BaP-DNA Adducts Research Articles

Vitamin D Receptor Activation Enhances Benzo[a]pyrene Metabolism via CYP1A1 Expression in Macrophages

Benzo[a]pyrene (BaP) activates the aryl hydrocarbon (AHR) and induces the expression of genes involved in xenobiotic metabolism, including CYP1A1. CYP1A1 is involved not only in BaP detoxification but also in metabolic activation, which results in DNA adduct formation. Vitamin D receptor (VDR) belongs to the NR1I subfamily of the nuclear receptor superfamily, which also regulates expression of xenobiotic metabolism genes. We investigated the cross-talk between AHR and VDR signaling pathways and found that 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], a potent physiological VDR agonist, enhanced BaP-induced transcription of CYP1A1 in human monocytic U937 cells and THP-1 cells, breast cancer cells, and kidney epithelium-derived cells. 1,25(OH)(2)D(3) alone did not induce CYP1A1, and 1,25(OH)(2)D(3) plus BaP did not increase CYP1A2 or CYP1B1 mRNA expression in U937 cells. The combination of 1,25(OH)(2)D(3) and BaP increased CYP1A1 protein levels, BaP hydroxylation activity, and BaP-DNA adduct formation in U937 cells and THP-1 cells more effectively than BaP alone. The combined effect of 1,25(OH)(2)D(3) and BaP on CYP1A1 mRNA expression in U937 cells and/or THP-1 cells was inhibited by VDR knockdown, VDR antagonists, and α-naphthoflavone, an AHR antagonist. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that VDR directly bound to an everted repeat (ER) 8 motif in the human CYP1A1 promoter. Thus, CYP1A1 is a novel VDR target gene involved in xenobiotic metabolism. Induction of CYP1A1 by the activation of VDR and AHR may contribute to BaP-mediated toxicity and the physiological function of this enzyme.

Exposure to benzo[a]pyrene of Hepatic Cytochrome P450 Reductase Null (HRN) and P450 Reductase Conditional Null (RCN) mice: Detection of benzo[a]pyrene diol epoxide-DNA adducts by immunohistochemistry and 32P-postlabelling

Benzo[a]pyrene (BaP) is a widespread environmental carcinogen activated by cytochrome P450 (P450) enzymes. In Hepatic P450 Reductase Null (HRN) and Reductase Conditional Null (RCN) mice, P450 oxidoreductase (Por) is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic P450 function. Treatment of HRN mice with a single i.p. or oral dose of BaP (12.5 or 125mg/kgbody weight) resulted in higher DNA adduct levels in liver (up to 10-fold) than in wild-type (WT) mice, indicating that hepatic P450s appear to be more important for BaP detoxification in vivo. Similar results were obtained in RCN mice. We tested whether differences between hepatocytes and non-hepatocytes in P450 activity may underlie the increased liver BaP-DNA binding in HRN mice. Cellular localisation by immunohistochemistry of BaP-DNA adducts showed that HRN mice have ample capacity for formation of BaP-DNA adducts in liver, indicating that the metabolic process does not result in the generation of a reactive species different from that formed in WT mice. However, increased protein expression of cytochrome b5 in hepatic microsomes of HRN relative to WT mice suggests that cytochrome b5 may modulate the P450-mediated bioactivation of BaP in HRN mice, partially substituting the function of Por.

Abstract 2643: Interactions between polycyclic aromatic hydrocarbon exposure and genetic polymorphisms on benzo(a)pyrene-DNA adducts in a Polish cohort of mothers and newborns

