Genetic association of aromatic hydrocarbon receptor (AHR) and cytochrome P450, family 1, subfamily A,polypeptide 1 (CYP1A1) polymorphisms with dioxin blood concentrations among pregnant Japanese women

Dioxins are metabolized by cytochrome P450, family 1 (CYP1) via the aromatic hydrocarbon receptor (AHR). We determined whether different blood dioxin concentrations are associated with polymorphisms in AHR (dbSNP ID: rs2066853), AHR repressor (AHRR; rs2292596), CYP1 subfamily A polypeptide 1 (CYP1A1; rs4646903 and rs1048963), CYP1 subfamily A polypeptide 2 (CYP1A2; rs762551), and CYP1 subfamily B polypeptide 1 (CYP1B1; rs1056836) in pregnant Japanese women. These six polymorphisms were detected in 421 healthy pregnant Japanese women. Differences in dioxin exposure concentrations in maternal blood among the genotypes were investigated. Comparisons among the GG, GA, and AA genotypes of AHR showed a significant difference (genotype model: P = 0.016 for the mono-ortho polychlorinated biphenyl concentrations and toxicity equivalence quantities [TEQs]). Second, we found a significant association with the dominant genotype model ([TT + TC] vs. CC: P = 0.048 for the polychlorinated dibenzo-p-dioxin TEQs; P = 0.035 for polychlorinated dibenzofuran TEQs) of CYP1A1 (rs4646903). No significant differences were found among blood dioxin concentrations and polymorphisms in AHRR, CYP1A1 (rs1048963), CYP1A2, and CYP1B1. Thus, polymorphisms in AHR and CYP1A1 (rs4646903) were associated with maternal dioxin concentrations. However, differences in blood dioxin concentrations were relatively low.     

Relationship between miRNAs polymorphisms and peripheral blood leukocyte DNA telomere length in coke oven workers: A cross-sectional study

ObjectiveThe purpose of this study was to investigate the factors affecting telomere length (TL) in coke oven workers by analyzing the interaction between miRNAs polymorphisms and coke oven emissions (COEs) exposure.MethodsA total of 544 coke oven workers and 238 healthy controls were recruited. Peripheral blood was collected from the subjects, genomic DNA was extracted, leukocyte TL was detected by real-time quantitative polymerase chain reaction, and fifteen polymorphisms of eight miRNAs were genotyped by flight mass spectrometry.ResultsStatistical analysis showed that the peripheral blood DNA TL in the exposure group was shorter than that in the control group (P < 0.001). Generalized linear model found that COEs-exposure [β (95%CI) = -0.427 (−0.556, −0.299), P < 0.001], genotype CC+CT for miR-612 rs1144925 [β (95%CI) = −0.367 (−0.630, −0.104), P = 0.006], and the interaction of miR-181B1 rs12039395 TT genotype and COEs-exposure [β (95% CI) = 0.564 (0.108, 1.020), P = 0.015] were associated with the shortened TL.ConclusionCOEs-exposure and miR-612 rs1144925 TT could promote telomere shortening in coke oven workers. The interaction of miR-181B1 rs12039395 TT genotype and COEs-exposure could protect telomere. This provides clues for further mechanistic studies between miRNA and telomere damage.     

Influence of GSTs, CYP2E1 and mEH polymorphisms on 1, 3-butadiene-induced micronucleus frequency in Chinese workers

1,3-butadiene (BD) has been classified as a human carcinogen, however, the relationship between chromosomal damage and its metabolic polymorphisms is not clear. The present study used the CBMN assay to detect chromosomal damage in the peripheral lymphocytes of 166 exposed workers and 41 non-exposed healthy individuals. PCR and PCR-RFLP were applied to detect GSTT1, GSTM1, CYP2E1 c1c2 and mEH Tyr113His, His139Arg polymorphisms. The results demonstrated that the micronucleus (MN) frequency of the exposed workers was significantly higher than controls (P < 0.01). Among the exposed workers, the individuals with high BD exposures are more susceptible to chromosomal damage than those with low exposures (FR = 1.30, 95% CI 1.14-1.53; P < 0.05). Gender-difference was also found in our study: males got lower micronucleus frequency than females. Workers who carried the genotypes of GSTM1 (+), CYP2E1 (c1c2/c2c2) and mEH intermediate (I) group had significantly higher MN frequency than those carrying the genotypes of GSTM1 (−) (FR = 1.29, 95% CI 1.05-1.59; P < 0.05), CYP2E1 (c1c1) (FR = 1.55, 95% CI 1.24-1.93; P < 0.01) or mEH high (H) group (FR = 1.57, 95% CI 1.08-2.34; P < 0.05), respectively. Our data indicated that the current BD exposure level could cause significantly higher MN frequency in workers than controls. Polymorphisms of GSTM1, CYP2E1 and mEH are susceptible to altered chromosome damage.     

