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Published in final edited form as: J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Feb 7;1141:122023. doi: 10.1016/j.jchromb.2020.122023

Quantitation of Phenanthrene Dihydrodiols in the Urine of Smokers and Non-smokers by Gas Chromatography-Negative Ion Chemical Ionization-Tandem Mass Spectrometry

Kai Luo 1, J Bradley Hochalter 1, Steven G Carmella 1, Stephen S Hecht 1,*
PMCID: PMC7377154  NIHMSID: NIHMS1606850  PMID: 32109746

Abstract

Polycyclic aromatic hydrocarbons (PAH) are well-established environmental carcinogens likely to be causative agents for some human cancers. Bay-region diol epoxides are ultimate carcinogenic metabolites of multiple PAH. Dihydrodiols are the important intermediate products of this pathway and can be further oxidized to form diol epoxides. We quantified two dihydrodiol metabolites of phenanthrene (Phe), the simplest PAH with a bay-region, in the 6 h urine of smokers (N=25) and non-smokers (N=25) using a newly developed and validated analytical method. After hydrolysis by ß-glucuronidase and sulfatase, and solid phase extraction, the sample was silylated and analyzed by gas chromatography-negative ion chemical ionization-tandem mass spectrometry (GC-NICI-MS/MS). Levels (nmol/6 h urine) of Phe-1,2-dihydrodiol (Phe-1,2-D) and Phe-3,4-dihydrodiol (Phe-3,4-D) were 2.04±1.52 and 0.51±0.35, respectively, in smokers, significantly higher than those in non-smokers (1.35±1.11 of Phe-1,2-D, p<0.05; 0.27±0.25 of Phe-3,4-D, p<0.005). Cigarette smoking also influenced the regioselective metabolism of Phe, presenting as a significant difference in the urinary distribution pattern of Phe-1,2-D and Phe-3,4-D between smokers and non-smokers: the ratio Phe-3,4-D: Phe-1,2-D increased from 0.20 in non-smokers to 0.28 in smokers (p<0.01), which can be explained by the induction of the phenanthrene metabolizing enzymes CYP1A2 and CYP1B1 by cigarette smoke. The method described here is the first example of facile quantitation of an intact human dihydrodiol metabolite of any PAH with three or more aromatic rings and will be applicable in clinical and molecular epidemiology studies of PAH metabolism and cancer susceptibility.

Keywords: Phenanthrene, Dihydrodiol, Bioactivation, Cigarette Smoke

1. Introduction

Polycyclic aromatic hydrocarbons (PAH) are important environmental carcinogens believed to be among the causes of cancer induced by environmental pollutants and cigarette smoke. One member of this class of compounds, benzo[a]pyrene (BaP), is considered “carcinogenic to humans” by the International Agency for Research on Cancer [1]. Phenanthrene (Phe) is a main component of environmental PAH mixtures and occurs in various sources such as air and particulate matter [2–4], cigarette smoke [5, 6], soil and house dust [7–9], water and sediment [10, 11], diesel emissions [12, 13], as well as foodstuffs [14–16]. Phe metabolites have notable strengths as PAH exposure biomarkers. Although Phe is a non-carcinogenic PAH, it has structural features such as a bay region and an enzymology profile similar to that of carcinogenic PAH including BaP and may thus mirror the bioactivation and detoxification pathways of BaP. Phe metabolites are excreted in relatively large quantities in human urine and are easier to quantify than those of BaP. For example, the concentrations of Phe-tetraols are about 10,000 times higher than those of BaP-tetraol in human urine [17]. Thus Phe is of potentially high diagnostic value in the biomonitoring of occupational and epidemiological PAH exposures by providing information on both exposure and metabolic activation.

The metabolism of Phe by the PAH diol epoxide metabolic activation pathway (Scheme 1) yields dihydrodiols, diol epoxides, and tetraols, whereas phenanthrols signify detoxification [18–21]. Phe can be metabolized at three different molecular regions (1,2-; 3,4- and 9,10- positions) depending on various cytochrome P450 isoforms involved in the oxidation process [22]. Of particular interest is (1R,2R)-dihydroxy-1,2-dihydrophenanthrene (Phe-1,2-D), which is formed along with two other isomeric dihydrodiols (3R,4R)-dihydroxy-3,4-dihydrophenanthrene (Phe,3,4-D) and (9R,10R)-dihydroxy-9,10-dihydrophenanthrene (Phe-9,10-D) by metabolic steps competing with phenol formation. Phe-9,10-D is not representative of a metabolic activation pathway. Phe-1,2-D can be further oxidized to the bay-region (1R,2S)-dihydroxy-(3S,4R)-epoxy-1,2,3,4-tetrahydrophenanthrene (Phe-1,2-D-3,4-E), structurally analogous to the highly carcinogenic bay region (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) of BaP, while Phe-3,4-D can only form the reverse diol epoxide (3S,4R)-dihydroxy-(1R,2S)-epoxy-1,2,3,4-tetrahydrophenanthrene (Phe-3,4-D-1,2-E) (Scheme 1), analogous to less carcinogenic diol epoxides [23]. Phe-1,2-D-3,4-E undergoes hydrolysis to produce (1R,2S,3R,4S)-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (Phe-(1R,2S,3R,4S)-tetraol), which was significantly correlated with Phe-(1S,2R,3S,4R)-tetraol and BaP-tetraol (hydrolysis product of BPDE) in human urine (r = 0.72, p < 0.0001) [17]. Thus we propose that Phe-1,2-D, the precursor of the “ultimate reactive metabolite” Phe-1,2-D-3,4-E, can be used as a proxy for the carcinogenic metabolism pathway of BaP and other PAH metabolically activated to bay region diol epoxides.

