Detoxification Of Polycyclic Aromatic Hydrocarbons Research Articles

Combined exposure to pyrene and fluoranthene and their molecular effects on the Sydney rock oyster, Saccostrea glomerata

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitously detected in the water column, associated with particulate matter or in the tissue of marine organisms such as molluscs. PAH exposure and their resultant bioaccumulation in molluscs can cause a range of serious physiological effects in the affected animals. To examine the molecular response of these xenobiotics in bivalves, Sydney rock oysters (Saccostrea glomerata) were exposed to pyrene and fluoranthene for seven days. Chemical analysis of the soft-tissue of PAH stressed S. glomerata confirmed that pyrene and fluoranthene could be bioaccumulated by these oysters. RNA-Seq analysis of PAH-exposed S. glomerata showed a total of 765 transcripts differentially expressed between control and PAH-stressed oysters. Closer examination of the transcripts revealed a range genes encoding enzymes involved in PAH detoxification (e.g. cytochrome P450), innate immune responses (e.g. pathogen recognition, phagocytosis) and protein synthesis. Overall, pyrene and fluoranthene exposure appears to have resulted in a suppression of pathogen recognition and some protein synthesis processes, whereas transcripts of genes encoding proteins involved in clearance of cell debris and some transcripts of genes involved in PAH detoxification were induced in response to the stressors. Pyrene and fluoranthene exposure thus invoked a complex molecular response in S. glomerata, with results suggesting that oysters focus on removing the stressors from their system and dealing with the downstream effects of PAH exposure, potentially at the exclusion of other, less immediate concerns (e.g. protection from infection).

Cyclodextrin-promoted Diels Alder reactions of a polycyclic aromatic hydrocarbon under mild reaction conditions

Reported herein is the effect of cyclodextrins on the rates of aqueous Diels Alder reactions of 9-anthracenemethanol with a variety of N-substituted maleimides. These reactions occurred under mild reaction conditions (aqueous solvent, 40°C) and were most efficient for the reaction of N-cyclohexylmaleimide with a methyl-β-cyclodextrin additive (94% conversion in 24h). These results can be explained on the basis of a model wherein the cyclodextrins bind the hydrophobic substituents on the maleimides and activate the dienophile via electronic modulation of the maleimide double bond. The results reported herein represent a new mechanism for cyclodextrin-promoted Diels Alder reactions, and have significant potential applications in the development of other cyclodextrin-promoted organic transformations. Moreover, the ability to deplanarize polycyclic aromatic hydrocarbons (PAHs) under mild conditions, as demonstrated herein, has significant applications for PAH detoxification.

Metal–PAH mixtures in the aquatic environment: A review of co-toxic mechanisms leading to more-than-additive outcomes

Mixtures of metals and polycyclic aromatic hydrocarbons (PAHs) occur ubiquitously in aquatic environments, yet relatively little is known regarding their combined toxicities. Emerging reports investigating the additive mortality in metal–PAH mixtures have indicated that more-than-additive effects are equally as common as strictly-additive effects, raising concern for ecological risk assessment typically based on the summation of individual toxicities. Moreover, the current separation of focus between in vivo and in vitro studies, and fine- and coarse-scale endpoints, creates uncertainty regarding the mechanisms of co-toxicity involved in more-than-additive effects on whole organisms. Drawing from literature on metal and PAH toxicity in bacteria, protozoa, invertebrates, fish, and mammalian models, this review outlines several key mechanistic interactions likely to promote more-than-additive toxicity in metal–PAH mixtures. Namely, the deleterious effects of PAHs on membrane integrity and permeability to metals, the potential for metal–PAH complexation, the inhibitory nature of metals to the detoxification of PAHs via the cytochrome P450 pathway, the inhibitory nature of PAHs towards the detoxification of metals via metallothionein, and the potentiated production of reactive oxygenated species (ROS) in certain metal (e.g. Cu) and PAH (e.g., phenanthrenequinone) mixtures. Moreover, the mutual inhibition of detoxification suggests the possibility of positive feedback among these mechanisms. The individual toxicities and interactive aspects of contaminant transport, detoxification, and the production of ROS are herein discussed.