Abstract Background Polycyclic aromatic hydrocarbons (PAHs) are a class of chemicals common in the environment. Major sources of PAHs include fossil fuel combustion and cigarette smoking. Certain PAHs are carcinogenic, though the degree to which genetic variation influences susceptibility to PAH exposure remains unclear. Benzo(a)pyrene (BaP) is a well-studied PAH that is classified as a probable human carcinogen. Here, we evaluated single nucleotide polymorphism (SNP) frequency in genes related to PAH metabolism and detoxification, and explored the interactions between PAH exposure and SNPs on BaP-DNA adducts, an established cancer risk marker, in umbilical cord white blood cells (WBCs). Methods Maternal and umbilical cord blood were collected at delivery from participants in a longitudinal cohort study of Polish mothers and newborns in Krakow, Poland. WBC DNA was isolated from cord blood, and genetic polymorphisms and BaP-DNA cord adducts were then measured in 503 mothers and 445 newborns. Maternal PAH exposure was measured during pregnancy with a personal air exposure monitor. For this study 7 maternal and newborn genes related to PAH metabolism, detoxification and repair were selected for SNP analyses: CYP1A1, CYP1A2, CYP1B1, GSTM3, GSTT2, NQO1 and XRCC1. SNP x PAH interactions on logarithmic (ln)-transformed BaP-DNA adduct levels were explored. Results We observed a number of significant interactions between high PAH exposure and SNPs in both mothers and newborns on BaP-DNA adducts in cord blood. For example, the interaction between a CYP1A2 SNP (rs2069514) and high PAH exposure on cord BaP-DNA adducts in newborns was significant (α=−1.34, p<0.01). We will present significant interaction results from analyses performed on both maternal and newborn CYP1A1, CYP1A2, GSTM3, GSTT2, NQO1 and XRCC1 SNPs. Discussion It is biologically plausible that an individual's abilities to metabolically activate and detoxify BaP and repair DNA damage play a role in susceptibility to DNA adduct formation following prenatal PAH exposure. Ultimately, this susceptibility could correlate with future risk of cancer development. Our findings of several SNP x PAH exposure interactions on cord BaP-DNA adducts support the hypothesis that genetic variability can contribute to susceptibility and potential cancer risk from prenatal PAH exposures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2643. doi:1538-7445.AM2012-2643

Acute Treatment with Kerosene Damages the Dermal Barrier and Alters the Distribution of Topically Applied Benzo(a)pyrene in Mice

The dermal route is important in many occupational exposures. Some materials may reduce the barrier function of the skin to enhance absorption and effect on internal organs. We have reported previously that kerosene cleaning following treatment with used engine oil increased DNA adduct levels in the lungs of mice compared with animals treated with used oil alone. To investigate what other physiological parameters might be affected by kerosene, we conducted in vitro and in vivo measurements of skin barrier function. We also topically applied 3H-BAP(100 nM in 25 μL acetone) and washed half the mice with 25 μL kerosene 1 hr after carcinogen application. Groups of four mice were euthanized from 1 to 72 hr after treatment. Skin, lungs, and livers were harvested from each animal and stored separately. Kerosene application reduced the barrier function of the skin in vitro beyond the effect of the acetone vehicle and the vehicle plus BAP. In vivo studies indicated that kerosene treatment reduced the barrier function at 4 and 8 hr post application and that the barrier function recovered at 24 hr after a single treatment. The fraction of the radiolabel remaining in the skin of animals treated with 3H-BAP and washed with kerosene was significantly less than those not washed, beginning at 24 hr (p< 0.05). Fractional distribution to the lungs and livers of these animals became significantly elevated at this time. Kerosene treatment compromises dermal barrier function and the ability of the skin to retain water, enhances carcinogen absorption, and alters organ distribution. This appears to contribute to the increase in BAP DNA adducts we reported earlier.

Relationship between villus BaP-DNA adducts level and blighted ovum in early pregnancy

Objective To investigate relations between villus Benzo (a)pyrene (BaP)-DNA adducts and blighted ovum in early pregnancy, and to explore possible environmental factors influencing embryo development. Methods One hundred and two pregnant women with blighted ovum were selected into this study as research group; and 102 normal pregnant women were taken as control group; the age, gravidity, parity and gestational weeks of the two groups were matched. After artificial abortion, villi of the patients were collected and washed by normal saline. Then, the tissue was homogenated and genome DNA was extracted to detect quantity of the tissue. BaP-DNA adducts levels were examined by high-performance liquid chromatography-fluorescence method (HPLC). The personal information of pregnant women was collected by questionnaire. Logistic regression model was used to investigate the association between BaP-DNA adducts and blighted ovum. Results BaP-DNA adducts level in villi of research group [(8. 9±8. 2) adducts/108 nucleotides] was significantly higher than that of control group [(2. 0±1. 4) adducts/108 nucleotides], P＜0.05. The higher the BaP-DNA adducts in villi, the higher risk the blighted ovum; when the BaP-DNA adducts level increased to 6.06 adducts/108 nucleotides, the risk of blighted ovum might increase 59.39 times (95% CI:15.50-227.55). Maternal education level was a protective factor (OR=-0.21, 95%CI:-0. 19--0.03) after controlling potential confounders. Conclusions High level of villi BaP-DNA adducts might increase the risk of blighted ovum in early pregnancy, and could have an adverse effect on embryo development. Key words: Embryonic development; Pregnancy trimester, first; Chorionic villi; Benzo(a)pyrene; DNA Adducts; Chromatography, high pressure liquid