Scheme 1.

Scheme 1.

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Metabolic activation of benzo[a]pyrene and phenanthrene via the diol epoxide pathway.

Most previous studies quantified Phe-1,2-D in human urine using an indirect method involving conversion of the dihydrodiol to phenols after 2–16 h acid treatment, then calculating the dihydrodiol concentration as the difference between the total phenols analyzed before and after acid treatment [18, 21, 24]. One direct method using methyl iodide as a derivatization reagent was reported, but this required a large urine volume (10 mL), tedious analysis time (16 h for derivatization) and relatively high consumption of organic solvents (110 mL per sample) during sample preparation, and consequently has not been widely applied [25].

The aim of this study was to develop a rapid and sensitive analytical method for the quantitation of Phe-1,2-D and Phe-3,4-D in human urine to investigate: 1) the concentration of these compounds in the urine of smokers and non-smokers; 2) their potential use as biomarkers of exposure to PAH; and 3) the effect of cigarette smoking on regioselectivity in the metabolism of Phe.

2. Materials and Methods

2.1. Chemicals, Enzymes and Sample Preparation Supplies

Phe (purity ≥ 99.5%) and [1,2,3,4,4a,9a-13C6]Phe (purity ~95%) were obtained from Millipore Sigma (St. Louis, MO, USA) and Toronto Research Chemicals (North York, ON, Canada) respectively. Standards of Phe-1,2-D and [13C6]Phe-1,2-D were prepared by incubation of Phe and [13C6]Phe with cytochrome P4501A1 and cofactors, followed by HPLC purification, as described [26]. Purity (>95%) and concentration of Phe-1,2-D were determined by quantitative nuclear magnetic resonance (QNMR) (1H NMR of Phe-1,2-D (500 MHz, DMSO-d6) δ 8.17 (d, J = 8.6 Hz, 1H), 7.84 (d, J = 7.9 Hz, 1H), 7.79 (d, J = 8.3 Hz, 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.45 (d, J = 7.5 Hz, 1H), 7.20 (d, J = 10.2 Hz, 1H), 6.09 (d, J = 10.2 Hz, 1H), 5.53 (s, 1H), 5.19 (s, 1H), 4.65 (s, 1H), 4.28 (s, 1H).) and HPLC-UV. Standard Phe-3,4-D was provided by H. Yagi[27]. RC-2 [bis-Trimethylsilyltrifluoroacetamide (BSTFA) + 1% trimethylchlorosilane (TMCS)] was purchased from Regis Technologies (Morton Grove, IL, USA). β-Glucuronidase and arylsulfatase (from Helix pomatia) were obtained from Roche Diagnostics Corp. (Indianapolis, IN, USA). Strata SDBL styrene-divinylbenzene SPE cartridges (50 mg/1 mL, #8B-S014-DAK) were obtained from Phenomenex (Torrance, CA, USA). Oasis MAX (Mixed-mode Anion eXchange sorbent) SPE cartridges (60 mg/1 mL/# 186000378) were from Waters (Milford, MA, USA). All solvents used in this study including methanol and acetonitrile were HPLC grade and from Fisher Scientific (Fair Lawn, NJ, USA). Ultrapure H2O from Millipore Milli-Q Advantage A10 Water Purification System was used throughout the study.

2.2. Urine Samples

Urine samples from 50 volunteers (25 smokers: 15 males / 10 females; 25 non-smokers: 13 males / 12 females) were obtained from ongoing studies at the Tobacco Research Programs, University of Minnesota, approved by the Institutional Review Board. The volume of their 6 h urine was measured and aliquots were stored at −20 ˚C until analysis. Smoking status of all the subjects were determined by measurement of urinary cotinine [28]. Those who had cotinine ≥ 30 ng/mL were classified as smokers. Our previous results have shown that 6-h urinary excretion of racemic [D10]Phe-1,2,3,4-tetraol, the major end product of the Phe diol epoxide pathway, was sufficient to reflect the extent of metabolism of [D10]Phe to this metabolite in smokers who received an oral or inhalation dose of [D10]Phe, because it was highly correlated with 48-h excretion [29].