Identification and expression profile of a new cytochrome P450 isoform (CYP414A1) in the hepatopancreas of Venerupis (Ruditapes) philippinarum exposed to benzo[a]pyrene, cadmium and copper

With the objective to identify promising molecular biomarkers for marine pollution monitoring, a new cytochrome P450 gene was identified from Venerupis (Ruditapes) philippinarum and classified as a member of a new subfamily, CYP414A1. Phylogenetic analysis showed that CYP414A1 was closely related to members of the CYP2 family. CYP414A1 mRNA expression was significantly induced by 50 μg/L B[a]P at 96 h, while no significant change was found in 5 μg/L B[a]P-exposed samples. For heavy metals exposure, the expression of CYP414A1 was significantly up-regulated by Cd but sharply depressed by Cu exposure. These results suggested that CYP414A1 responded to various xenobiotics stresses, and could be used as a candidate biomarker of heavy metals and B[a]P.     

Genetic variants in SMARC genes are associated with DNA damage levels in Chinese population

The switching defective/sucrose nonfermenting (SWI/SNF) related, matrix associated, actin dependent regulators of chromatin (SMARC) are components of human SWI/SNF like chromatin remodeling protein complexes, which are essential in the process of DNA damage repair. In this study, we hypothesized that genetic variants in SMARC genes may modify the capacity of DNA repair to damage. To test this hypothesis, we genotyped a total of 20 polymorphisms in five key SMARC genes (SMARCA5, SMARCC2, SMARCD1, SMARCD2, SMARCD3) to evaluate their associations with DNA damage levels in 307 subjects. The DNA damage levels were measured with comet assay. The multiple linear regression was used to assess the relationship between each polymorphism and DNA damage levels in additive model. We found that the genotypes of rs6857360 (β = 0.23, 95% CI = 0.06–0.40, P = 0.008) in SMARCA5, rs6919 (β = 0.20, 95% CI = 0.05–0.34, P = 0.008) and rs2727280 (β = 0.18, 95% CI = 0.04–0.33, P = 0.013) in SMARCD2, and rs17173769 (β = −0.27, 95% CI = −0.52 to −0.01, P = 0.045) in SMARCD3 were significantly associated with DNA damage levels. After combining these four polymorphisms, we found that the more unfavorable alleles the subjects carried, the heavier DNA damage they suffered, suggesting a locus-dosage effect between combined genotypes and DNA damage levels (P for trend = 0.006). These findings suggest that genetic variants in SMARC genes may contribute the individual variations of DNA damage levels in Chinese population. Further larger and functional studies are warranted to confirm our findings.     

An acellular assay to assess the genotoxicity of complex mixtures of organic pollutants bound on size segregated aerosol. Part I: DNA adducts