A light in the darkness: New biotransformation genes, antioxidant parameters and tissue-specific responses in oysters exposed to phenanthrene

Phenanthrene (PHE), a major component of crude oil, is one of the most abundant polycyclic aromatic hydrocarbons (PAHs) in aquatic ecosystems, and is readily bioavailable to marine organisms. Understanding the toxicity of PAHs in animals requires knowledge of the systems for xenobiotic biotransformation and antioxidant defence and these are poorly understood in bivalves. We report, for the first time, new transcripts and tissue-specific transcription in gill and digestive gland from the oyster Crassostrea brasiliana following 24h exposure to 100 and 1000μgL−1 PHE, a model PAH. Six new cytochrome P450 (CYP) and four new glutathione S-transferase (GST) genes were analysed by means of quantitative reverse transcription PCR (qRT-PCR). Different antioxidant endpoints, including both enzymatic and non-enzymatic parameters, were assessed as potential biomarkers of oxidative stress. GST activity was measured as an indicator of phase II biotransformation. Rapid clearance of PHE was associated with upregulation of both phase I and II genes, with more pronounced effects in the gill at 1000μgL−1 PHE. After 24h of exposure, PHE also caused impairment of the antioxidant system, decreasing non-protein thiols and glutathione levels. On the other hand, no change in antioxidant enzymes was observed. PHE treatment (100μgL−1) significantly decreased GST activity in the gill of exposed oysters. Both CYP and GST were transcribed in a tissue-specific manner, reflecting the importance of the gill in the detoxification of PAHs. Likewise, the antioxidant parameters followed a similar pattern. The data provide strong evidence that these genes play key roles in C. brasiliana biotransformation of PHE and highlight the importance of gill in xenobiotic metabolism.

Induction of CYP1A1, CYP1A2, CYP1B1, increased oxidative stress and inflammation in the lung and liver tissues of rats exposed to incense smoke

Incense smoke is increasingly being recognized as a potential environmental contaminant and is linked to malignant and non-malignant respiratory diseases. The detoxification of environmental contaminants including polycyclic aromatic hydrocarbons (PAHs) involves the induction of cytochrome P-450 family enzymes (CYPs) by PAHs. However, the detoxification of PAHs also results in the generation of reactive and unstable intermediary metabolites which are implicated in the oxidative stress, DNA damage, and inflammation. It is unclear whether CYPs are similarly induced by incense smoke, which incidentally contains substantial amounts of PAHs. Here, we examined the impact of long-term incense smoke exposure on the induction of CYPs in male Wister Albino rats. Incense smoke exposure significantly induced the expression of CYP1A1, CYP1A2, and CYP1B1 mRNAs in both lung and liver tissues. The extent of CYP1A1 and CYP1B1 induction was significantly higher in the liver compared to that in the lung, while that of CYP1A2 was greater in the lung than in liver. Incense smoke exposure also increased malondialdehyde and reduced glutathione levels in lung and liver tissues, and the catalase activity in the liver tissues to significant levels. Furthermore incense smoke exposure led to a marked increase in TNF-α and IL-4 levels. The data demonstrate for the first time the capacity of incense smoke to induce CYP1 family enzymes in the target and non-target tissues. Induction of CYPs increased oxidative stress and inflammation appear to be intimately linked to promote the carcinogenesis and health complications in people chronically exposed to incense smoke.