2.3. Sample Preparation

The analytical method is summarized in Scheme 2. An aliquot of 1.0 mL urine was added to a 2.0 mL glass vial containing 1.0 mL of 0.5 M NaOAc buffer (pH 5), β-glucuronidase (1000 units) and arylsulfatase (8000 units), and 5 pmol of [13C6]Phe-1,2-D as internal standard. The mixture was incubated overnight with shaking at 37 °C. After incubation, the sample was purified by loading onto a Strata SDBL cartridge preconditioned with 1.0 mL of CH3OH and 1.0 mL of H2O. The cartridge was then washed in order with 1.0 mL of 0.28% NH4OH (wt%) /10% CH3OH (vol%) in H2O, 1.0 mL of H2O, 1.0 mL of freshly prepared 1% formic acid (vol%)/10% CH3OH (vol%) in H2O and 1.0 mL of H2O, and the analyte was eluted with 0.8 mL×2 of CH3OH. The Strata SDBL we used is from Phenomenex. The packing material is styrene-divinylbenzene copolymer, which is used in organic trace analysis for solid phase extraction preconcentration. It has been successfully used in previous studies for the assay of phenanthrene tetraol. It can give a good recovery of the dihydrodiols and remove most of the urine matrix with a basic and acid wash. The solvents of the analyte fraction from the Strata SDBL were removed using a Speedvac concentrator (Thermo Scientific, San Jose, CA). The residue was redissolved in 1.0 mL of 10% CH3OH (vol%) in H2O with sonication and loaded onto an Oasis MAX cartridge preconditioned with 1.0 mL of CH3OH and 1.0 mL of 0.84% NH4OH (wt%) in H2O. The cartridge was washed with 1.0 mL of 0.84% NH4OH (wt%) in H2O and the dihydrodiols were eluted with 1mL CH3OH. The solvent was removed on a Speedvac and the residue was dissolved in 100 μL × 2 times of CH3OH with sonication, transferred to an insert vial (Chrom Tech, 300 μL insert/vial, CP-0952–03SIL), concentrated to dryness, and dissolved in 10 μL of BSTFA plus 1% TMCS with sonication and vortexing. The samples were heated at 60 ˚C for 60 min to complete the derivatization, and 3 μL was analyzed by GC-NICI-MS/MS.

Scheme 2.

Scheme 2.

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Outline of the method for the analysis of Phe-1,2-D and Phe-3,4-D, and fragmentation in the daughter ion spectrum of di-trimethylsilyl-Phe-1,2-D; TMS = (CH3)3Si.

2.4. GC-NICI-MS/MS Analysis

Gas chromatography-negative ion chemical ionization- tandem mass spectrometry (GC-NICI-MS/MS) was carried out with a TSQ Quantum instrument (Thermo Scientific, San Jose, CA). Phe-1,2-D and [13C6]Phe-1,2-D were detected as their trimethylsilyl derivatives. The GC was equipped with a 0.25 mm i.d. × 0.15 μm film thickness × 30 m DB17-MS column (Agilent Technologies, Palo Alto, CA). The oven temperature program was 80 °C for 1 min, then 80–200 °C at 35 °C /min, then 200–215 °C at 3 °C /min, then 215–320 °C at 35 °C /min, and then held for 2 min. The carrier gas was He at a constant flow rate of 1.0 ml/min. The programmed temperature vaporizer (PTV) injection port (Topaz 2.0 mm ID Baffled Inlet Liner for Thermo TRACE GCs equipped with PTV inlets, 2 mm × 2.75 × 120, Cat.# 23438, operated in the splitless mode) was set up as follows: 60 °C for 0.5 min, then 60–90 °C at 0.5 °C /sec, hold for 1 min, then 90–250 °C at 3 °C /sec, and then held for 15.5 min. The use of a PTV injector starting at a low temperature was found to be critical in preventing the decomposition of the silylated Phe-1,2-D and Phe-3,4-D. The MS transfer line was kept at 320 °C. The NICI-MS/MS conditions were as follows: CI gas, argon at 2.0 ml/min; source temperature, 250 °C; and emission current 70 mA. Selected reaction monitoring (SRM) with a collision energy of 30 eV, electron energy of 50 eV, scan width of m/z 0.02, and dwell-time 0.03 sec was used to detect Phe-1,2-D and [13C6]Phe-1,2-D at m/z 193 → m/z 165 and m/z 199 → m/z 171, respectively (Scheme 2).

2.5. Statistical Analysis

The t-Test: Paired Two Sample for Means (Excel 16.27) was used to analyze the difference of analytes between smokers and non-smokers, and p<0.05 was considered statistically significant.

3. Results

The method used in this study for the analysis of Phe-1,2-D and Phe-3,4-D was different from those reported previously, because the silylated analyte was quantified directly using a smaller amount of urine and a shorter analysis time. A typical chromatogram obtained upon analysis of a 6 h urine sample from a smoker is illustrated in Figure 1, showing clear quantifiable peaks for Phe-1,2-D, [13C6]Phe-1,2-D, and Phe-3,4-D as their TMS derivatives.

Figure 1.

Figure 1.

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Typical GC-NICI-MS/MS chromatograms obtained upon analysis of a 6 h urine sample from a smoker. A, Phe-1,2-D (3.33 nM) and Phe-3,4-D (1.13 nM), as their di-TMS derivatives, observed in the urine sample; B, internal standard [13C6]Phe-1,2-D-di-TMS.

The average recovery of [13C6]Phe-1,2-D spiked into the samples was 56.3±6.81% (N=3). The method limit of detection (LOD) calculated from the peak signal-to-noise ratio of real samples with a minimal value of 3 was 0.29±0.036 nM for Phe-1,2-D and 0.084±0.0067 nM for Phe-3,4-D. The analyte showed excellent linearity (r2 > 0.99) in the range of 1–400 nM in the instrumental calibration curve.

