BaP-DNA Adducts Research Articles

DNA adducts as link between in vitro and in vivo carcinogenicity – A case study with benzo[a]pyrene

To reduce the need for animal tests, in vitro assays are often used as alternative methods. To derive toxic doses for higher tier organisms from in vitro assay results, quantitative in vitro-in vivo extrapolation (qIVIVE) based on physiological-based toxicokinetic (PBTK) models is typically the preferred approach. Such PBTK models require many input parameters to address the route from dose to target site concentration. However, respective data is very often not available. Hence, our aim is to call attention to an alternative way to build a link between animal (in vivo) and cell-derived (in vitro) toxicity data. To this end, we selected the carcinogenic chemical benzo[a]pyrene (BaP) for our study. Our approach relates both in vitro assay and in vivo data to a main intermediate marker structure for carcinogenicity on the subcellular level – the BaP-DNA adduct BaP-7,8-dihydrodiol-9,10-epoxide-deoxyguanosine. Thus, BaP dose is directly linked to a measure of the toxicity-initiating event. We used Syrian hamster embryo (SHE) and Balb/c 3T3 cell transformation assay as in vitro data and compared these data to outcomes of in vivo carcinogenicity tests in rodents. In vitro and in vivo DNA adduct levels range within three orders of magnitude. Especially metabolic saturation at higher doses and interspecies variabilities are identified and critically discussed as possible sources of errors in our simplified approach. Finally, our study points out possible routes to overcome limitations of the envisaged approach in order to allow for a reliable qIVIVE in the future.

Effects of Benzo[a]pyrene-DNA adducts, dietary vitamins, folate, and carotene intakes on preterm birth: a nested case–control study from the birth cohort in China

BackgroundPolycyclic aromatic hydrocarbons (PAHs) and its DNA adducts has been suggested to increase the risk of preterm birth (PB). Yet, few studies have been conducted to investigate this association, and the role of dietary nutrients intakes including vitamins, folate, and carotene during pre- and post-conception on this association has not been studied.MethodsBuilding upon a birth cohort in Taiyuan China, we conducted a nested case control study including 83 PB and 82 term births. Benzo[a]pyrene (BaP)-DNA adducts were measured by an improved LC-MC/MC analytic method. Dietary nutrient intakes were estimated from food frequency questionnaire using the Chinese Standard Tables of Food Consumption. Multivariable logistic regression model was used to examine the associations.ResultsIncreased risk of PB was observed as per interquartile increase in maternal BaP-DNA adduct level (OR = 1.27, 95%CI 0.95–1.67). Compared to low level (below mean) of maternal adducts, high level (above mean) of adducts was associated with the risk of PB (OR = 2.05, 95%CI 1.05–4.01). After stratified by dietary nutrients intakes, high adducts levels were associated with approximately 2–fourfold times increases in risk of PB among women with low vitamin A, C, E, folate, and carotene intakes during pre- and/or post-conception. Stronger stratified associations were consistently seen during preconception. Similar patterns were observed after additional adjustment for supplementation.ConclusionsOur study supports the hypothesis that high level of maternal PAHs exposure was significantly associated with increased risk of PB, and provides the first evidence that dietary vitamins, carotene, and folate intake levels may modify this association during different pregnancy windows. Our findings are relevant to identify recommendation for environment management and prenatal nutrition regarding pregnant women and newborns. Further investigation in other populations is warranted.

Oral benzo[a]pyrene administration attenuates dextran sulfate sodium-induced colitis in mice

Benzo[a]pyrene (BaP) is an environmental pollutant produced by combustion processes and is present in grilled foods as well as in tobacco smoke. BaP acts as an agonist for the aryl hydrocarbon receptor (AHR), and is metabolized by AHR-inducing enzymes. BaP metabolism can result in either detoxification or metabolic activation, the latter leads to an increased risk of disease, particularly lung cancer and cardiovascular disease, in a context-dependent manner. Although AHR activation has been thought to protect against inflammatory bowel disease, it remains unknown whether BaP exerts a protective or deleterious effect on colitis. In this study, we examined the effect of oral BaP administration on colitis induced by dextran sulfate sodium (DSS) in mice, an animal model of inflammatory bowel disease. BaP administration attenuated weight loss, shortening of the colon, disease activity index scores, and histological damage in DSS-induced colitis mice. BaP also suppressed colonic expression of inflammation-associated genes and plasma interleukin-6 secretion induced by DSS treatment. BaP-DNA adduct formation, a marker of BaP metabolic activation, was not enhanced in the colon after DSS treatment. Thus, oral BaP exerts an anti-inflammatory effect on DSS-induced colitis, without the toxicity associated with metabolic activation. The results provide insights into the disease-specific roles of BaP.