Abstract 2753: Interactions between PAH exposure and genetic polymorphisms on BaP-DNA adducts in a NYC cohort of African American and Dominican mothers and newborns

Abstract Background: Polycyclic aromatic hydrocarbons (PAHs) are a class of chemicals common in the environment. Major sources of PAHs include fossil fuel combustion and cigarette smoking. Certain PAHs are carcinogenic, though the degree to which genetic variation influences susceptibility to PAH exposure remains unclear. Benzo(a)pyrene (BaP) is a well-studied PAH that is classified as a probable human carcinogen. Here, we evaluated haplotype frequency in genes related to PAH metabolism and detoxification, and explored the interactions between PAH exposure and haplotype on BaP-DNA adducts, an established cancer risk marker, in umbilical cord white blood cells (WBCs). Methods: Maternal and umbilical cord blood were collected at delivery from participants in a longitudinal cohort study of African American (AA) and Dominican (D) mothers and newborns in New York City. WBC DNA was isolated from cord blood, and genetic polymorphisms and BaP-DNA cord adducts were then measured in 516 mothers and 323 newborns. Maternal PAH exposure was measured during pregnancy with a personal air exposure monitor. A total of 18 genes, selected for biologic relevance, were analyzed for SNPs. For this study, a subset of 7 maternal and newborn genes related to PAH metabolism, detoxification and repair were selected for haplotype analyses: CYP1A1, CYP1A2, CYP1B1, GSTM, GSTT, NQO1 and XRCC. Haplotype × PAH interactions on logarithmic-transformed BaP-DNA adduct levels were explored. Results: Notable differences in haplotype frequency were observed between AAs and Ds and between mothers and newborns. We observed a number of significant interactions between high PAH exposure and polymorphic haplotypes in both mothers and newborns on BaP-DNA adducts in cord blood. To give one example, the haplotype CACGCG of CYP1B1 in AA mothers had a frequency of 10.4%, versus 0.3% in D mothers (p&amp;lt;0.0001). By comparison, the reference haplotype TAGCTG had a frequency of 19.9%. We observed a significant interaction between the CYP1B1 haplotype and high PAH exposure on cord BaP-DNA adducts in AA newborns (β=−0.67, p=0.01) but not Ds (β=−0.13, p=0.72). We will present results for both maternal and newborn CYP1A1, CYP1A2, GSTM, GSTT, NQO1 and XRCC haplotypes in both AAs and Ds. Discussion: It is biologically plausible that an individual's ability to metabolically activate and detoxify BaP and repair DNA damage plays a role in susceptibility to DNA adduct formation following prenatal PAH exposure. Ultimately, this susceptibility could correlate with future risk of cancer development. We have identified haplotype patterns that differ between ethnic groups and also between mothers and newborns. We also observed several haplotype × PAH exposure interactions on cord BaP-DNA adducts, supporting the hypothesis that genetic variability can contribute to susceptibility and potential cancer risk from prenatal PAH exposures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2753. doi:10.1158/1538-7445.AM2011-2753

A novel in vitro pancreatic carcinogenesis model

Environmental factors (e.g., BaP) have been pointed out as one of the etiologies of pancreatic cancer. However, very limited experimental assays are available to identify pancreatic specific environmental mutagens or susceptibility genes. In this study, we have developed a simple in vitro cell culture model system that can be used to study the molecular and biochemical aspects of carcinogenesis in a near-normal immortalized pancreatic ductal epithelial cell lines. In order to demonstrate that xenobiotic stress response is intact in these cells, we employed standard molecular biology techniques. For examples, luciferase reporter and/or real-time quantitative PCR assays were used to determine stress-induced CYP1A1 and CYP1B1 gene expression. Western blotting and immunocytochemistry assays were used to demonstrate that TCDD or BaP could activate AhR signaling. For exploring the carcinogenesis mechanism, we incubated cells with [3H]BaP and determined BaP–DNA binding activity by measuring its radioactivity. BaP–DNA adduct formation was further confirmed by [32P]-postlabeling assay. Finally, we demonstrated the effects of endogenous AhR or BRCA1 in BaP–DNA adduct accumulation in our cell system. As results, no apparent BaP–DNA adduct accumulation by [32P]-postlabeling assay was found in either control-siRNA or AhR-siRNA pretreated cells. On the other hand, a significant increase of BaP–DNA adduct accumulation was found in BRCA1 knockdown cells. In conclusion, we suggest that this in vitro model may provide the feasibility for future studies on the molecular basis of pancreatic ductal cell carcinogenesis caused by dietary mutagens.