The accuracy was determined by adding various amounts of Phe-1,2-D and Phe-3,4-D to the urine of a nonsmoker. As shown in Table 1, there was excellent agreement between the added and quantified amounts, the regression equation was y=0.80x+0.85 (y: determined level (nM), x: spiked level (nM) (R2=0.9992) for Phe-1,2-D and y=1.04x+0.10 (R2=0.9998) for Phe-3,4-D, and the y intercept, corresponding to 0.85 nM and 0.10 nM for Phe-1,2-D and Phe-3,4-D, respectively, agreed with the level determined in the urine of this nonsmoker, 0.77±0.06 nM and 0.09±0.01 nM. The intra-day precision was determined by analyzing six aliquots of a nonsmoker’s urine. The results were 0.84±0.10 nM for Phe-1,2-D (CV=12.3%) and 0.10±0.01 nM for Phe-3,4-D (CV=8.7%). The inter-day precision was determined by analyzing 3 aliquots of a spiked (5.00 nM of Phe-1,2-D and 0.50 nM of Phe-3,4-D) nonsmoker’s urine on 3 different days. The results were 4.76±0.11 nM, 4.50±0.41 nM and 4.63±0.24 nM for Phe-1,2-D (CV=2.8%), and 0.60±0.02 nM, 0.57±0.03 nM and 0.55±0.03 nM for Phe-3,4-D (CV=4.2%).

Table 1.

Precision and accuracy a, levels (nmol/6 h urine) and ratios of Phe-1,2-D and Phe-3,4-D in human urine.
Spiked Concentration (nM) Determined Concentration (nM) Precision (CV, %) Accuracy (%) b Smokers (N=25)
 Non-smokers (N=25)
mean±SD median (interquartile) mean±SD median (interquartile)
Phe-1,2-D 0.00 0.766±0.063 8.16 2.04±1.52 1.69 (0.95∼2.56) 1.35±1.11c 0.93 (0.58∼1.85)
0.200 0.920±0.071 7.72 77.1
1.00 1.75±0.239 13.6 98.8
5.00 4.76±0.105 2.21 79.9
10.0 9.19±0.159 1.73 84.2
20.0 16.8±1.04 6.24 79.9
Phe-3,4-D 0.00 0.090±0.008 9.26 0.51±0.35 0.45 (0.28∼0.64) 0.27±0.25d 0.21 (0.11∼0.29)
0.0500 0.132±0.014 10.4 84.0
0.100 0.192±0.015 7.89 101
0.500 0.600±0.022 3.63 102
1.00 1.19±0.054 4.56 110
5.00 5.28±0.050 0.95 104
Ratio=Phe–3,4–DPhe–1,2–D 0.28±0.11 0.26 (0.20∼0.34) 0.20±0.08e 0.18 (0.14∼0.24)
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a

Phe-1,2-D and Phe-3,4-D were added to 1 mL aliquots of a nonsmoker’s urine (N=3 for each concentration point).

b

The accuracy was reported as recovery (%) = (determined concentration − basal concentration) / spiked concentration × 100 (%).

c

p<0.05, comparing non-smokers and smokers

d

p<0.005, comparing non-smokers and smokers

e

p<0.01 comparing non-smokers and smokers.

The concentrations of Phe-1,2-D and Phe-3,4-D were 1.87±1.27 nM (0.43–5.69 nM) and 0.36±0.26 nM (0.08–0.90 nM) in nonsmokers, and 4.05±3.70 nM (0.54–17.65 nM) and 1.03±0.92 nM (0.17–4.28 nM) in smokers. Levels of Phe-1,2-D and Phe-3,4-D in the 6 h urine of 50 subjects (25 smokers and 25 non-smokers) are summarized in Table 1. The overall mean and median amounts of Phe-1,2-D were 2.04±1.52 and 1.69 nmol/ 6 h urine in smokers, significantly higher than those of non-smokers (1.35±1.11 and 0.93 nmol/ 6 h urine, p<0.05). The mean and median amounts of Phe-3,4-D were also significantly higher in smokers (0.51±0.35 and 0.45 nmol/ 6 h urine) than in non-smokers (0.27±0.25 and 0.21 nmol/ 6 h urine) to (p<0.005). To characterize the regioselectivity of the metabolism of Phe by the diol epoxide pathway, the concentration ratio of [Phe-3,4-D/Phe-1,2-D] was calculated, and found to be significantly higher in smokers (0.28±0.11) than in non-smokers (0.20±0.08, p<0.01).

4. Discussion

The simple, accurate, and reproducible method developed in this study allowed sensitive and rapid analysis of Phe dihydrodiols using smaller volumes of urine compared with previous studies, and has the potential to be applied in large-scale studies. This is critical for clinical and molecular epidemiology studies using Phe metabolite analysis to predict cancer risk.

Several metabolites including 1-hydroxypyrene (1-OHP), Phe phenols (OHPhe) and Phe tetraols have been used as biomarkers to assess human exposure to PAH via cigarette smoking. Levels of urinary 1-OHP were about twice (1.5–2.7) as great in cigarette smokers as in non-smokers [30–32]. Levels of 1-, 2-, 3- and 4-OHPhe in smokers were 1.7–2.9 times higher in smokers than nonsmokers [32]. And smokers had a significantly higher PheT level (~ 2.6 fold, p=0.0073) than nonsmokers [33]. The total urinary concentrations of Phe-1,2-D and Phe-3,4-D were 1.6-fold higher in smokers than non-smokers in this study (p<0.05), generally consistent with the other biomarkers mentioned above.