An acellular assay consisting of calf thymus DNA with/without rat liver microsomal S9 fraction was used to study the genotoxicity of complex mixtures of organic air pollutants bound to size segregated aerosols by means of DNA adduct analysis. We compared the genotoxicity of the organic extracts (EOMs) from three size fractions of aerosol ranging from 0.17 μm to 10 μm that were collected by high volume cascade impactors in four localities of the Czech Republic differing in the extent of the environmental pollution: (1) small village in proximity of a strip mine, (2) highway, (3) city center of Prague and (4) background station. The total DNA adduct levels induced by 100 μg/ml of EOMs were analyzed by ³²P-postlabelling analysis with a nuclease P1 method for adduct enrichment. The main finding of the study was most of the observed genotoxicity was connected with a fine particulate matter fraction (<1 μm). The concentrations of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) in EOMs indicate that fine fractions (0.5–1 μm) bound the highest amount of c-PAHs in all aerosol sampling sites, which might be related to the higher specific surface of this fraction as compared with a course fraction (1–10 μm) and higher mass as compared with a condensational fraction (0.17–0.5 μm). As for aerosol mass, both fine and condensational fractions are effective carriers of c-PAHs. Similarly, the DNA adduct levels per m³ of air were highest for the fine fraction, while the condensational fraction (strip mine site and city center) revealed the highest DNA adduct levels in cases where aerosol mass is taken into consideration. A strong correlation was found between the c-PAHs and DNA adduct levels induced by EOMs in all the localities and for various size fractions (R² = 0.98, p < 0.001). It may be concluded that the analysis of total DNA adducts induced in an acellular assay with/without metabolic activation represents a relatively simple method to assess the genotoxic potential of various complex mixtures.     

Crosstalk between activated forms of the aryl hydrocarbon receptor and glucocorticoid receptor

Pyrene, benzo[a]pyrene (BaP), and indeno[1,2,3-cd]pyrene (IND) are poly cyclic aromatic hydrocarbons (PAHs) with four to six annealed phenyl rings. Dexamethasone (Dex) is a synthetic agonist of glucocorticoids. The aryl hydrocarbon receptor (AhR) ligands, BaP and IND, did not directly activate the glucocorticoid receptor (GR), and Dex did not activate the AhR either. Whenever BaP and IND were added to Dex-treated cultures, they were present with Dex for longer periods, and higher enhancement of Dex-induced transactivation of the GR was found, which indicates that the freshly activated AhR is essential for synergistic interactions with the activated GR. The degree of enhancement of Dex-induced transactivation of the GR by PAHs, BaP ≈ IND > pyrene, paralleled the potency of PAHs in activating the AhR. This synergistic interaction was more distinct in ovarian granulosa cells (HO23) than in HepG2, 293T, or HeLa cells. In contrast, Dex suppressed AhR-mediated expressions, including AhR and cytochrome P450 (CYP) 1 A1 expressions. Dex also counteracted the BaP-induced decrease in cell viability. Crosstalk between the AhR and GR was independent of their expression levels. We concluded that the AhR functionally cross-reacts with the GR, through which transactivation activity of the GR is further enhanced, and in contrast, transactivation activity of the AhR is inhibited. This report shows the significance of in vitro endocrine-related results, which provide a clue for molecular studies of an interactive mechanism between the AhR and GR, and should be confirmed by future in vivo studies.     

Multidrug resistance protein (MRP) 4 attenuates benzo[a]pyrene-mediated DNA-adduct formation in human bronchoalveolar H358 cells

Multi-drug resistance protein (MRP) 4, an ATP-binding cassette (ABC) transporter, has broad substrate specificity. It facilitates the transport of bile salt conjugates, conjugated steroids, nucleoside analogs, eicosanoids, and cardiovascular drugs. Recent studies in liver carcinoma cells and hepatocytes showed that MRP4 expression is regulated by the aryl hydrocarbon receptor (AhR) and nuclear factor E2-related factor 2 (Nrf2). The AhR has particular importance in the lung and is most commonly associated with the up-regulation of cytochrome P-450 (CYP)-mediated metabolism of benzo[a]pyrene (B[a]P) to reactive intermediates. Treatment of H358, human bronchoalveolar, cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or (−)-benzo[a]pyrene-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol), the proximate carcinogen of B[a]P, revealed that MRP4 expression was increased compared to control. This suggested that MRP4 expression might contribute to the paradoxical decrease in (+)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-2′-deoxyguanosine ((+)-anti-trans-B[a]PDE-dGuo) DNA-adducts observed in TCDD-treated H358 cells. We have now found that decreased MRP4 expression induced by a short hairpin RNA (shRNA), or chemical inhibition with probenecid, increased (+)-anti-trans-B[a]PDE-dGuo formation in cells treated with (−)-B[a]P-7,8-dihydrodiol, but not the ultimate carcinogen (+)-anti-trans-B[a]PDE. Thus, up-regulation of MRP4 increased cellular efflux of (−)-B[a]P-7,8-dihydrodiol, which attenuated DNA-adduct formation. This is the first report identifying a specific MRP efflux transporter that decreases DNA damage arising from an environmental carcinogen.     