Oxidative stress responses of gulf killifish exposed to hydrocarbons from the Deepwater Horizon oil spill: Potential implications for aquatic food resources

Ecosystem effects of polycyclic aromatic hydrocarbons (PAHs) remain under investigation following the Gulf of Mexico Deepwater Horizon oil spill. Fundulus grandis, an established indicator of aquatic ecosystem health, was investigated because this species shares genes and biochemical pathways with higher trophic-level fish and plays an important role in the gulf food chain. Oxidative stress responses including hepatic cytochrome P4501A (CYP1A) and serum antioxidant capacity were evaluated in fish exposed to PAHs. Fish were exposed to water-accommodated fractions (WAFs) of crude oil (7.0 ± 0.10 mg/L C6-C28) after which solutions were diluted below the level of detection over 8 h using 15 ppt aerated artificial seawater. Before euthanasia, fish remained in aquaria for 12 h, 24 h, or 48 h. Three replicate experiments were conducted at each time point using unexposed fish as experimental controls. Significant differences (p < 0.05) in CYP1A induction were observed in exposed versus control fish at 24 h. Expression of CYP1A increased by 25%, 66%, and 23% in exposed fish at 12 h, 24 h, and 48 h, respectively. Significant increases were observed in antioxidant capacity of nonenzymatic antioxidants in exposed versus control fish at each time point. Given the activity of CYP1A, radicals formed during PAH detoxification likely resulted in increased oxidant load requiring elevated antioxidant defenses. Research is needed to determine the duration of oxidative stress responses considering the potential for lipid oxidation in exposed fish or species feeding on exposed fish.

PAH metabolites, GST and EROD in European eel (Anguilla anguilla) as possible indicators for eel habitat quality in German rivers

The stock of the European eel (Anguilla anguilla L.) continues to decline and has reached a new minimum in 2011. Poor health status of the spawners due to organic contaminants is one of the possible causes for this dramatic situation. Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants, which are rapidly metabolized in vertebrates. EROD (ethoxyresorufin-O-deethylase) and GST (glutathione-S-transferase) are two enzymes involved in PAH detoxification in fish. In this study, PAH metabolites as well as EROD and GST activity in a large, comprising dataset of more than 260 migratory and pre-migratory eels from five large German river basin districts were used to describe PAH exposure and its metabolism as possible indicators for the habitat quality for eels. Eel from the river Elbe appear to be moderately contaminated with PAH. Highest mean values of PAH metabolites were analysed in fish from the river Rhine. However, the results suggest that contaminants such as PAH are metabolized in the fish and may have contributed to EROD activity in eels caught from the Elbe estuary to 600 km upstream. Since the eel's onset of cessation of feeding is closely linked to maturation and migration, we propose bile pigments as new indicators contributing to identify the proportion of migratory eel, which is crucial information for eel management plans. We showed that PAH metabolites normalized to bile pigments as well as EROD could be used to describe the habitat quality and might be suitable parameters in search for suitable stocking habitats.

The influence of CYP1A1 and GSTM1 polymorphism on the concentration of urinary 1-hydroxypyrene in cPAHs exposed Iranian anode plant workers

Polycyclic aromatic hydrocarbons (PAHs) are a group of environmental pollutants in the occupational settings which are associated with an increased risk of cancer. CYP and GST enzymes have an important role in metabolism of PAHs. Genetic polymorphisms of these enzymes may affect their capacity for oxidation and detoxification of PAHs which will result in variation in PAHs metabolites. The aim of this study was to evaluate the association of urinary 1-hydroxypyrene levels with genetic polymorphisms of CYP1A1 and GSTM1 polymorphism in the Iranian anode plant workers. The study population consisted of 42 workers working in carbon anode plant in an aluminum production industry and matched control group consisted of 43 office workers. Personal air sampling was performed to assess workers atmospheric exposure to carcinogenic PAHs. Blood samples were taken for DNA extraction and determination of GSTM1 and CYP1A1 polymorphism. Urine samples were taken immediately after the end of the working shift on the last working day for analysis of urinary 1-hydroxypyrene using High Performance Liquid Chromatography (HPLC). Statistical analysis was performed using SPSS ver. 16 software. The mean concentration of occupational exposure to cPAHs in the exposed group was 11.42 μg/m3 ranging from 3.6 to 31.5 μg/m3. Mean level of urinary 1-hydroxypyrene in the exposed and control groups was 6.32±4.9 μmole/mole creatinine and 0.54±.48 μmole/mole creatinine respectively. According to the statistical analysis we found no influence of CYP1A1 MspI polymorphism on the urinary 1-hydroxypyrene levels of exposed and control subjects before and after stratification according to smoking habit. Our results also showed that, GSTM1 null genotype is associated with significantly higher levels of urinary 1-hydroxypyrene before stratification according to smoking habit in the exposed subjects (P=0.003) and after stratification the association remained statistically significant only in the non smoker subjects (P= 0.003). Association of GSTM1 null genotype with higher levels of urinary 1-hydroxypyrene shows higher susceptibility of these genotype carriers to genotoxic effects of PAHs and employment of GSTM1 null carriers in the occupational settings with high levels of PAHs pollution should be reconsidered. Regarding the CYP1A1 polymorphism, it seems more studies, especially with large sample sizes, are necessary for achieving conclusive results.