Benzo[a]pyrene-Induced Genotoxicity in Rats Is Affected by Co-Exposure to Sudan I by Altering the Expression of Biotransformation Enzymes.

The environmental pollutant benzo[a]pyrene (BaP) is a human carcinogen that reacts with DNA after metabolic activation catalysed by cytochromes P450 (CYP) 1A1 and 1B1 together with microsomal epoxide hydrolase. The azo dye Sudan I is a potent inducer of CYP1A1/2. Here, Wistar rats were either treated with single doses of BaP (150 mg/kg bw) or Sudan I (50 mg/kg bw) alone or with both compounds in combination to explore BaP-derived DNA adduct formation in vivo. Using 32P-postlabelling, DNA adducts generated by BaP-7,8-dihydrodiol-9,10-epoxide were found in livers of rats treated with BaP alone or co-exposed to Sudan I. During co-exposure to Sudan I prior to BaP treatment, BaP-DNA adduct levels increased 2.1-fold in comparison to BaP treatment alone. Similarly, hepatic microsomes isolated from rats exposed to Sudan I prior to BaP treatment were also the most effective in generating DNA adducts in vitro with the activated metabolites BaP-7,8-dihydrodiol or BaP-9-ol as intermediates. DNA adduct formation correlated with changes in the expression and/or enzyme activities of CYP1A1, 1A2 and 1B1 in hepatic microsomes. Thus, BaP genotoxicity in rats in vivo appears to be related to the enhanced expression and/or activity of hepatic CYP1A1/2 and 1B1 caused by exposure of rats to the studied compounds. Our results indicate that the industrially employed azo dye Sudan I potentiates the genotoxicity of the human carcinogen BaP, and exposure to both substances at the same time seems to be hazardous to humans.

Benzo(a)pyrene-induced cytotoxicity, cell proliferation, DNA damage, and altered gene expression profiles in HT-29 human colon cancer cells.

In the US alone, around 60,000 lives/year are lost to colon cancer. In order to study the mechanisms of colon carcinogenesis, in vitro model systems are required in addition to in vivo models. Towards this end, we have used the HT-29 colon cancer cells, cultured in Dulbecco's Modified Eagle Medium (DMEM), which were exposed to benzo(a)pyrene (BaP), a ubiquitous and prototypical environmental and dietary toxicant at 1, 10, 100nM and 1, 5, 10, and 25μM concentrations for 96h. Post-BaP exposure, growth, cytotoxicity, apoptosis, and cell cycle changes were determined. The BaP metabolite concentrations in colon cells were identified and measured. Furthermore, the BaP biotransformation enzymes were studied at the protein and mRNA levels. The BaP exposure-induced damage to DNA was assessed by measuring the oxidative damage to DNA and the concentrations of BaP-DNA adducts. To determine the whole repertoire of genes that are up- or downregulated by BaP exposure, mRNA transcriptome analysis was conducted. There was a BaP exposure concentration (dose)-dependent decrease in cell growth, cytotoxicity, and modulation of the cell cycle in the treatment groups compared to untreated or dimethylsulfoxide (DMSO: vehicle for BaP)-treated categories. The phase I biotransformation enzymes, CYP1A1 and 1B1, showed BaP concentration-dependent expression. On the other hand, phase II enzymes did not exhibit any marked variation. Consistent with the expression of phase I enzymes, elevated concentrations of BaP metabolites were generated, contributing to the formation of DNA lesions and stable DNA adducts, which were also BaP concentration-dependent. In summary, our studies established that biotransformation of BaP contributes to cytotoxicity, proliferation of tumor cells, and alteration of gene expression by BaP. • Benzo(a)pyrene (BaP) is an environmental and dietary toxicant. • BaP causes cytotoxicity in cultured HT-29 colon cancer cells. • mRNA transcriptome analyses revealed that BaP impacts cell growth, cell cycle, biotransformation, and DNA damage.