Diminished Carcinogen Detoxification Is a Novel Mechanism for Hypoxia-inducible Factor 1-mediated Genetic Instability

The hypoxia-inducible factor 1 (HIF-1) pathway is induced in many tumors and associated with poorer outcome. The hypoxia-responsive transcription factor HIF-1alpha dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT), which is also an important binding partner for the aryl hydrocarbon receptor (AhR). AhR is an important mediator in the metabolic activation and detoxification of carcinogens, such as the environmental pollutant benzo[a]pyrene (BaP). We hypothesized that HIF-1alpha activation attenuates BaP-induced AhR-mediated gene expression, which may lead to increased genetic instability and malignant progression. Human lung carcinoma cells (A549) were simultaneously stimulated with CoCl(2), which leads to HIF-1alpha stabilization and varying concentrations of BaP. Both quantitative PCR and immunoblot analysis indicated that induction of the hypoxia response pathway significantly reduced the levels of AhR downstream targets CYP1A1 and CYP1B1 and AhR protein binding to ARNT. We further demonstrate that the BaP-induced hypoxanthine-guanine phosphoribosyltransferase mutation frequency and gamma-H2AX foci were markedly amplified when the HIF-1 pathway was induced. BaP-DNA adducts were only marginally increased, and transient strand breaks were diminished by HIF-1 induction, indicating changes in DNA repair. These data indicate that concurrent exposure of tumor cells to hypoxia and exogenous genotoxins can enhance genetic instability.

Abstract 3448: Synergistic effect of benzo(a)pyrene (BaP) and dietary fat on biotransformation enzymes, DNA adducts and colon tumors in ApcMin mice

Abstract Studies in our laboratory thus far have shown formation of colon tumors in ApcMin mice subsequent to ingestion of fat containing BaP, an environmental carcinogen. These findings have human health relevance in that around 60,000 lives/year are lost to colon cancer in US alone. Diet and environment have been implicated in the development of sporadic colon tumors. Since biotransformation of toxicants is one of the factors that trigger carcinogenesis, the objective of this study was to determine how dietary fat potentiates the development of colon tumors through altered BaP biotransformation, using the ApcMin mouse model. Benzo(a)pyrene was administered to ApcMin mice in unsaturated- (peanut oil) and saturated- (coconut oil) fats at doses of 50 and 100 μg/kg via oral gavage over a 60-day period. Blood, colon and liver were collected at the end of exposure period. The expression of BaP biotransformation enzymes (CYP1A1, CYP1B1 and GST) in liver and colon were assayed at the level of mRNA and activities. Tissue samples were analyzed by reverse phase-HPLC for BaP metabolites, and 32P-postlabeling method for BaP- DNA adducts. BaP exposure through dietary fat altered its biotransformation in a dose-dependent manner, with 100 μg/kg dose group registering an elevated expression of BaP biotransformation enzymes, greater concentration of BaP metabolites, BaP-DNA adducts and more adenomas compared to the 50 μg/kg dose group (p &amp;lt; 0.05). This effect was more pronounced for saturated fat group compared to unsaturated fat group (p &amp;lt; 0.05). These findings establish that saturated fat contributes to sustained induction of BaP biotransformation enzymes and extensive metabolism of this carcinogen. As a consequence, the reactive metabolites such as epoxides and quinones generated in colon bind with DNA, and form adducts resulting in colon tumors in a long term exposure situation (funded by NIH grants S11ES014156, 1F31ES017391-01, 5T32 HL007735-14 and RO3CA130112-01). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3448.

Deficiency of glycine N‐methyltransferase results in deterioration of cellular defense to stress in mouse liver

Previously, we reported that glycine N-methyltransferase (GNMT) interacts with benzo[a]pyrene (BaP) and inhibits BaP-DNA adducts formation. In addition, Gnmt knockout (Gnmt(-/-)) mice developed chronic hepatitis and hepatocellular carcinoma (HCC). The aims of this study were to understand the gene expression profile of Gnmt(-/-) mice and to study the interaction between BaP and GNMT deficiency in vivo. Gene expression profiles of Gnmt(-/-) mice were analyzed by 2-D PAGE and real-time PCR. Both wild-type and Gnmt(-/-) mice were challenged with BaP and sacrificed at the age of 13 months. Compared with the wild-type mice, proteins involved in the anti-oxidation/detoxification response, glycolytic energy metabolism and one-carbon metabolism pathways were down-regulated significantly in Gnmt(-/-) mice. Malondialdehyde assay showed that lipid peroxidation was significantly increased in the Gnmt(-/-) mice liver. H(2)O(2) treatment demonstrated that the survival rate of HuH-7 cells overexpressing GNMT was significantly higher than the controls. BaP challenge experiments showed that 71.4% (5/7) of male and all (7/7) female Gnmt(-/-) mice developed HCC, while only 16.7% (1/6) of male and 20% (1/5) of female wild-type mice had HCC. GNMT regulates genes related to detoxification and anti-oxidation pathways. BaP is a liver cancer carcinogen especially during GNMT deficiency.