The Beat

The Beat

Abstract 3579: Mechanistic roles of cytochrome P4501A1 and 1A2 in lung carcinogenesis mediated by polycyclyc aromatic hydrocarbons: Implications for lung cancer in humans .

Abstract Humans are frequently exposed to polycyclic aromatic hydrocarbons (PAHs). Cytochrome P4501A (CYP1A) enzymes play important roles in the activation of PAHs such as 3-methylcholanthrene (MC) to DNA-binding metabolites, which in turn mediate carcinogenesis in target organs such as lung, breast, and skin. In this study, we tested the hypothesis that CYP1A1 and 1A2 play mechanistic roles in PAH-mediated carcinogenesis and tumorigenesis. Eight week-old female wild type (WT) (A/J) mice or mice lacking the gene for CYP1A1 or CYP1A2 on the A/J background were treated with a single dose of MC (40 μmol/kg), benzo[a]pyrene (BP), or vehicle (corn oil), and liver and lung tumors were studied after 28 weeks. While 100% of WT or Cyp1a2-null mice exposed to MC showed lung tumors after 28 weeks, about 80% of the Cyp1a1-null mice showed lung tumors. However, there were striking differences in the Cyp1a1-null and Cyp1a2-null mice in regard to lung tumor multiplicities. The WT mice treated with MC had about 15 lung tumors/animal. On the other hand, the Cyp1a2-null mice displayed about 40 lung tumors/animal, while the Cyp1a1-null mice showed about 2-3 tumors/mouse. Although BP was less potent in inducing lung tumors in the WT mice compared to MC, it was similar to MC In that tumors were suppressed in the Cyp1a1-null mice and augmented in the Cyp1a2-null mice. DNA adduct studies at early time points showed increased MC or BP adducts in lungs of Cyp1a2-null mice compared to WT mice, and decreased adduct formation in the Cyp1a1-null mice, supporting the hypothesis that DNA adducts are early biomarkers of PAH-mediated carcinogenesis. Immunohistochemical studies showed that CYP1A1 was overexpressed in the tumorous portions of the lung in the Cyp1a2-null or WT mice, compared to non-tumorous regions. These results suggest that CYP1A1 contributes to the formation of tumorigenesis by PAHs, while CYP1A2 protects against carcinogenesis mediated by PAHs, presumably through it role in PAH detoxification. In conclusion, our results strongly suggest that CYP1A1 and 1A2 could be novel candidates for cancer prevention and therapy though either inhibition of CYP1A1 or induction of CYP1A2. (Supported by NIH grant ES009132.) Citation Format: Bhagavatula Moorthy, Guodong Zhou, Lihua Wang, Weiwu Jiang. Mechanistic roles of cytochrome P4501A1 and 1A2 in lung carcinogenesis mediated by polycyclyc aromatic hydrocarbons: Implications for lung cancer in humans . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3579. doi:10.1158/1538-7445.AM2013-3579

Three novel cytochrome P450 genes identified in the marine polychaete Perinereis nuntia and their transcriptional response to xenobiotics