Enhanced DNA adduct formation by benzo[a]pyrene in human liver cells lacking cytochrome P450 oxidoreductase

Diet is a major source of human exposure to polycyclic aromatic hydrocarbons (PAHs), of which benzo[a]pyrene (BaP) is the most commonly studied and measured. BaP has been considered to exert its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes whose activity can be modulated by cytochrome P450 oxidoreductase (POR), the electron donor to CYP enzymes. Previous studies showed that BaP-DNA adduct formation was greater in the livers of Hepatic Reductase Null (HRN) mice, in which POR is deleted specifically in hepatocytes, than in wild-type (WT) mice. In the present study we used human hepatoma HepG2 cells carrying a knockout (KO) in the POR gene as a human in vitro model that can mimic the HRN mouse model. Treatment to BaP for up to 48 h caused similar cytotoxicity in POR KO and WT HepG2 cells. However, levels of BaP activation (i.e. BaP-7,8-dihydrodiol formation) were higher in POR KO HepG2 cells than in WT HepG2 cells after 48 h. This also resulted in substantially higher BaP-DNA adduct formation in POR KO HepG2 cells indicating that BaP metabolism is delayed in POR KO HepG2 cells thereby prolonging the effective exposure of cells to unmetabolized BaP. As was seen in the HRN mouse model, these results suggest that cytochrome b5, another component of the mixed-function oxidase system, which can also serve as electron donor to CYP enzymes along with NADH:cytochrome b5 redutase, contributes to the bioactivation of BaP in POR KO HepG2 cells. Collectively, these findings indicate that CYPs play a more important role in BaP detoxication as opposed to activation.

Effect of Benzo[a]pyrene-DNA Adduct in Cord Blood on the Neurodevelopment of 12-Month-Old Infants in Qingdao City.

IntroductionThe study was designed to explore the possible adverse effects of prenatal polycyclic aromatic hydrocarbons (PAHs) on the neurodevelopment of the infants at the age of 12 months in a birth cohort in Qingdao of China. Benzo[a]pyrene (BaP)-DNA adduct level in umbilical cord blood was measured by enzyme immunoassay.MethodsChild neurodevelopment was assessed at both 6 months and 12 months of age using the Gesell Development Inventory (GDI).ResultsThis study results reveal that multivariate linear analysis, cord BaP-DNA adduct level was inversely associated with developmental quotient score in the adaptive domain [β = −0.08; 95% CI: (−0.16, −0.003); p = 0.04], gross motor domain [β = −0.10; 95% CI: (−0.20, −0.01); p = 0.02], fine motor domain [β = −0.15; 95% CI: (−0.25, −0.05); p = 0.01], language domain [β = −0.12; 95% CI: (−0.21, −0.03); p = 0.02], and personal–social domain [β = −0.13; 95% CI: (−0.22, −0.04); p<0.01]. Further, multivariate logistic regression analysis showed increased cord BaP-DNA adduct levels associated with increased odds of delayed in language domain.ConclusionIn conclusion, the study suggested that prenatal PAH exposure monitored by umbilical cord blood BaP-DNA adducts may adversely affect the neurodevelopment of the infants at 12 months of age.

Deletion of cytochrome P450 oxidoreductase enhances metabolism and DNA adduct formation of benzo[a]pyrene in Hepa1c1c7 cells.

The environmental carcinogen benzo[a]pyrene (BaP) is presumed to exert its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes. However, studies using the Hepatic Reductase Null (HRN) mouse model, in which cytochrome P450 oxidoreductase (POR), the electron donor to CYP enzymes, is deleted specifically in hepatocytes, have shown that loss of hepatic POR-mediated CYP function leads to greater BaP-DNA adduct formation in livers of these mice than in wild-type (WT) mice. Here, we used CRISPR/Cas9 technology to knockout (KO) POR expression in mouse hepatoma Hepa1c1c7 cells to create an in vitro model that can mimic the HRN mouse model. Western blotting confirmed the deletion of POR in POR KO Hepa1c1c7 cells whereas expression of other components of the mixed-function oxidase system including cytochrome b5 (Cyb5) and NADH:cytochrome b5 reductase (which can also serve as electron donors to CYP enzymes), and CYP1A1 was similar in BaP-exposed WT and POR KO Hepa1c1c7 cells. BaP exposure caused cytotoxicity in WT Hepa1c1c7 cells but not in POR KO Hepa1c1c7 cells. In contrast, CYP-catalysed BaP-DNA adduct levels were ~10-fold higher in POR KO Hepa1c1c7 cells than in WT Hepa1c1c7 cells, in concordance with the presence of higher levels of BaP metabolite (e.g. BaP-7,8-dihydrodiol) in the medium of cultured BaP-exposed POR KO Hepa1c1c7 cells. As was seen in the HRN mouse model, these results suggest that Cyb5 contributes to the bioactivation of BaP in POR KO Hepa1c1c7 cells. These results indicate that CYP enzymes may play a more important role in the detoxication of BaP, as opposed to its bioactivation.