Although the number of subjects was relatively small, we observed that males had higher levels of Phe-1,2-D and Phe-3,4-D than females (Table 2), particularly among smokers. Male smokers had 2.6- and 1.6-times higher levels of Phe-1,2-D and Phe-3,4-D than females, while among non-smokers, the levels of Phe-1,2-D and Phe-3,4-D in males were only 1.1- and 1.04-times higher than those of females. Further research with larger sample sizes is needed to investigate potential gender differences in PAH metabolism, health risks, and related mechanisms. The U.S. CDC’s National Health and Nutrition Examination Survey (NHANES) (2001–2014) investigated 1-, 2-, 3- and 4-OHPhe, the main metabolites of the Phe detoxification pathway, in males and females, and found that males had higher levels of all the analytes (1.1–1.5 times) than females [34].

Table 2.

Levels of Phe-1,2-D and Phe-3,4-D in males and females (nmol/6 h urine).

Smokers (N=25) Non-smokers (N=25)
Males (N=15) Females (N=10) Males (N=13) Females (N=12)
mean±SD mean±SD mean±SD mean±SD
Phe-1,2-D 2.71±1.61 1.05±0.53 1.42±1.00 1.28±1.25
Phe-3,4-D 0.60±0.38 0.37±0.26 0.27±0.19 0.26±0.31
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Oxidation of Phe can occur at three different molecular regions (1,2-; 3,4- and 9,10- positions) mainly catalyzed by different cytochrome P450 isoforms. The ratios of Phe-3,4-oxidation to 1,2-oxidation were only 0.07 and 0.17 for human CYP1A1 in different studies, but 0.91 and 0.77 for human CYP1A2- and 0.56 for human CYP1B1, indicating that 3,4-oxidation is more favored in the case of CYP1A2 and CYP1B1 than with CYP1A1 [35, 36]. Activities and polymorphisms of human CYP1A2 and CYP1B1 have been confirmed in studies of caffeine and 17β-estradiol (E2) at their different positions, therefore affecting their metabolite ratios and individual risk of toxicity and disease caused by these compounds [37–42]. Cigarette smoke is a potent inducer of CYP1A2 and CYP1B1 enzyme activity in both animals and humans [36, 43–46]. The ratio [3-OHPhe+4-OHPhe] / [1-OHPhe+2-OHPhe] increased from 0.39 to 0.57 with cigarette consumption in NHANES (2011–2012) with a large sample size [32]. In the present study, the ratio of Phe-3,4-D to Phe-1,2-D significantly increased from 0.20 in non-smokers to 0.28 in smokers (p<0.05), consistent with the observation that cigarette smoke can promote 3,4-oxidation of Phe in humans via a CYP-mediated pathway.

The results of this study showed that mean urinary Phe-1,2-D was 4.01 and 5.08 times higher than Phe-3,4-D in smokers and non-smokers, respectively. Previous studies found contrasting results for their downstream products: urinary levels of Phe-(1S,2R,3S,4R)-tetraol (the tetraol enantiomer resulting mainly from the reverse diol epoxide Phe-3,4-D-1,2-E that would be formed from Phe-3,4-D, Figure 1) were 17.7 and 6.77 times higher than those of Phe-(1R,2S,3R,4S)-tetraol (the tetraol enantiomer mainly resulting from the bay-region diol epoxide Phe-1,2-D-3,4-E that would be formed from Phe-1,2-D) in smokers (N=30) and creosote workers (N=26) [17]. Incubations of human hepatocytes with Phe metabolites also demonstrated that conversion of Phe-3,4-D to tetraols was 2.45–29.5 times more than that of Phe-1,2-D [47] which can explain the predominance of Phe-(1S,2R,3S,4R)-tetraol in spite of the higher concentration of Phe-1,2-D than Phe-3,4-D.

In summary, we have developed a straightforward GC-NICI-MS/MS method for the quantitation of Phe-1,2-D and Phe-3,4-D, as their trimethylsilyl ethers, in human urine. This method has the potential to be applied in clinical and molecular epidemiology studies of PAH metabolism. While Phe is non-carcinogenic, these metabolites are important because they represent the first step in the diol epoxide and reverse diol epoxide metabolic activation pathways of carcinogenic PAH. The results demonstrate that urinary levels of Phe-1,2-D, the metabolite on the bay region diol epoxide pathway of Phe metabolism, are higher in smokers than non-smokers, indicating its potential use as a biomarker of both exposure to, and metabolic activation of PAH. This is the first example of facile quantitation of an intact human dihydrodiol metabolite of any PAH with three or more aromatic rings.

Acknowledgements

This study was supported by grants CA-138338 and CA-203851 from the National Cancer Institute and the FDA Center for Tobacco Products. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the Food and Drug Administration. We thank Bob Carlson for his editorial assistance. Mass spectrometry was carried out in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center, supported in part by Cancer Center Support Grant CA-77598.