Exposure to polycyclic aromatic hydrocarbons and missed abortion in early pregnancy in a Chinese population

Background Polycyclic aromatic hydrocarbons (PAHs) are formed during incomplete burning of fossil fuels, wood, and tobacco products. High PAH exposure has been associated with low birth weight, intrauterine growth restriction, and preterm birth, but little is known about its impact on adverse outcomes in early pregnancy such as in-utero fetal death. Objectives To examine associations between exposure to PAHs and missed abortion in which the embryo has died but a miscarriage has not yet occurred during early pregnancy in a Chinese population in Tianjin. Methods A case–control study was conducted from April to November, 2007 in Tianjin, China. Cases experienced a missed abortion while controls underwent elective abortions before 14 weeks of pregnancy. Eighty-one cases were recruited from four hospitals, with the same number of controls matched on hospital, maternal age (± 8 years), gravidity (1 or >1), and gestational age (± 30 days). Two maternal measures of PAH exposures were obtained based on benzo[ a]pyrene (BaP) DNA adducts in 1) aborted tissues and 2) maternal blood (for a subset of subjects). In addition, proxy measures for PAH exposures from different sources were derived from maternal interviews. Results In conditional logistic regression analyses, we estimated more than 4-fold increase in risk of having experienced a missed abortion in women with above the median levels of blood BaP-DNA adducts (adjusted OR = 4.27; 95% CI, 1.41–12.99); but no increase with adduct levels in aborted tissues (adjusted OR = 0.76; 95% CI, 0.37–1.54). BaP-DNA adduct levels in maternal blood and aborted tissues were poorly correlated ( r = − 0.12; n = 102). Missed abortion risk also was higher among women reporting traffic congestion near the residence, commuting by walking, and performing regular cooking activities during pregnancy. Conclusion High levels of maternal PAH exposures may contribute to an increased risk of experiencing a missed abortion during early pregnancy.

Pregnane X Receptor Protects HepG2 Cells from BaP-Induced DNA Damage

Pregnane X receptor (PXR) is a nuclear receptor that coordinately regulates transcriptional expression of both phase I and phase II metabolizing enzymes. PXR plays an important role in the pharmacokinetics of a broad spectrum of endogenous and xenobiotic compounds and appears to have evolved in part to protect organisms from toxic xenobiotics. Metabolism of benzo[a]pyrene (BaP), a well-established carcinogen and ubiquitous environmental contaminant, can result in either detoxification or bioactivation to its genotoxic forms. Therefore, PXR could modulate the genotoxicity of BaP by changing the balance of the metabolic pathways in favor of BaP detoxification. To examine the role of PXR in BaP genotoxicity, BaP-DNA adduct formation was measured by 32P-postlabeling in BaP-treated parental HepG2 cells and human PXR-transfected HepG2 cells. The presence of transfected PXR significantly reduced the level of adducts relative to parental cells by 50-65% (p < 0.001), demonstrating that PXR protects liver cells from genotoxicity induced by exposure to BaP. To analyze potential PXR-regulated detoxification pathways in liver cells, a panel of genes involved in phase I and phase II metabolism and excretion was surveyed with real-time quantitative reverse transcription PCR. The messenger RNA levels of CYP1A2, GSTA1, GSTA2, GSTM1, UGT1A6, and BCRP (ABCG2) were significantly higher in cells overexpressing PXR, independent of exposure to BaP. In addition, the total GST enzymatic activity, which favors the metabolic detoxification of BaP, was significantly increased by the presence of PXR (p < 0.001), independent of BaP exposure. Taken together, these results suggest that PXR plays an important role in protection against DNA damage by polycyclic aromatic hydrocarbons (PAHs) such as BaP, and that these protective effects may be through a coordinated regulation of genes involved in xenobiotic metabolism.