Alteration of benzo(a)pyrene biotransformation by resveratrol in ApcMin/+ mouse model of colon carcinogenesis.

Epidemiological surveys have revealed that environmental and dietary factors contribute to most of the human cancers. Our earlier studies have shown that resveratrol (RVT), a phytochemical reduced the tumor number, size and incidence of dysplasias induced by benzo(a)pyrene (BaP), an environmental toxicant in the ApcMin/+ mouse model of colon cancer. In this study we investigated to ascertain whether the preventive effects of RVT on BaP-induced colon carcinogenesis is a result of altered BaP biotransformation by RVT. For the first group of mice, 100μg BaP/kg bw was administered in peanut oil via oral gavage over a 60day period. For the second group, 45μg RVT/kg bw was co-administered with BaP. For the third group, RVT was administered for 1week prior to BaP exposure. Blood, colon and liver were collected from control and BaP/RVT-treated mice at 60days post-BaP & RVT exposure. We have assayed activities and expression (protein & mRNA) of drug metabolizing enzymes such as cytochrome P4501A1 (CYP1A1), CYP1B1, and glutathione-S-transferase (GST) in colon and liver samples from the treatment groups mentioned above. An increased expression of CYP1A1 in liver and colon and of CYP1B1 in liver of BaP-treated mice was seen, while RVT inhibited the extent of biotransformation mediated by these enzymes in the respective tissue samples. In the case of GST, an increased expression in colon of BaP alone-treated mice was noted when RVT was administered prior to BaP or simultaneously with BaP. However, there is no change in liver GST expression between BaP and RVT treatment groups. The concentrations of BaP aqueous (phase II) metabolites were found to be greater than the organic (phase I) metabolites, suggesting that RVT slows down the phase I metabolism (metabolic activation) of BaP, while enhancing phase II metabolism (detoxification). Additionally, the BaP-DNA adduct concentrations measured in colon and liver of BaP + RVT-treated mice were low relative to their BaP counterparts. Taken together, our findings strongly suggest that RVT alleviates BaP-induced colon carcinogenesis by impairing biotransformation pathways and DNA adduct formation, and therefore holds promise as a chemopreventive agent.

Exposure to endocrine disruptors 17alpha-ethinylestradiol and estradiol influences cytochrome P450 1A1-mediated genotoxicity of benzo[a]pyrene and expression of this enzyme in rats

Endocrine disruptors (EDs) are compounds that interfere with the balance of the endocrine system by mimicking or antagonising the effects of endogenous hormones, by altering the synthesis and metabolism of natural hormones, or by modifying hormone receptor levels. The synthetic estrogen 17α-ethinylestradiol (EE2) and the environmental carcinogen benzo[a]pyrene (BaP) are exogenous EDs whereas the estrogenic hormone 17β-estradiol is a natural endogenous ED. Although the biological effects of these individual EDs have partially been studied previously, their toxicity when acting in combination has not yet been investigated. Here we treated Wistar rats with BaP, EE2 and estradiol alone or in combination and studied the influence of EE2 and estradiol on: (i) the expression of cytochrome P450 (CYP) 1A1 and 1B1 in rat liver on the transcriptional and translational levels; (ii) the inducibility of these CYP enzymes by BaP in this rat organ; (iii) the formation of BaP-DNA adducts in rat liver in vivo; and (iv) the generation of BaP-induced DNA adducts after activation of BaP with hepatic microsomes of rats exposed to BaP, EE2 and estradiol and with recombinant rat CYP1A1 in vitro. BaP acted as a strong and moderate inducer of CYP1A1 and 1B1 in rat liver, respectively, whereas EE2 or estradiol alone had no effect on the expression of these enzymes. However, when EE2 was administered to rats together with BaP, it significantly decreased the potency of BaP to induce CYP1A1 and 1B1 gene expression. For EE2, but not estradiol, this also correlated with a reduction of BaP-induced CYP1A1 enzyme activity in rat hepatic microsomes. Further, while EE2 and estradiol did not form covalent adducts with DNA, they affected BaP-derived DNA adduct formations in vivo and in vitro. The observed decrease in BaP-DNA adduct levels in rat liver in vivo resulted from the inhibition of CYP1A1-mediated BaP bioactivation by EE2 and estradiol. Our results indicate that BaP genotoxicity mediated through its activation by CYP1A1 in rats in vivo is modulated by estradiol and its synthetic derivative EE2.