Polychaetes have previously been used as bioindicators of environmental pollution. Their ability to eliminate organic pollutants such as polycyclic aromatic hydrocarbons (PAH) has been extensively analyzed. However, the cytochrome P450 monooxygenases (CYP) genes in polychaetes, which catalyze the first step of oxidative degradation of PAHs, have received little attention. Based on the partial sequences of three CYP genes that were enriched by subtractive cDNA libraries of the polychaete Perinereis nuntia, we amplified and sequenced the full-length cDNA of these novel CYP genes. These genes were named CYP4BB2, CYP423A1 and CYP424A1 by the Cytochrome P450 Nomenclature Committee. The deduced amino acid sequence of CYP4BB2 in P. nuntia showed 68% sequence identity to CYP4BB1 in Nereis virens, and was listed as a new member of the CYP4BB subfamily. The sequence of CYP423A1 and CYP424A1 both share less than 40% sequence identity to all known CYP enzymes and were classed into new CYP families. CYP family members are composite parts of a larger group called a clan. CYP4BB2 and CYP424A1 are listed as CYP4 clan members, while CYP423A1 is of the CYP2 clan. The 3D structures of these P. nuntia CYPs were successfully predicted by homology-modeling using the SWISS-MODEL workspace. The models of CYP424A1 and CYP4BB2 were created using 1jpzB (CYP102A) as a template, while CYP423A1 utilized 3czhB (CYP2R1) as its template. The presence of characteristic CYP superfamily motifs, such as the F-G⋯C-G amino acid sequence, and the conservation of the three-dimensional CYP structure shown by the modeling, suggested that these novel P. nuntia CYP genes may contain conserved functional domains of CYP monooxygenases. To examine the effect of xenobiotics on living organisms, we analyzed the transcriptional levels of these three new CYP genes in sandworms (P. nuntia) exposed to seawater artificially contaminated with benzo[a]pyrene (BaP). We also exposed individuals to industrial wastewater collected from Quanzhou Bay, Fujian, China, which was known to be contaminated with PAHs. Worms exposed to BaP had significantly higher levels of CYP4BB2, CYP423A1 and CYP424A1 mRNA. Transcription was up-regulated 5.9-, 5.3- and 12.3-folds respectively compared with the control worms living in clean seawater. The transcriptional levels of CYPs in worms cultured in the diluted wastewater collected from Quanzhou Bay, all positively correlated with the levels of PAHs detected in the water. The transcriptional up-regulation of the three CYP genes observed in this study, suggest the monooxygenases encoded by these CYP genes may play an important role in the detoxification of PAHs in this polychaete worm. These CYPs maybe essential for the adaptation of worms to contaminated environments and may be useful in the assessment of xenobiotic exposure.

Association between human glutathione S-transferase omega rs4925 polymorphism and bladder cancer

Glutathione S-transferases (GSTs) play an important role in the detoxification of polycyclic aromatic hydrocarbons and aromatic amines, the toxic substances contained in cigarettes. GST Omega 1 (GSTO1) not only utilizes glutathione in conjugation reaction but also contributes to the biotransformation of several xenobiotics. A single nucleotide polymorphism (Ala-140Asp) of GSTO1 gene causing variations in enzyme activity may influence individual susceptibility to bladder cancer (BC). It is hypothesized that genetic polymorphism of GSTO1 gene has an effect on BC risk in particular by interacting with cigarette smoking. A total of histopathologically confirmed 300 BC patients and 300 cancer-free controls were recruited from February 2002 to February 2009. Genotyping of the GSTO1 Ala140Asp polymorphism was determined using a polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) method. The odds ratio (OR) and 95% confidence interval (CI) were calculated as a measure of the combined effect of cigarette smoking and the GSTO1 Ala140Asp polymorphism on BC risk. We found that study subjects with the GSTO1 Ala/Ala genotype have a significantly increased BC risk (OR = 1.5; 95% CI = 1.1 - 2.7). A statistically significant increased BC risk was also found in ever smokers with the GSTO1 Ala/Ala genotype (OR = 4.9; 95%CI = 2.8 - 9.7). In conclusion, this study provides an epidemiologic evidence of a significantly increased BC risk among ever smokers with the GSTO1 Ala/Ala genotype.