The role of human cytochrome P4503A4 in biotransformation of tissue-specific derivatives of 7H-dibenzo[c,g]carbazole

The environmental pollutant 7H-dibenzo[c,g]carbazole (DBC) and its derivative, 5,9-dimethylDBC (DiMeDBC), produced significant and dose-dependent levels of micronuclei followed by a substantial increase in the frequency of apoptotic cells in the V79MZh3A4 cell line stably expressing the human cytochrome P450 (hCYP) 3A4. In contrast, neither micronuclei nor apoptosis were found in cells exposed to the sarcomagenic carcinogen, N-methylDBC (N-MeDBC). A slight but significant level of gene mutations and DNA adducts detected in V79MZh3A4 cells treated with N-MeDBC, only at the highest concentration (30 μM), revealed that this sarcomagenic carcinogen was also metabolized by hCYP3A4. Surprisingly, DBC increased the frequency of 6-thioguanine resistant (6-TG^r) mutations only at the highest concentration (30 μM), while DiMeDBC failed to increase the frequency of these mutations. The resistance to 6-thioguanine is caused by the mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene. The molecular analysis of the coding region of Hprt gene showed a deletion of the entire exon 8 in DiMeDBC-induced 6-TG^r mutants, while no changes in the nucleotide sequences were identified in 6-TG^r mutants produced by DBC and N-MeDBC. Based on our results, we suggest that hCYP3A4 is involved in the metabolism of DBC and its tissue-specific derivatives. While hCYP3A4 probably plays an important role in biotransformation of the liver carcinogens, DBC and DiMeDBC, it might only have a marginal function in N-MeDBC metabolism.     

eNOS gene polymorphisms modify the association of PM10 with oxidative stress

Previous studies have suggested that air pollution increases various health outcomes through oxidative stress and oxidative stress-related genes modify the relationship between air pollution and health outcomes. Therefore, we evaluated the effect of PM₁₀ on the levels of malondialdehyde (MDA), oxidative stress biomarker, and the effect modification by genetic polymorphisms of eNOS, oxidative stress-related gene, in the 560 Korean elderly. We obtained urine samples repeatedly from participants during five medical examinations between 2008 and 2010 and all ambient air pollutant concentration data from the Korea National Institute of Environmental Research air quality monitoring system. We measured urinary levels of MDA to assess oxidative stress and genotyped eNOS (rs1799983, rs2853796, and rs7830). Mixed-effect model was used to estimate the effect of PM₁₀ on the level of oxidative stress biomarker and their modification by genotypes. PM₁₀ showed apparent positive effect on MDA level after adjusting for age, sex, BMI, cotinine level, temperature, dew point, levels of SO₂, O₃, NO₂, and CO, and season (p = 0.0133). Moreover, the association of PM₁₀ with MDA was found only in participants with eNOS GG genotype for rs1799983 (p = 0.0107), TT genotype for rs2853796 (p = 0.0289), or GT genotype for rs7830 (p = 0.0158) and in participants with a set of risky haplotypes (GTT, GTG, GGT, and TGT) (p = 0.0093). Our results suggest that PM₁₀ affect oxidative stress in the elderly and eNOS genotype affect the oxidative stress level in regard of exposure to PM₁₀.     