Combined treatment with benzo[a]pyrene and 1α,25-dihydroxyvitamin D3 induces expression of plasminogen activator inhibitor 1 in monocyte/macrophage-derived cells

Benzo[a]pyrene (BaP) is an environmental pollutant found in cigarette smoke and is implicated as a causative agent of tobacco-related diseases, such as arteriosclerosis. In contrast, vitamin D signaling, which is principally mediated by conversion of vitamin D to the active form, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], decreases cardiovascular disease risk. However, combined treatment with BaP and 1,25(OH)2D3 enhances BaP toxicity, including BaP-DNA adduct formation. We further investigated the cross-talk between BaP and 1,25(OH)2D3 signaling pathways, and found that combined treatment with these compounds induces mRNA and protein expression of plasminogen activator inhibitor 1 (PAI-1) in monocyte/macrophage-derived THP-1 and U937 cells. Protein synthesis inhibitor treatment did not inhibit induction of the PAI-1 gene (SERPINE1) in these cells. BaP plus 1,25(OH)2D3 induced differentiation markers, inhibited cellular proliferation, and induced apoptosis and oxidative stress in these cells. Reactive oxygen species scavenger treatment suppressed apoptosis but not SERPINE1 induction in cells treated with BaP plus 1,25(OH)2D3. Thus, combined treatment with BaP and 1,25(OH)2D3 induced SERPINE1 mRNA expression in these cells through a mechanism that does not require de novo protein synthesis or reactive oxygen species production. These findings suggest that induction of the proinflammatory factor PAI-1 plays a role in BaP toxicity. Interestingly, PAI-1 knockdown decreased expression of the cell surface antigen CD14, a monocytic differentiation marker, in THP-1 cells treated with BaP plus 1,25(OH)2D3. PAI-1 induction may also be related to a function of monocytes/macrophages in response to xenobiotic and vitamin D signaling.

Polymethoxyflavones prevent benzo[a]pyrene/dextran sodium sulfate-induced colorectal carcinogenesis through modulating xenobiotic metabolism and ameliorate autophagic defect in ICR mice.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental carcinogenic pollutants and they have become an important issue in food contamination. Dietary intake of PAHs has been recognized as a major route of human exposure. However, the mechanisms behind dietary PAH-induced colorectal cancer (CRC) remain unclear. Several studies have shown that polymethoxyflavones (PMFs) are effective in preventing carcinogen-induced CRC or colitis. In this study, we investigated the preventive effect of PMFs on benzo[a]pyrene/dextran sulfate sodium (BaP/DSS)-induced colorectal tumorigenesis in ICR mice. We found that PMFs significantly prevented BaP/DSS-induced colorectal tumor formation. BaP mutagenic metabolite and DNA adducts were found to be reduced in colonic tissue in the PMFs-treated groups through the modulation of BaP metabolism. At the molecular level, the results of RNA-sequencing indicated that PMFs ameliorated BaP/DSS-induced abnormal molecular mechanism change including activated inflammation, downregulated anti-oxidation targets, and induced metastasis genes. The autophagic defect caused by BaP/DSS-induced tumorigenesis was improved by pretreatment with PMFs. We found BaP/DSS-induced CRC may be a Wnt/β-catenin independent process. Additionally, consumption of PMFs extracts also altered the composition of gut microbiota and made it similar to that in the control group by increasing butyrate-producing probiotics and decreasing CRC-related bacteria. BaP in combination with DSS significantly induced colorectal tumorigenesis through induced DNA adduct formation, abnormal gene expression, and imbalanced gut microbiota composition. PMFs were a powerful preventive agent that suppressed BaP/DSS-induced CRC via modulating multiple pathways as well as ameliorating autophagic defect. These results demonstrated for the first time the chemopreventive efficacy and comprehensive mechanisms of dietary PMFs for preventing BaP/DSS-induced colorectal carcinogenesis.

B(a)P adduct levels and fertility: A cross‑sectional study in a Sicilian population.