Influence of TCDD and natural Ah receptor agonists on benzo[a]pyrene-DNA adduct formation in the Caco-2 human colon cell line

Several compounds originating from cruciferous vegetables and citrus fruits bind to and activate the aryl hydrocarbon receptor (AhR). This receptor plays an important role in the toxicity of the known tumour promoter and potent AhR-agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, vegetables and fruits are generally considered as healthy. Therefore, besides the AhR activation, the natural AhR agonists (NAhRAs) are assumed to show other health-concerning effects. AhR activation induces several cytochrome P450 phase I enzymes involved, e.g. in the bioactivation of carcinogenic polycyclic aromatic hydrocarbons, like benzo[a]pyrene (BaP), and may as such stimulate DNA adduct formation of those compounds. Therefore, the influence of TCDD, indolo[3,2-b]carbazole (ICZ, an NAhRA originating from cruciferous vegetables) and an NAhRA-containing extract of grapefruit juice (GJE) on BaP-DNA adduct formation in the human Caco-2 cell line was studied. Also, we investigated if different effects of TCDD, ICZ and GJE on adduct formation could be related to the modulation of transcription of biotransformation- and DNA-repair enzymes. Co-exposure to high AhR-activating concentrations of both TCDD and ICZ significantly reduced the amount of BaP-DNA adducts at 0.1 microM BaP, while at higher concentrations of BaP no influence was observed. In contrast, exposure to 0.1 microM BaP combined with GJE showed a significant increase in BaP-DNA adducts, and a significant decrease at 0.3 and 1 microM BaP. These differences could not be related to transcription of the phase I and II enzymes CYP1A1, CYP1B1, NQO1, GSTP1 and UGT1A6 or to transcription of the nucleotide excision repair enzymes ERCC1, XPA, XPC, XPF and XPG. We conclude that ICZ showed a similar effect on BaP-DNA adduct formation than TCDD, while GJE influenced the adduct formation in a different way. The difference in the influence on adduct formation may be due to effects at the level of enzyme activity, rather than gene expression.

Identification through microarray gene expression analysis of cellular responses to benzo( a )pyrene and its diol-epoxide that are dependent or independent of p53

Human colon carcinoma cells (HCT116) differing in p53 status were exposed to benzo(a)pyrene (BaP) or anti-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE) and their gene expression responses compared by complementary DNA microarray technology. Exposure of cells to BPDE for up to 24 h resulted in gene expression profiles more distinguishable by duration of exposure than by p53 status, although a subset of genes were identified that had significantly different expression in p53 wild-type (WT) cells relative to p53-null cells. Apoptotic signalling genes were up-regulated in p53-WT cells but not in p53-null cells and, consistent with this, reduced viability and caspase activity were also p53 dependent. BPDE modulated cell cycle and histone genes in both cell lines and, in agreement with this, both cell lines accumulated in S phase. In p53-WT cells, G(2) arrest was also evident, which was associated with accumulation of CDKN1A. Regardless of p53 status, exposure to BaP for up to 48 h had subtle effects on gene transcription and had no influence on cell viability or cell cycle. Interestingly, DNA adduct formation after BaP, but not BPDE, exposure was p53 dependent with 10-fold lower levels detected in p53-null cells. Other cell lines were investigated for BaP-DNA adduct formation and in these the effect of p53 knockdown was also to reduce adduct formation. Taken together, these results give further insight into the role of p53 in the response of human cells to BaP and BPDE and suggest that loss of this tumour suppressor can influence the metabolic activation of BaP.

Relationship between Polycyclic Aromatic Hydrocarbon–DNA Adducts, Environmental Tobacco Smoke, and Child Development in the World Trade Center Cohort

BackgroundPolycyclic aromatic hydrocarbons (PAHs), including benzo[a]pyrene (BaP), are air pollutants released by the World Trade Center (WTC) fires and urban combustion sources. BaP–DNA adducts provide a measure of PAH-specific genetic damage, which has been associated with increased risk of adverse birth outcomes and cancer. We previously reported that levels of BaP–DNA adducts in maternal and umbilical cord blood obtained at delivery were elevated among subjects who had resided within 1 mile of the WTC site during the month after 9/11; and that elevated blood adducts in combination with in utero exposure to environmental tobacco smoke (ETS) were significantly associated with decreased fetal growth.ObjectiveOur aim was to assess possible effects of prenatal exposure to WTC pollutants on child development.MethodsAfter 11 September 2001, we enrolled a cohort of nonsmoking pregnant women who delivered at three lower Manhattan hospitals. We have followed a subset of children through their third birthdays and measured cognitive and motor development using the Bayley-II Scales of Child Development (BSID-II).ResultsIn multivariate analyses, we found a significant interaction between cord blood adducts and in utero exposure to ETS on mental development index score at 3 years of age (p = 0.02, n = 98) whereas neither adducts nor ETS alone was a significant predictor of (BSID-II) cognitive development.ConclusionAlthough limited by small numbers, these results suggest that exposure to elevated levels of PAHs in conjunction with prenatal ETS exposure may have contributed to a modest reduction in cognitive development among cohort children.