Comparison of DNA adduct induction in vitro by PAHs and nitro-pahs in freshly isolated rat hepatocytes

Polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs have been identified widely in occupational and environmental pollution, such as diesel engine emissions and other combustion products. In most cases, hepatic biotransformation is involved in converting these chemicals to their carcinogenic metabolites. It has been demonstrated that isolated hepatocytes possess substantial amounts of the enzymes responsible for metabolizing xenobiotics and are therefore a convenient model for studying chemicals that require activation to exert their carcinogenic effects. In this study, rat hepatocytes were isolated by collagenase digestion and then exposed to benzo[a]pyrene (B) [a]P), benzo[a]anthracene (B[a]A), 1-nitropyrene (1-NP) and 1,6-dinitropyrene (1,6-DNP) at different doses and/or times so that DNA adducts levels, as measured with the ³²P-postlabelling technique, could be compared. Each of the four compounds tested induced significant increases of total DNA adducts with clear dose-related responses. One or more individual adducts were identified as major adducts for each compound. Time-related increases of DNA adducts were also observed from 1 to 4 hr of incubation. Greater amounts of DNA adducts were induced by B[a]P or 1,6-DNP than by B[a]A or 1-NP, with potency being in the order 1,6-DNP > B[a]P > 1-NP B[a]A. These results demonstrate that freshly isolated hepatocytes can be used as an effective in vitro system for the detection of DNA adducts using ³²P-postlabelling, and have shown 1,6-DNP to be the most potent of the tested constituents of diesel emissions.     

Occupational exposure to low levels of benzene: Biomarkers of exposure and nucleic acid oxidation and their modulation by polymorphic xenobiotic metabolizing enzymes

This study investigated nucleic acid oxidation associated with exposure to benzene at low levels in 239 workers recruited among traffic policemen, taxi drivers and gasoline pump attendants of the city of Parma (Italy). Biomarkers of exposure, namely urinary t,t-muconic acid (t,t-MA) and S-phenylmercapturic acid (S-PMA), urinary cotinine, and urinary biomarkers of nucleic acid oxidation, namely 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo), 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 8-oxo-7,8-dihydroguanine (8-oxoGua) were determined by liquid chromatography–tandem mass spectrometry. Relevant polymorphisms of NAD(P)H:quinone oxidoreductase (NQO1), glutathione S-transferases M1-1 (GSTM1), T1-1 (GSTT1), and A1 (GSTA1) were characterized by polymerase chain reaction-based methods in a subgroup of subjects. Biomarkers of nucleic acid oxidation were correlated with each other (r ≥ 0.32, p < 0.0001) and with exposure biomarkers (r ≥ 0.28, p < 0.0001). Multiple linear regression models including age, sex and smoking habits as independent variables demonstrated that benzene exposure is associated with oxidation damage to nucleic acid, particularly to RNA (p < 0.0001) and is modulated by the NQO1 polymorphism. The study confirmed a significant modulating effect of GSTM1 (p = 0.010), GSTT1 (p = 0.023) and GSTA1 (p = 0.048) polymorphisms on S-PMA excretion, with a significant interaction between GSTM1 and both GSTT1 and GSTA1 (p = 0.006 and p = 0.037, respectively).     

Role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to organophosphate pesticides

Organophosphate pesticides (OPs) are primarily metabolized by several xenobiotic metabolizing enzymes (XMEs). Very few studies have explored genetic polymorphisms of XMEs and their association with DNA damage in pesticide-exposed workers. The present study was designed to determine the role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to OPs. We examined 284 subjects including 150 workers occupationally exposed to OPs and 134 normal healthy controls. The DNA damage was evaluated using the alkaline comet assay and genotyping was done using PCR-RFLP. The results revealed that the PONase activity toward paraoxonase and AChE activity was found significantly lowered in workers as compared to control subjects (p < 0.001). Workers showed significantly higher DNA damage compared to control subjects (14.37 ± 2.15 vs. 6.24 ± 1.37 tail% DNA, p < 0.001). Further, the workers with CYP2D6*3 PM and PON1 (QQ and MM) genotypes were found to have significantly higher DNA damage when compared to other genotypes (p < 0.05). In addition, significant increase in DNA damage was also observed in workers with concomitant presence of certain CYP2D6 and PON1 (Q192R and L55M) genotypes which need further extensive studies. In conclusion, the results indicate that the PON1 and CYP2D6 genotypes can modulate DNA damage elicited by some OPs possibly through gene-environment interactions.     

