoxic risk.39 Nevertheless, comprehensive studies are needed to systematically assess the physiological and pathological impacts of DHM on CYP1A1/2 and other drug-metabolizing enzymes.

Supplementary Material

supplement

ACKNOWLEDGMENTS

We thank the Masonic Cancer Center’s Analytical Biochemistry Core Facility for their assistance with LC-MS analyses.

Funding

This work was funded by Grant R01 CA 193278 (C.X.) from the National Cancer Institute of the National Institutes of Health, in part by National Cancer Institute Cancer Center Support Grant CA 077598, and by Canadian Institutes of Health Research (CIHR) Grant 123467 (C.J.B.). C.J.B. was supported by a salary award from the Fonds de recherche du Quebec-Sante (FRQ-S).

ABBREVIATIONS

DHM

dihydromethysticin

NNK

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

AhR

aryl hydrocarbon receptor

NNAL

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol

O6-mG

O6-methylguanine

DHK

dihydrokavain

NNAL-O-Gluc

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol glucuronide

UGT

UDP-glucuronosyltransferase

PXR

pregnane X receptor

CAR

constitutive androstane receptor

XRE

xenobiotic response element

BaP

benzo[a]pyrene

PhIP

2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

NADPH

reduced form of nicotinamide adenine dinucleotide phosphate

EROD

7-ethoxyresorufin O-deethylation

qRT-PCR

quantitative reverse transcription polymerase chain reaction

ANOVA

analysis of variance

Footnotes

The authors declare no competing financial interest.

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemres-tox.6b00203.

Confirmation of the Ahr status of lung and liver tissues from representative mice (PDF)

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