Measurement of DNA Adducts as Biomarkers of Genotoxic Exposure to Benzo[A]Pyrene Using a LC-MS/MS Technique

P-133 Introduction: Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants representing the first family of carcinogenic pollutants in professional environment. It is now well established that some of these molecules including the tumorigenic benzo[a]pyrene (BaP) are responsible for the apparition of lung, skin and bladder cancers. It is therefore of major importance to evaluate individual exposure to carcinogenic PAHs in order to improve health risk assessment. In addition to the classical determination of atmospheric concentrations of gaseous and particulate PAHs and 1-hydroxyprene excreted in urine, we are interested in measuring specific urinary metabolites and DNA adducts of BaP. Methods: A high performance liquid chromatography coupled to fluorescence detection (HPLC-Fluo) method has been set-up for the urinary determination of 3-hydroxybenzo[a]pyrene (3-OHBaP) and BaP tetrol, and compared to a HPLC coupled to tandem mass spectrometry (HPLC-MS/MS) method. This technique already used for the quantification of 8-oxo-7,8-dihydro-2′-désoxyguanosine was also developed for the measurement of several DNA adducts of BaP. Results: The limit of detection (LOD) obtained for 3-OHBaP is 0.4 ng/L of urine thanks to a double-step procedure of purification and concentration of urinary samples on Sep-Pak cartridges followed by an on-line extraction on the HPLC-Fluo system, and is not improved using a tandem mass spectrometric detection. The LOD of BaP tetrol is currently determined. HPLC-MS/MS allows a highly sensitive and specific detection and quantification of eight stable and depurinating DNA adducts of BaP. These include five DNA adducts of the carbocation pathway and three adducts arising from the BaP-diol-epoxyde pathway. The corresponding limits of detection range from 1 to 40 injected fmoles. Discussion and Conclusions: In the weeks coming, our developed analytical tools will be applied to measure BAP metabolites, DNA adducts and DNA oxidative damages in urinary samples obtained from volunteers professionally exposed to PAHs. The results should be of main importance to identify pertinent biomarkers in order to measure exposure to BaP and assess health risks of workers which may orientate preventing actions.

PAH–DNA Adducts in Cord Blood and Fetal and Child Development ina Chinese Cohort

Polycyclic aromatic hydrocarbons (PAHs) are an important class of toxic pollutants released by fossil fuel combustion. Other pollutants include metals and particulate matter. PAH–DNA adducts, or benzo[a]pyrene (BaP) adducts as their proxy, provide a chemical-specific measure of individual biologically effective doses that have been associated with increased risk of cancer and adverse birth outcomes. In the present study we examined the relationship between prenatal PAH exposure and fetal and child growth and development in Tongliang, China, where a seasonally operated coal-fired power plant was the major pollution source. In a cohort of 150 nonsmoking women and their newborns enrolled between 4 March 2002 and 19 June 2002, BaP–DNA adducts were measured in maternal and umbilical cord blood obtained at delivery. The number of gestational months occurring during the period of power plant operation provided a second, more general measure of exposure to plant emissions, in terms of duration. High PAH–DNA adduct levels (above the median of detectable adduct level) were associated with decreased birth head circumference (p = 0.057) and reduced children’s weight at 18 months, 24 months, and 30 months of age (p < 0.05), after controlling for potential confounders. In addition, in separate models, longer duration of prenatal exposure was associated with reduced birth length (p = 0.033) and reduced children’s height at 18 (p = 0.001), 24 (p < 0.001), and 30 months of age (p < 0.001). The findings suggest that exposure to elevated levels of PAHs, with the Tongliang power plant being a significant source, is associated with reduced fetal and child growth in this population.