Footnotes

Final version of this manuscript is available online at https://www.sciencedirect.com/science/article/pii/S1570023219314710

References

  • [1].International Agency for Research on Cancer, Some Non-Heterocyclic Polycyclic Aromatic Hydrocarbons and Some Related Exposures, IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, v. 92, IARC, Lyon, France, 2010. [PMC free article] [PubMed] [Google Scholar]
  • [2].Pratt GC, Herbrandson C, Krause MJ, Schmitt C, Lippert CJ, McMahon CR, Ellickson KM, Measurements of gas and particle polycyclic aromatic hydrocarbons (PAHs) in air at urban, rural and near-roadway sites, Atmos Environ, 179 (2018) 268–278. [Google Scholar]
  • [3].Naumova YY, Eisenreich SJ, Turpin BJ, Weisel CP, Morandi MT, Colome SD, Totten LA, Stock TH, Winer AM, Alimokhtari S, Kwon J, Shendell D, Jones J, Maberti S, Wall SJ, Polycyclic aromatic hydrocarbons in the indoor and outdoor air of three cities in the US, Environ Sci Technol, 36 (2002) 2552–2559. [DOI] [PubMed] [Google Scholar]
  • [4].Guo H, Lee SC, Ho KF, Wang XM, Zou SC, Particle-associated polycyclic aromatic hydrocarbons in urban air of Hong Kong, Atmos Environ, 37 (2003) 5307–5317. [Google Scholar]
  • [5].Vu AT, Taylor KM, Holman MR, Ding YS, Hearn B, Watson CH, Polycyclic aromatic hydrocarbons in the mainstream smoke of popular U.S. cigarettes, Chem Res Toxicol, 28 (2015) 1616–1626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [6].Yershova K, Yuan JM, Wang RW, Valentin L, Watson C, Gao YT, Hecht SS, Stepanov I, Tobacco-specific N-nitrosamines and polycyclic aromatic hydrocarbons in cigarettes smoked by the participants of the Shanghai Cohort Study, Int J Cancer, 139 (2016) 1261–1269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [7].Yu HY, Li TJ, Liu Y, Ma LM, Spatial distribution of polycyclic aromatic hydrocarbon contamination in urban soil of China, Chemosphere, 230 (2019) 498–509. [DOI] [PubMed] [Google Scholar]
  • [8].Yadav IC, Devi NL, Li J, Zhang G, Polycyclic aromatic hydrocarbons in house dust and surface soil in major urban regions of Nepal: Implication on source apportionment and toxicological effect, Sci Total Environ, 616 (2018) 223–235. [DOI] [PubMed] [Google Scholar]
  • [9].Wang CH, Zhou SL, Song J, Wu SH, Human health risks of polycyclic aromatic hydrocarbons in the urban soils of Nanjing, China, Sci Total Environ, 612 (2018) 750–757. [DOI] [PubMed] [Google Scholar]
  • [10].Xiang N, Jiang CX, Yang TH, Li P, Wang HH, Xie YL, Li SN, Zhou HL, Diao XP, Occurrence and distribution of polycyclic aromatic hydrocarbons (PAHs) in seawater, sediments and corals from Hainan Island, China, Ecotox Environ Safe, 152 (2018) 8–15. [DOI] [PubMed] [Google Scholar]
  • [11].Manoli E, Samara C, Polycyclic aromatic hydrocarbons in natural waters: sources, occurrence and analysis, Trac-Trend Anal Chem, 18 (1999) 417–428. [Google Scholar]
  • [12].Zheng X, Wu Y, Zhang SJ, Hu JN, Zhang KM, Li ZH, He LQ, Hao JM, Characterizing particulate polycyclic aromatic hydrocarbon emissions from diesel vehicles using a portable emissions measurement system, Sci Rep, 7 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [13].Zielinska B, Sagebiel J, Arnott WP, Rogers CF, Kelly KE, Wagner DA, Lighty JS, Sarofim AF, Palmer G, Phase and size distribution of polycyclic aromatic hydrocarbons in diesel and gasoline vehicle emissions, Environ Sci Technol, 38 (2004) 2557–2567. [DOI] [PubMed] [Google Scholar]
  • [14].Jiang DF, Wang GL, Li LL, Wang XL, Li W, Li X, Shao LJ, Li FH, Occurrence, dietary exposure, and health risk estimation of polycyclic aromatic hydrocarbons in grilled and fried meats in Shandong of China, Food Sci Nutr, 6 (2018) 2431–2439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Masuda M, Wang Q, Tokumura M, Miyake Y, Amagai T, Simultaneous determination of polycyclic aromatic hydrocarbons and their chlorinated derivatives in grilled foods, Ecotox Environ Safe, 178 (2019) 188–194. [DOI] [PubMed] [Google Scholar]
  • [16].Kipopoulou AM, Manoli E, Samara C, Bioconcentration of polycyclic aromatic hydrocarbons in vegetables grown in an industrial area, Environ Pollut, 106 (1999) 369–380. [DOI] [PubMed] [Google Scholar]