AHR2 knockdown prevents PAH-mediated cardiac toxicity and XRE- and ARE-associated gene induction in zebrafish (Danio rerio)

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants often present in aquatic systems as complex mixtures. Embryonic fish are sensitive to the developmental toxicity of some PAHs, but the exact mechanisms involved in this toxicity are still unknown. This study explored the role of the aryl hydrocarbon receptor (AHR) in the oxidative stress response of zebrafish to the embryotoxicity of select PAHs. Embryos were exposed to two PAHs, benzo[k]fluoranthene (BkF; a strong AHR agonist) and fluoranthene (FL; a cytochrome P4501A (CYP1A) inhibitor), alone and in combination. CYP1A, CYP1B1, CYP1C1, and redox-responsive genes glutathione s-transferase pi 2 (GSTp2), glutathione peroxidase 1 (GPx1), the glutamate-cysteine ligase catalytic subunit (GCLc), MnSOD and CuZnSOD mRNA expression was examined. CYP1 activity was measured via an in vivo ethoxyresorufin-O-deethlyase (EROD) activity assay, and the area of the pericardium was measured as an index of cardiotoxicity. BkF or FL alone caused no deformities whereas BkF + FL resulted in extreme pericardial effusion. BkF induced CYP activity above controls and co-exposure with FL inhibited this activity. BkF induced expression of all three CYPs, GSTp2, and GCLc. BkF + FL caused greater than additive induction of the three CYPs, GSTp2, GPx1, and GCLc but had no effect on MnSOD or CuZnSOD. AHR2 knockdown protected against the cardiac deformities caused by BkF + FL and significantly inhibited the induction of the CYPs, GSTp2, GPx1, and GCLc after BkF + FL compared to non-injected controls. These results further show the protective role of AHR2 knockdown against cardiotoxic PAHs and the role of AHR2 as a mediator of redox-responsive gene induction.     

Renin–angiotensin–aldosterone system related gene polymorphisms and urinary total arsenic is related to chronic kidney disease

A recent study demonstrated that an increased risk of chronic kidney disease (CKD) was associated with high urinary total arsenic levels. However, whether genomic instability is related to CKD remains unclear. An association between CKD and genetic polymorphisms of regulation enzymes of the renin–angiotensin–aldosterone system (RAAS), such as angiotensin-converting enzyme (ACE), angiotensinogen (AGT), angiotensin II type I receptor (AT1R), and aldosterone synthase (CYP11B2) has not been shown. The aim of the present study was to investigate the relationship between arsenic, genetic polymorphisms of RAAS enzymes and CKD. A total of 233 patients and 449 age- and gender-matched controls were recruited from the Taipei Medical University Hospital, Taipei Municipal Wan Fang Hospital and the Shin Kong Wu Ho-Su Memorial Hospital. Concentrations of urinary arsenic were determined by a high-performance liquid chromatography-linked hydride generator, and atomic absorption spectrometry. Polymorphisms of ACE(I/D), AGT(A[− 20]C), (T174M), (M235T), AT1R(A1166C) and CYP11B2(C[− 344]T) were examined by polymerase chain reaction and restriction fragment length polymorphism. Subjects carrying the CYP11B2 TT genotype had a higher odds ratio (OR), 1.39 (0.96–2.01), of CKD; while those with the AGT(A[− 20]C) CC genotype had an inverse OR of CKD (0.20 (0.05–0.81)), and a high-risk genotype was defined as A/A + A/C for AGT(A[− 20C]) and T/T for CYP11B2(C[− 344]T). The trend test showed a higher OR for CKD in patients who had either high urinary total arsenic levels or carried the high-risk genotype, or both, compared to patients with low urinary total arsenic levels, who carried the low-risk genotype, and could also be affected by the hypertension or diabetes status.     