EPHX1 A139G polymorphism and lung cancer risk: a meta-analysis

Microsomal epoxide hydrolase 1 (EPHX1) plays an important role in both the activation and the detoxification of polycyclic aromatic hydrocarbons and aromatic amines. Polymorphisms at exon 4 of the EPHX1 gene have been reported to be associated with variations in EPHX1 activity. Many studies have investigated the association between EPHX1 A139G polymorphism and lung cancer risk, but the impact of EPHX1 A139G polymorphism on lung cancer risk is not clear owing to the apparent inconsistence among those studies. This study aimed to identify the association between EPHX1 A139G polymorphism and lung cancer risk by performing a meta-analysis. We used the pooled odds ratio (OR) with its corresponding 95% confidence interval (95% CI) to explore the association. Finally, 26 studies with a total of 14,494 subjects were included into this meta-analysis. Meta-analyses of total studies showed the EPHX1 A139G polymorphism was associated with lung cancer risk under three genetic models (OR (G versus A) = 1.17, 95% CI 1.04-1.31, P (OR) = 0.01; OR (AG versus AA) = 1.21, 95% CI 1.06-1.37, P (OR) = 0.004; OR (AG + GG versus AA) = 1.22, 95% CI 1.06-1.39, P (OR) = 0.005). Sensitivity analyses and subgroup analyses further identified the significant association between the EPHX1 A139G polymorphism and lung cancer risk. No evidence of publication bias was observed. Meta-analyses of available data supported the concept of EPHX1 A139G polymorphism as a genetic susceptibility factor for lung cancer.

In Silico Modeling of UDP-Glucuronosyltransferase 1A10 Substrates Using the Volsurf Approach

UDP-glucuronosyltransferase 1A10 (UGT1A10) catalyzes glucuronidation of a wide range of chemicals including many drugs. Here, we report the first in silico model quantifying the substrate selectivity and binding affinity (as K(m)) for UGT1A10. The training set for model construction comprises 32 structurally diverse compounds, which are known substrates for UGT1A10. The model was derived by applying the standard VolSurf method involving calculation of VolSurf descriptors and partial least square (PLS) analyses. The yielded PLS model with two components shows statistical significance in both fitting and internal predicting (r(2) = 0.827, q(2) = 0.774). The model predictability was further validated against a test set of 11 external compounds. The activity values for all test substrates were predicted within 1 log unit. Moreover, the model reveals an overlay of chemical features influencing the enzyme-substrate binding. Those include the size and shape, capacity factors, hydrophilic regions, hydrophobic regions, and polarizability. In conclusion, the VolSurf approach is successfully utilized to establish a predictive model for UGT1A10. The derived model should be an efficient tool for high-throughput prediction of UGT1A10 metabolism.

Microsomal epoxide hydrolase polymorphisms, cigarette smoking, and risk of colorectal cancer: The Fukuoka Colorectal Cancer Study

Microsomal epoxide hydrolase (EPHX1) plays an important role in the activation and detoxification of polycyclic aromatic hydrocarbons, carcinogens found in cigarette smoke. Polymorphisms in exon 3 (Y113H) and exon 4 (H139R) of the EPHX1 have been associated with enzyme activity. We investigated the risk of colorectal cancer in relation to the EPHX1 Y113H and H139R polymorphisms and assessed effect modifications of cigarette smoking and the other covariates. The interaction between the EPHX1 polymorphisms and selected genetic polymorphisms was also examined. We used data from Fukuoka Colorectal Cancer Study, a community-based case-control study, including 685 cases and 778 controls. In-person interviews were conducted to assess lifestyle factors. The EPHX1 Y113H and H139R polymorphisms were determined by the TaqMan assay and the polymerase chain reaction-restriction fragment length polymorphism, respectively. Neither of the two polymorphisms nor the imputed EPHX1 phenotype was associated with colorectal cancer risk. Cigarette smoking and alcohol intake showed no effect modification on the association with the EPHX1 polymorphisms or the imputed EPHX1 phenotype. Increased risks of colorectal cancer associated with the 113Y allele and imputed EPHX1 phenotype were observed among individuals with high body mass index (BMI; ≥25.0 kg/m(2)), but not among those with low BMI (<25.0 kg/m(2)). The risk decreased with an increasing number of the 139R allele in the null genotypes of GSTM1/GSTT1. It is unlikely that the EPHX1 polymorphisms play an important role in colorectal carcinogenesis. The observed interactions of the EPHX1 polymorphisms with BMI and the GSTM1/GSTT1 genotypes warrant further investigation.