Benzo(a)pyrene (BaP) is a carcinogenic polycyclic aromatic hydrocarbon for human tissues. Still today it is not fully investigated if BaP can affect negatively the male fertility through the BaP-DNA adducts production. In the present study, BaP Tetrol I-1 (TI-1) and BaP Tetrol II-2 (TII-2) BaP-DNA adducts were investigated in spermatozoa of a Sicilian male population. Semen samples from 86 volunteers in two eastern Sicilian cities (Regalbuto and Melilli) were collected. The quality of semen was evaluated in all samples according to the World Health Organization (WHO) guidelines. We analyzed BaP-DNA adducts in extracted sperm cell DNA using the modified high-performance liquid chromatography-fluorescence method to detects both Tetrols. Differences between Tetrol levels were assessed by the Wilcoxon signed-rank test and the Mann-Whitney U test, as appropriate. Correlation between semen quality parameters and Tetrol concentrations were analyzed using the Spearman's correlation coefficient. Σ(TI-1+TII-2) were significantly higher in spermatozoa of volunteers from Regalbuto. Furthermore, a greater dispersion of the levels of adducts was observed in these specimens. TI-1 adducts were higher than TII-2 in Melilli samples (95% CI) and TII-2 were higher than TI-1 in Regalbuto semen samples (95% CI). A significant inverse correlation between sperm progressive motility and both TI-1 and TII-2 adducts was observed. The present study showed that BaP negatively affects male fertility by TI-1 and TII-2 DNA-adduct production. These results suggest that DNA adducts could be used as biomarker to assess BaP exposure by air pollution. Further studies are needed to confirm if these findings could affect male fertility because of the growing impairment of this function observed in recent years.

Environmental Pollutants and Neurodevelopment: Review of Benefits From Closure of a Coal-Burning Power Plant in Tongliang, China.

Background. Understanding preventable causes of neurodevelopmental disorders is a public health priority. Polycyclic aromatic hydrocarbons (PAH) from combustion of fossil fuel, lead, and mercury are among known neurodevelopmental toxicants. Method. For the first time, we comprehensively review the findings from a study by the Columbia Center for Children’s Environmental Health and Chinese partners that followed 2 groups of mother-child pairs, one from 2002 and another from 2005, in Tongliang County, China. Pregnant mothers in the 2 cohorts experienced different exposure to PAH because a local coal-burning power plant was shut down in 2004. Investigators assessed change in prenatal PAH exposure, measured using a biomarker (benzo[a]pyrene [BaP]-DNA adducts in cord blood). Developmental quotients were measured using the Gesell Developmental Scales at age 2 and IQ was assessed using the Wechsler Intelligence Scale for Children at age 5. Biologic markers of preclinical response were measured in cord blood: methylation status of long interspersed nuclear elements (LINE1), an indicator of genomic stability, and brain-derived neurotrophic factor (BDNF), a neuronal growth promoter. Analyses accounted for co-exposure to lead and mercury. Results. BaP-DNA adducts were significantly inversely associated with Gesell Developmental Scales scores in the first cohort but not in the second cohort; and levels of BDNF and LINE1 methylation were higher in the second cohort. Conclusion. In this study, reduced exposure to PAH was associated with beneficial effects on neurodevelopment as well as molecular changes related to improved brain development and health. These benefits should encourage further efforts to limit exposure to these toxic pollutants.

NADH:Cytochrome b5 Reductase and Cytochrome b5 Can Act as Sole Electron Donors to Human Cytochrome P450 1A1-Mediated Oxidation and DNA Adduct Formation by Benzo[a]pyrene.

Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after activation by cytochrome P450 (P450). Here, we investigated whether NADH:cytochrome b5 reductase (CBR) in the presence of cytochrome b5 can act as sole electron donor to human P450 1A1 during BaP oxidation and replace the canonical NADPH:cytochrome P450 reductase (POR) system. We also studied the efficiencies of the coenzymes of these reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of CBR, to mediate BaP oxidation. Two systems containing human P450 1A1 were utilized: human recombinant P450 1A1 expressed with POR, CBR, epoxide hydrolase, and cytochrome b5 in Supersomes and human recombinant P450 1A1 reconstituted with POR and/or with CBR and cytochrome b5 in liposomes. BaP-9,10-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol, BaP-3-ol, a metabolite of unknown structure, and two BaP-DNA adducts were generated by the P450 1A1-Supersomes system, both in the presence of NADPH and in the presence of NADH. The major BaP-DNA adduct detected by 32P-postlabeling was characterized as 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP (assigned adduct 1), while the minor adduct is probably a guanine adduct derived from 9-hydroxy-BaP-4,5-epoxide (assigned adduct 2). BaP-3-ol as the major metabolite, BaP-9-ol, BaP-1,6-dione, BaP-3,6-dione, an unknown metabolite, and adduct 2 were observed in the system using P450 1A1 reconstituted with POR plus NADPH. When P450 1A1 was reconstituted with CBR and cytochrome b5 plus NADH, BaP-3-ol was the predominant metabolite too, and an adduct 2 was also generated. Our results demonstrate that the NADH/cytochrome b5/CBR system can act as the sole electron donor both for the first and second reduction of P450 1A1 during the oxidation of BaP in vitro. They suggest that NADH-dependent CBR can replace NADPH-dependent POR in the P450 1A1-catalyzed metabolism of BaP.