  • [17].Hochalter JB, Zhong Y, Han S, Carmella SG, Hecht SS, Quantitation of a minor enantiomer of phenanthrene tetraol in human urine: correlations with levels of overall phenanthrene tetraol, benzo[a]pyrene tetraol, and 1-hydroxypyrene, Chem Res Toxicol, 24 (2011) 262–268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Jacob J, Grimmer G, Dettbarn G, Profile of urinary phenanthrene metabolites in smokers and non-smokers, Biomarkers, 4 (1999) 319–327. [DOI] [PubMed] [Google Scholar]
  • [19].Asahi M, Kawai M, Toyama T, Kumagai Y, Chuesaard T, Tang N, Kameda T, Hayakawa K, Toriba A, Identification and quantification of in vivo metabolites of 9,10-phenanthrenequinone in human urine associated with producing reactive oxygen species, Chem Res Toxicol, 27 (2014) 76–85. [DOI] [PubMed] [Google Scholar]
  • [20].Hecht SS, Chen ML, Yagi H, Jerina DM, Carmella SG, r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene in human urine: A potential biomarker for assessing polycyclic aromatic hydrocarbon metabolic activation, Cancer Epidemiol Biomarkers Prev, 12 (2003) 1501–1508. [PubMed] [Google Scholar]
  • [21].Grimmer G, Dettbarn G, Jacob J, Biomonitoring of polycyclic aromatic-hydrocarbons in highly exposed coke plant workers by measurement of urinary phenanthrene and pyrene metabolites (phenols and dihydrodiols), Int Arch Occup Environ Health, 65 (1993) 189–199. [DOI] [PubMed] [Google Scholar]
  • [22].Jacob J, Raab G, Soballa V, Schmalix WA, Grimmer G, Greim H, Doehmer J, Seidel A, Cytochrome P450-mediated activation of phenanthrene in genetically engineered V79 Chinese hamster cells, Environ Toxicol Phar, 1 (1996) 1–11. [DOI] [PubMed] [Google Scholar]
  • [23].Hecht SS, Carmella SG, Villalta PW, Hochalter JB, Analysis of phenanthrene and benzo[a]pyrene tetraol enantiomers in human urine: relevance to the bay region diol epoxide hypothesis of benzo[a]pyrene carcinogenesis and to biomarker studies, Chem Res Toxicol, 23 (2010) 900–908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [24].Seidel A, Spickenheuer A, Straif K, Rihs HP, Marczynski B, Scherenberg M, Dettbarn G, Angerer J, Wilhelm M, Bruning T, Jacob J, Pesch B, New biomarkers of occupational exposure to polycyclic aromatic hydrocarbons, J Toxicol Environ Health A, 71 (2008) 734–745. [DOI] [PubMed] [Google Scholar]
  • [25].Polanska K, Hanke W, Dettbarn G, Sobala W, Gromadzinska J, Magnus P, Seidel A, The determination of polycyclic aromatic hydrocarbons in the urine of non-smoking Polish pregnant women, Sci Total Environ, 487 (2014) 102–109. [DOI] [PubMed] [Google Scholar]
  • [26].Nordqvist M, Thakker DR, Vyas KP, Yagi H, Levin W, Ryan DE, Thomas PE, Conney AH, Jerina DM, Metabolism of chrysene and phenanthrene to bay-region diol epoxides by rat-liver enzymes, Mol Pharmacol, 19 (1981) 168–178. [PubMed] [Google Scholar]
  • [27].Jerina DM, Selander H, Yagi H, Wells MC, Davey JF, Mahadevan V, Gibson DT, Dihydrodiols from anthracene and phenanthrene, J Am Chem Soc, 98 (1976) 5988–5996. [DOI] [PubMed] [Google Scholar]
  • [28].Murphy SE, Park SS, Thompson EF, Wilkens LR, Patel Y, Stram DO, Le Marchand L, Nicotine N-glucuronidation relative to N-oxidation and C-oxidation and UGT2B10 genotype in five ethnic/racial groups, Carcinogenesis, 35 (2014) 2526–2533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [29].Zhong Y, Wang J, Carmella SG, Hochalter JB, Rauch D, Oliver A, Jensen J, Hatsukami DK, Upadhyaya P, Zimmerman C, Hecht SS, Metabolism of [D-10]phenanthrene to tetraols in smokers for potential lung cancer susceptibility assessment: Comparison of oral and inhalation routes of administration, J Pharmacol Exp Ther, 338 (2011) 353–361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [30].Hecht SS, Carmella SG, Kotandeniya D, Pillsbury ME, Chen ML, Ransom BWS, Vogel RI, Thompson E, Murphy SE, Hatsukami DK, Evaluation of toxicant and carcinogen metabolites in the urine of e-cigarette users versus cigarette smokers, Nicotine Tob Res, 17 (2015) 704–709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [31].Etemadi A, Poustchi H, Chang CM, Blount BC, Calafat AM, Wang LQ, De Jesus VR, Pourshams A, Shakeri R, Shiels MS, Inoue-Choi M, Ambrose BK, Christensen CH, Wang BG, Murphy G, Ye XY, Bhandari D, Feng J, Xia BY, Sosnoff CS, Kamangar F, Brennan P, Boffetta P, Dawsey SM, Abnet CC, Malekzadeh R, Freedman ND, Urinary biomarkers of carcinogenic exposure among cigarette, waterpipe, and smokeless tobacco users and never users of tobacco in the Golestan cohort study, Cancer Epidemiol Biomarkers Prev, 28 (2019) 337–347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [32].Centers for Disease Control and Prevention, Fourth Report on Human Exposure to Environmental Chemicals, Updated Tables, (January 2019, Vol. 2), U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, Atlanta, GA, 2019. [Google Scholar]