Effects of Dietary Fish Oil on the Depletion of Carcinogenic PAH-DNA Adduct Levels in the Liver of B6C3F1 Mouse

Many carcinogenic polycyclic aromatic hydrocarbons (PAHs) and their metabolites can bind covalently to DNA. Carcinogen-DNA adducts may lead to mutations in critical genes, eventually leading to cancer. In this study we report that fish oil (FO) blocks the formation of DNA adducts by detoxification of PAHs. B6C3F1 male mice were fed a FO or corn oil (CO) diet for 30 days. The animals were then treated with seven carcinogenic PAHs including benzo(a)pyrene (BaP) with one of two doses via a single intraperitoneal injection. Animals were terminated at 1, 3, or 7 d after treatment. The levels of DNA adducts were analyzed by the 32P-postlabeling assay. Our results showed that the levels of total hepatic DNA adducts were significantly decreased in FO groups compared to CO groups with an exception of low PAH dose at 3 d (P = 0.067). Total adduct levels in the high dose PAH groups were 41.36±6.48 (Mean±SEM) and 78.72±8.03 in 109 nucleotides (P = 0.011), respectively, for the FO and CO groups at 7 d. Animals treated with the low dose (2.5 fold lower) PAHs displayed similar trends. Total adduct levels were 12.21±2.33 in the FO group and 24.07±1.99 in the CO group, P = 0.008. BPDE-dG adduct values at 7 d after treatment of high dose PAHs were 32.34±1.94 (CO group) and 21.82±3.37 (FO group) in 109 nucleotides with P value being 0.035. Low dose groups showed similar trends for BPDE-dG adduct in the two diet groups. FO significantly enhanced gene expression of Cyp1a1 in both the high and low dose PAH groups. Gstt1 at low dose of PAHs showed high levels in FO compared to CO groups with P values being 0.014. Histological observations indicated that FO played a hepatoprotective role during the early stages. Our results suggest that FO has a potential to be developed as a cancer chemopreventive agent.

Persistent induction of cytochrome P4501A1 in human hepatoma cells by 3-methylcholanthrene: evidence for sustained transcriptional activation of the CYP1A1 promoter.

Cytochrome P450 (P450)1A1 plays a critical role in the metabolic activation and detoxification of polycyclic aromatic hydrocarbons (PAHs), many of which are potent human carcinogens. In this investigation, we tested the hypothesis that MC elicits persistent induction of CYP1A1 expression in human hepatoma cells (HepG2) and that this phenomenon is mediated by sustained transcriptional activation of the CYP1A1 promoter. Treatment of HepG2 cells with MC resulted in marked induction (8-20-fold) of ethoxyresorufin O-de-ethylase activities, CYP1A1 apoprotein contents, and mRNA levels, which persisted for up to 96 h. MC also caused sustained transcriptional activation of the human CYP1A1 promoter for up to 96 h, as inferred from transient transfection experiments. Experiments with deletion constructs indicated that Ah response elements located at -886, -974, and -1047, but not -491, nucleotides from the start site, contributed to the sustained transcriptional activation of the CYP1A1 promoter. Electrophoretic mobility-shift and chromatin immunoprecipitation assays suggested that prolonged CYP1A1 induction was mediated by Ah receptor (AHR)-independent mechanisms. Experiments with [3H]MC and liquid chromatography-tandem mass spectrometry demonstrated rapid elimination of MC and its metabolites from the cells by 12 to 24 h, suggesting that these compounds did not elicit sustained CYP1A1 induction via the classical AHR-mediated pathway. In conclusion, the results of this study support the hypothesis that MC causes persistent induction of CYP1A1 in human hepatoma cells by mechanisms entailing sustained transcriptional activation of the CYP1A1 promoter via AHR-independent mechanisms. These observations have important implications for human carcinogenesis mediated by PAHs.