NADPH- and NADH-dependent metabolism of and DNA adduct formation by benzo[a]pyrene catalyzed with rat hepatic microsomes and cytochrome P450 1A1.

Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after metabolic activation by cytochrome P450 (CYP) enzymes. Here we investigated the efficiencies of rat hepatic microsomes and rat recombinant CYP1A1 expressed with its reductase, NADPH:CYP oxidoreductase (POR), NADH:cytochrome b5 reductase, epoxide hydrolase and/or cytochrome b5 in Supersomes™ to metabolize this carcinogen. We also studied the effectiveness of coenzymes of two of the microsomal reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of NADH:cytochrome b5 reductase, to mediate BaP metabolism in these systems. Up to eight BaP metabolites and two DNA adducts were generated by the systems, both in the presence of NADPH and NADH. Among BaP metabolites, BaP-9,10-dihydrodiol, BaP-4,5-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol, BaP-3-ol, and a metabolite of unknown structure were formed by hepatic microsomes and rat CYP1A1. One of two DNA adducts formed by examined enzymatic systems (rat hepatic microsomes and rat CYP1A1) was characterized to be 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-N2-BPDE), while another adduct has similar chromatographic properties on polyethylaneimine–cellulose thin layer chromatography to a guanine adduct derived from reaction with 9-hydroxy-BaP-4,5-oxide. In the presence of either of the reductase cofactors tested, NADPH or NADH, cytochrome b5 stimulated CYP1A1-mediated formation of both BaP-DNA adducts. The results demonstrate that NADH can act as a sole electron donor for both the first and the second reduction of CYP1A1 during its reaction cycle catalyzing oxidation of BaP, and suggest that the NADH:cytochrome b5 reductase as the NADH-dependent reductase might substitute POR in this enzymatic system.Graphical abstract

The impact of p53 on DNA damage and metabolic activation of the environmental carcinogen benzo[a]pyrene: effects in Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice.

The tumour suppressor p53 is one of the most important cancer genes. Previous findings have shown that p53 expression can influence DNA adduct formation of the environmental carcinogen benzo[a]pyrene (BaP) in human cells, indicating a role for p53 in the cytochrome P450 (CYP) 1A1-mediated biotransformation of BaP in vitro. We investigated the potential role of p53 in xenobiotic metabolism in vivo by treating Trp53(+/+), Trp53(+/–) and Trp53(−/−) mice with BaP. BaP-DNA adduct levels, as measured by 32P-postlabelling analysis, were significantly higher in liver and kidney of Trp53(−/−) mice than of Trp53(+/+) mice. Complementarily, significantly higher amounts of BaP metabolites were also formed ex vivo in hepatic microsomes from BaP-pretreated Trp53(−/−) mice. Bypass of the need for metabolic activation by treating mice with BaP-7,8-dihydrodiol-9,10-epoxide resulted in similar adduct levels in liver and kidney in all mouse lines, confirming that the influence of p53 is on the biotransformation of the parent compound. Higher BaP-DNA adduct levels in the livers of Trp53(−/−) mice correlated with higher CYP1A protein levels and increased CYP1A enzyme activity in these animals. Our study demonstrates a role for p53 in the metabolism of BaP in vivo, confirming previous in vitro results on a novel role for p53 in CYP1A1-mediated BaP metabolism. However, our results also suggest that the mechanisms involved in the altered expression and activity of the CYP1A1 enzyme by p53 in vitro and in vivo are different.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-015-1531-8) contains supplementary material, which is available to authorized users.

Pulmonary Inflammation Impacts on CYP1A1-Mediated Respiratory Tract DNA Damage Induced by the Carcinogenic Air Pollutant Benzo[a]pyrene.