  • [33].Hecht SS, Chen MA, Yoder A, Jensen J, Hatsukami D, Le C, Carmella SG, Longitudinal study of urinary phenanthrene metabolite ratios: Effect of smoking on the diol epoxide pathway, Cancer Epidemiol Biomarkers Prev, 14 (2005) 2969–2974. [DOI] [PubMed] [Google Scholar]
  • [34].Centers for Disease Control and Prevention, Fourth Report on Human Exposure to Environmental Chemicals, Updated Tables, (January 2019, Vol. 1), U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, Atlanta, GA, 2019. [Google Scholar]
  • [35].Jacob J, Doehmer J, Grimmer G, Soballa V, Raab G, Seidel A, Greim H, Metabolism of phenanthrene, benz[a]anthracene, benzo[a]pyrene, chrysene and benzo[c]phenanthrene by eight cDNA-expressed human and rat cytochromes P450, Polycycl Aromat Comp, 10 (1996) 1–9. [Google Scholar]
  • [36].Schober W, Pusch G, Oeder S, Reindl H, Behrendt H, Buters JTM, Metabolic activation of phenanthrene by human and mouse cytochromes P450 and pharmacokinetics in CYP1A2 knockout mice, Chem-Biol Interact, 183 (2010) 57–66. [DOI] [PubMed] [Google Scholar]
  • [37].Cornelis MC, El-Sohemy A, Kabagambe EK, Campos H, Coffee, CYP1A2 genotype, and risk of myocardial infarction, JAMA-J Am Med Assoc, 295 (2006) 1135–1141. [DOI] [PubMed] [Google Scholar]
  • [38].Kalow W, Tang BK, Use of caffeine metabolite ratios to explore CYP1A2 and xanthine-oxidase activities, Clin Pharmacol Ther, 50 (1991) 508–519. [DOI] [PubMed] [Google Scholar]
  • [39].Sinues B, Saenz MA, Lanuza J, Bernal ML, Fanlo A, Juste JL, Mayayo E, Five caffeine metabolite ratios to measure tobacco-induced CYP1A2 activity and their relationships with urinary mutagenicity and urine flow, Cancer Epidemiol Biomarkers Prev, 8 (1999) 159–166. [PubMed] [Google Scholar]
  • [40].Hanna IH, Dawling S, Roodi N, Guengerich FP, Parl FF, Cytochrome P450 1B1 (CYP1B1) pharmacogenetics: association of polymorphisms with functional differences in estrogen hydroxylation activity, Cancer Res, 60 (2000) 3440–3444. [PubMed] [Google Scholar]
  • [41].Paracchini V, Pedotti P, Raimondi S, Garte S, Bradlow HL, Sepkovic DW, Taioli E, A common CYP1B1 polymorphism is associated with 2-OHE1/16-OHE1 urinary estrone ratio, Clin Chem Lab Med, 43 (2005) 702–706. [DOI] [PubMed] [Google Scholar]
  • [42].Watanabe J, Shimada T, Gillam EM, Ikuta T, Suemasu K, Higashi Y, Gotoh O, Kawajiri K, Association of CYP1B1 genetic polymorphism with incidence to breast and lung cancer, Pharmacogenetics, 10 (2000) 25–33. [DOI] [PubMed] [Google Scholar]
  • [43].Schrenk D, Brockmeier D, Morike K, Bock KW, Eichelbaum M, A distribution study of CYP1A2 phenotypes among smokers and non-smokers in a cohort of healthy Caucasian volunteers, Eur J Clin Pharmacol, 53 (1998) 361–367. [DOI] [PubMed] [Google Scholar]
  • [44].Gumus ZH, Du B, Kacker A, Boyle JO, Bocker JM, Mukherjee P, Subbaramaiah K, Dannenberg AJ, Weinstein H, Effects of tobacco smoke on gene expression and cellular pathways in a cellular model of oral leukoplakia, Cancer Prev Res, 1 (2008) 100–111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [45].Boyle JO, Gumus ZH, Kacker A, Choksi VL, Bocker JM, Zhou XK, Yantiss RK, Hughes DB, Du BH, Judson BL, Subbaramaiah K, Dannenberg AJ, Effects of cigarette smoke on the human oral mucosal transcriptome, Cancer Prev Res, 3 (2010) 266–278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [46].Port JL, Yamaguchi K, Du BH, De Lorenzo M, Chang M, Heerdt PM, Kopelovich L, Marcus CB, Altorki NK, Subbaramaiah K, Dannenberg AJ, Tobacco smoke induces CYP1B1 in the aerodigestive tract, Carcinogenesis, 25 (2004) 2275–2281. [DOI] [PubMed] [Google Scholar]
  • [47].Hecht SS, Berg JZ, Hochalter JB, Preferential glutathione conjugation of a reverse diol epoxide compared to a bay region diol epoxide of phenanthrene in human hepatocytes: relevance to molecular epidemiology studies of glutathione-S-transferase polymorphisms and cancer, Chem Res Toxicol, 22 (2009) 426–432. [DOI] [PMC free article] [PubMed] [Google Scholar]

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