Effect of gene-environment Interactions on mental development in African American, Dominican, and Caucasian mothers and newborns.

The health impact of environmental toxins has gained increasing recognition over the years. Polycyclic aromatic hydrocarbons (PAHs) and environmental tobacco smoke (ETS) are known to affect nervous system development in children, but no studies have investigated how polymorphisms in PAH metabolic genes affect child cognitive development following PAH exposure during pregnancy. In two parallel prospective cohort studies of non-smoking African American and Dominican mothers and children in New York City and of Caucasian mothers and children in Krakow, Poland, we explored the effect of gene-PAH interaction on child mental development index (MDI). Genes known to play important roles in the metabolic activation or detoxification of PAHs were selected. Genetic variations in these genes could influence susceptibility to adverse effects of PAHs in polluted air. We explored the effects of interactions between prenatal PAH exposure and 21 polymorphisms or haplotypes in these genes on MDI at 12, 24, and 36 months among 547 newborns and 806 mothers from three different ethnic groups. Significant interaction effects between haplotypes and PAHs were observed in mothers and their newborns in all three ethnic groups after Bonferroni correction. The strongest and most consistent effect observed was between PAH and haplotype ACCGGC of the CYP1B1 gene.

Relationship between N-acetyltransferase 2 single-nucleotide polymorphisms and aromatic DNA adducts

Relationship between N-acetyltransferase 2 single-nucleotide polymorphisms and aromatic DNA adducts

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

Bay region diol epoxides are recognized ultimate carcinogens of polycyclic aromatic hydrocarbons (PAH), and in vitro studies have demonstrated that they can be detoxified by conjugation with glutathione, leading to the widely investigated hypothesis that individuals with low activity forms of glutathione-S-transferases are at higher risk of PAH induced cancer, a hypothesis that has found at most weak support in molecular epidemiology studies. A weakness in this hypothesis was that the mercapturic acids resulting from the conjugation of PAH bay region diol epoxides had never been identified in human urine. We recently analyzed smokers' urine for mercapturic acids derived from phenanthrene, the simplest PAH with a bay region. The only phenanthrene diol epoxide-derived mercapturic acid in smokers' urine was produced from the reverse diol epoxide, anti-phenanthrene-3,4-diol-1,2-epoxide (11), not the bay region diol epoxide, anti-phenanthrene-1,2-diol-3,4-epoxide (10), which does not support the hypothesis noted above. In this study, we extended these results by examining the conjugation of phenanthrene metabolites with glutathione in human hepatocytes. We identified the mercapturic acid N-acetyl-S-(r-4,t-2,3-trihydroxy-1,2,3,4-tetrahydro-c-1-phenanthryl)-L-cysteine (14a), (0.33-35.9 pmol/mL at 10 microM 8, 24 h incubation, N = 10) in all incubations with phenanthrene-3,4-diol (8) and the corresponding diol epoxide 11, but no mercapturic acids were detected in incubations with phenanthrene-1,2-diol (7), and only trace amounts were observed in incubations with the corresponding bay region diol epoxide 10. Taken together with our previous results, these studies clearly demonstrate that glutathione conjugation of a reverse diol epoxide of phenanthrene is favored over conjugation of a bay region diol epoxide. Since reverse diol epoxides of PAH are generally weakly or nonmutagenic/carcinogenic, these results, if generalizable to other PAH, do not support the widely held assumption that glutathione-S-transferases are important in the detoxification of PAH in humans.