Pulmonary inflammation can contribute to the development of lung cancer in humans. We investigated whether pulmonary inflammation alters the genotoxicity of polycyclic aromatic hydrocarbons (PAHs) in the lungs of mice and what mechanisms are involved. To model nonallergic acute inflammation, mice were exposed intranasally to lipopolysaccharide (LPS; 20 µg/mouse) and then instilled intratracheally with benzo[a]pyrene (BaP; 0.5 mg/mouse). BaP-DNA adduct levels, measured by 32P-postlabeling analysis, were approximately 3-fold higher in the lungs of LPS/BaP-treated mice than in mice treated with BaP alone. Pulmonary Cyp1a1 enzyme activity was decreased in LPS/BaP-treated mice relative to BaP-treated mice suggesting that pulmonary inflammation impacted on BaP-induced Cyp1a1 activity in the lung. Our results showed that Cyp1a1 appears to be important for BaP detoxification in vivo and that the decrease of pulmonary Cyp1a1 activity in LPS/BaP-treated mice results in a decrease of pulmonary BaP detoxification, thereby enhancing BaP genotoxicity (ie, DNA adduct formation) in the lung. Because less BaP was detoxified by Cyp1a1 in the lungs of LPS/BaP-treated mice, more BaP circulated via the blood to extrapulmonary tissues relative to mice treated with BaP only. Indeed, we observed higher BaP-DNA adduct levels in livers of LPS/BaP-treated mice compared with BaP-treated mice. Our results indicate that pulmonary inflammation could be a critical determinant in the induction of genotoxicity in the lung by PAHs like BaP. Cyp1a1 appears to be involved in both BaP bioactivation and detoxification although the contribution of other enzymes to BaP-DNA adduct formation in lung and liver under inflammatory conditions remains to be explored.

Cytochrome b5 and epoxide hydrolase contribute to benzo[a]pyrene-DNA adduct formation catalyzed by cytochrome P450 1A1 under low NADPH:P450 oxidoreductase conditions

In previous studies we had administered benzo[a]pyrene (BaP) to genetically engineered mice (HRN) which do not express NADPH:cytochrome P450 oxidoreductase (POR) in hepatocytes and observed higher DNA adduct levels in livers of these mice than in wild-type mice. To elucidate the reason for this unexpected finding we have used two different settings for in vitro incubations; hepatic microsomes from control and BaP-pretreated HRN mice and reconstituted systems with cytochrome P450 1A1 (CYP1A1), POR, cytochrome b5, and epoxide hydrolase (mEH) in different ratios. In microsomes from BaP-pretreated mice, in which Cyp1a1 was induced, higher levels of BaP metabolites were formed, mainly of BaP-7,8-dihydrodiol. At a low POR:CYP1A1 ratio of 0.05:1 in the reconstituted system, the amounts of BaP diones and BaP-9-ol formed were essentially the same as at an equimolar ratio, but formation of BaP-3-ol was ∼1.6-fold higher. Only after addition of mEH were BaP dihydrodiols found. Two BaP-DNA adducts were formed in the presence of mEH, but only one when CYP1A1 and POR were present alone. At a ratio of POR:CYP1A1 of 0.05:1, addition of cytochrome b5 increased CYP1A1-mediated BaP oxidation to most of its metabolites indicating that cytochrome b5 participates in the electron transfer from NADPH to CYP1A1 required for enzyme activity of this CYP. BaP-9-ol was formed even by CYP1A1 reconstituted with cytochrome b5 without POR. Our results suggest that in livers of HRN mice Cyp1a1, cytochrome b5 and mEH can effectively activate BaP to DNA binding species, even in the presence of very low amounts of POR.

The correlations between BRCA1 defect and environmental factors in the risk of breast cancer

The risk factors for breast cancer, the most common female malignant cancer, include environmental factors such as radiation, tobacco, a high-fat diet, and xenoestrogens as well as hormones. In addition, BRCA1 and BRCA2 are the most well-known genetic factors that increase risk for breast cancer. Coincidence of those environmental and genetic factors might augment the risk of tumorigenesis of breast. To verify this hypothesis, we briefly evaluated the carcinogenic potency of various environmental factors in the absence or presence of BRCA1 as a genetic factor in a normal mammary epithelial cell line, MCF10A. Many environmental factors tested increased cellular ROS level in the absence of other insult. In addition, TCDD, DMBA, 3MC, and BPA enhanced the BaP-induced ROS production. BRCA1 knockdown (BRCA1-KD) cells by siRNA significantly induced cellular accumulation of ROS compared to control cells. In this setting, the addition of paraquat, TCDD, DMBA, 2OHE2 or 4OHE2 significantly augmented ROS generation in BRCA1-KD MCF10A cells. Measurements of BaP-DNA adduct formation as a marker of DNA damage also revealed that BRCA1 deficiency leads increased DNA damage. In addition, TCDD and DMBA significantly increased BaP-DNA adduct formation in the absence of BRCA1. These results imply that elevated level of ROS is correlated with increase of DNA damage in BRCA1 defective cells. Taken together, our study suggests that several environmental factors might increase the risk of tumorigenesis in BRCA1 defective breast epithelial cells.