Comparison of gene expression in cell culture to that in the intact animal: relevance to drugs and environmental toxicants. Focus on “Development of a transactivator in hepatoma cells that allows expression of phase I, phase II, and chemical defense genes”

to study the effects of drugs and other environmental toxicants on the liver, many believe that the use of hepatocyte-derived cultures offers advantages over studies in the liver of intact animals. Cell cultures can be manipulated more easily, and they are far less expensive to maintain than a

Tissue specific induction of cytochrome P450 (CYP) 1A1 and 1B1 in rat liver and lung following in vitro (tissue slice) and in vivo exposure to benzo( a)pyrene

Cytochrome P-450s (CYPs) detoxify a wide variety of xenobiotics and environmental contaminants, but can also bioactivate carcinogenic polycyclic aromatic hydrocarbons, such as benzo( a)pyrene (BaP), to DNA-reactive species. The primary CYPs involved in the metabolism and bioactivation of BaP are CYP1A1 and CYP1B1. Furthermore, BaP can induce expression of CYP1A1 and CYP1B1 via the aryl hydrocarbon receptor. Induction of CYP1A1 and CYP1B1 by BaP in target (lung) and non-target (liver) tissues was investigated utilizing precision-cut rat liver and lung slices exposed to BaP in vitro. Tissue slices were also prepared from rats pretreated in vivo with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) to induce expression of CYP1A1 and CYP1B1. In addition, in vivo exposure studies were performed with BaP to characterize and validate the use of the in vitro tissue slice model. In vitro exposure of liver and lung slices to BaP resulted in a concentration-dependent increase in CYP1A1 and CYP1B1 mRNA and protein levels, which correlated directly with the exposure-related increase in BaP-DNA adduct levels observed previously in the tissue slices [Harrigan, J.A., Vezina, C.M., McGarrigle, B.P., Ersing, N., Box, H.C., Maccubbin, A.E., Olson, J.R., 2004. DNA adduct formation in precision-cut rat liver and lung slices exposed to benzo( a)pyrene. Toxicological Sciences 77, 307–314]. Pretreatment of animals in vivo with TCDD produced a marked induction of CYP1A1 and CYP1B1 expression in the tissue slices, which was similar to the levels of CYP1A1 and CYP1B1 mRNA achieved in liver and lung following in vivo treatment with BaP. Following in vitro exposure to BaP, the levels of CYP1A1 were greater in the lung than the liver, while following all exposures (in vitro and in vivo), the levels of CYP1B1 mRNA were greater in lung tissue compared to liver. The higher expression of CYP1A1 and CYP1B1 in the lung was associated with higher levels of BaP-DNA adducts in the lung slices (Harrigan et al.’s work) and together, these results may contribute to the tissue specificity of BaP-mediated carcinogenesis.

Preferential induction of CYP1B1 by benzo[ a ]pyrene in human oral epithelial cells: impact on DNA adduct formation and prevention by polyphenols

The roles of CYP1A1 and 1B1 in tobacco smoke carcinogen, e.g. benzo[a]pyrene (BaP), induced DNA binding and their inhibition by the dietary polyphenol 5,7-dimethoxyflavone (DMF), compared with 3',4'-dimethoxyflavone (3',4'-DMF) and resveratrol, were investigated in the human oral epithelial squamous cell carcinoma (SCC)-9 cells. A low concentration of BaP (1 microM) dramatically induced BaP-DNA adduct formation (approximately 40-fold) in a time-dependent manner, while it only increased CYP1A1/1B1 activities, as measured by ethoxyresorufin O-deethylation, approximately 3-fold. Furthermore, BaP induced both CYP1B1 and CYP1A1 mRNA and protein expression, as determined by the branched DNA assay and western blot analysis, but with considerably higher levels of CYP1B1. Combined treatment of SCC-9 cells with 1 microM BaP and 20 microM DMF inhibited BaP-DNA adduct formation. The mechanism of this effect appeared to be direct inhibition of CYP1B1 enzyme with a K(i) value of 0.58 microM, a highly potent inhibition considering the high cellular uptake of DMF in the SCC-9 cells. DMF also inhibited CYP1A1, but not CYP1B1 protein, and mRNA expression in the cells. In an extension to other polyphenols, the structural analog 3',4'-DMF, in contrast to DMF, inhibited the expression of CYP1B1 both at the mRNA and protein levels. Surprisingly, in contrast to previous studies in other cell types, resveratrol had no effect on CYP1B1 in the SCC-9 cells. Based on this study, CYP1B1 mRNA may be an early biomarker of oral cancer, being a sensitive signal for tobacco-carcinogen exposure. Methoxylated dietary flavonoids, e.g. DMF and 3',4'-DMF, may be potent chemoprotectants by direct inhibition of CYP1B1/1A1 function and/or their protein expression.