Toxicogenomic outcomes predictive of forestomach carcinogenesis following exposure to benzo(a)pyrene: Relevance to human cancer risk

Forestomach tumors are observed in mice exposed to environmental carcinogens. However, the relevance of this data to humans is controversial because humans lack a forestomach. We hypothesize that an understanding of early molecular changes after exposure to a carcinogen in the forestomach will provide mode-of-action information to evaluate the applicability of forestomach cancers to human cancer risk assessment. In the present study we exposed mice to benzo(a)pyrene (BaP), an environmental carcinogen commonly associated with tumors of the rodent forestomach. Toxicogenomic tools were used to profile gene expression response in the forestomach. Adult Muta™Mouse males were orally exposed to 25, 50, and 75 mg BaP/kg-body-weight/day for 28 consecutive days. The forestomach was collected three days post-exposure. DNA microarrays, real-time RT-qPCR arrays, and protein analyses were employed to characterize responses in the forestomach. Microarray results showed altered expression of 414 genes across all treatment groups (± 1.5 fold; false discovery rate adjusted P ≤ 0.05). Significant downregulation of genes associated with phase II xenobiotic metabolism and increased expression of genes implicated in antigen processing and presentation, immune response, chemotaxis, and keratinocyte differentiation were observed in treated groups in a dose-dependent manner. A systematic comparison of the differentially expressed genes in the forestomach from the present study to differentially expressed genes identified in human diseases including human gastrointestinal tract cancers using the NextBio Human Disease Atlas showed significant commonalities between the two models. Our results provide molecular evidence supporting the use of the mouse forestomach model to evaluate chemically-induced gastrointestinal carcinogenesis in humans.     

Inhibition of carcinogen-bioactivating cytochrome P450 1 isoforms by amiloride derivatives

We examined the effects of amiloride derivatives, especially 5-(N-ethyl-N-isopropyl)amiloride (EIPA), on the activity of cytochrome P450 (CYP) 1 isoforms, known to metabolize carcinogenic polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (BP), into mutagenic metabolites and whose cellular expression can be induced through interaction of PAHs with the arylhydrocarbon receptor. EIPA was found to cause a potent and dose-dependent inhibition of CYP1-related ethoxyresorufine O-deethylase (EROD) activity in both liver cells and microsomes. It also markedly reduced activity of human recombinant CYP1A1 enzyme through a competitive mechanism; activities of other human CYP1 isoforms, i.e. CYP1A2 and CYP1B1, were also decreased. However, EIPA did not affect BP-mediated induction of CYP1A1 mRNA and protein levels in rat liver cells, likely indicating that EIPA does not block activation of the arylhydrocarbon receptor by PAHs. Inhibition of CYP1 activity by EIPA was associated with a decreased metabolism of BP, a reduced formation of BP-derived DNA adducts and a diminished BP-induced apoptosis in liver cells. The present data suggest that amiloride derivatives, such as EIPA, may be useful for preventing toxicity of chemical carcinogens, such as PAHs, through inhibition of CYP1 enzyme activity.     

In vitro inhibitory effects of asiaticoside and madecassoside on human cytochrome P450

The inhibitory effects and types of inhibition of asiaticoside and madecassoside on human CYPs were studied in vitro using recombinant human CYPs. The median inhibitory concentrations (IC₅₀) of asiaticoside and madecassoside were determined for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. Asiaticoside inhibited CYP2C19 (IC₅₀ = 412.68 ± 15.44 μM) and CYP3A4 (IC₅₀ = 343.35 ± 29.35 μM). Madecassoside also inhibited CYP2C19 (IC₅₀ = 539.04 ± 14.18 μM) and CYP3A4 (IC₅₀ = 453.32 ± 39.33 μM). Asiaticoside and madecassoside had no effect on the activities of CYP1A2, CYP2C9 and CYP2D6 and CYP2E1. Assessment of mechanism-based inhibition and the type of inhibition were performed for asiaticoside and madecassoside with CYP2C19 and CYP3A4. These results suggested that madecassoside is a mechanism-based inhibitor of CYP2C19 and CYP3A4. Assessment of mechanism-based inhibition by asiaticoside was limited by its low solubility. Asiaticoside exhibited non-competitive inhibition of CYP2C19 (K_i = 385.24 ± 8.75 μM) and CYP3A4 (K_i = 535.93 ± 18.99 μM). Madecassoside also showed non-competitive inhibition of CYP2C19 (K_i = 109.62 ± 6.14 μM) and CYP3A4 (K_i = 456.84 ± 16.43 μM). These results suggest that asiaticoside and madecassoside could cause drug-drug interactions via inhibition of CYP2C19 and CYP3A4. An in vivo study is needed to examine this further.