Genotoxic Metabolites Research Articles

Acrylamide in children – exposure assessment via urinary acrylamide metabolites as biomarkers

Acrylamide (AA), a substance classified as probably carcinogenic to humans, was detected for the first time in food products in 2002. AA can be primarily found in foods containing carbohydrates and proteins, where it is formed during the heating process. Exposure assessment based on food consumption data revealed an average daily intake of AA between 0.3 and 0.8 microg/kg BW/day. These data have been confirmed by human biomonitoring using haemoglobin adducts of AA in blood or the specific mercapturic acids in urine. However, human biomonitoring data on the internal exposure of children were only sporadically available. Especially data about the excretion of both relevant mercapturic acids were missing. The mercapturic acids other than the haemoglobin adducts give the recent AA exposure of the last 24h. In this study, we quantify the internal exposure of AA and the genotoxic metabolite glycidamide (GA) in 110 children with regard to their exposure through diet and/or environmental tobacco smoke. Hundred and ten 5-6-year-old children were randomly selected. Their dietary habits as well as their exposure to the environmental tobacco smoke were assessed by means of a questionnaire. By means of spot urine samples, mercapturic acids of acrylamide (AAMA) and mercapturic acids of glycidamide (GAMA) were analysed with LC-ESI-MS/MS. Median (95th percentile) urinary levels were 36.0 (152.7) microg AAMA/l and 13.4 (55.9) microg GAMA/l. Based on the metabolite levels, the median uptake of acrylamide was calculated to be 0.54 microg/kg BW/d. A number of associations with the consumption of French fries, various potato products, as well as fried cereals could be found. Significant results were found for French fries. No correlations between the exposure to environmental smoke and cotinine levels in urine were found. This is the first study to show the presence of AAMA and GAMA in urine specimens of 110 children, thus providing evidence for a background exposure by nutrition. Median (95th percentile) uptake of AA in children was 0.54 (1.91) microg/kg bodyweight and day, exceeding exposure in adults by 50%. These findings support the efforts to minimize AA formation and contamination in food. Comparing our findings with that of other human studies, there are hints that children have a higher AA intake than adults and that children more effectively oxidize AA. Both findings indicate that children might be the most vulnerable group of the population.

Physical activity as a negative modulator of estrogen-induced breast cancer

Physical activity is a protective factor for breast cancer. Exposure to estrogen is an important determinant of breast cancer risk and exercise reduces estrogen levels, with the level of evidence being stronger for post-menopausal women. Possible mechanisms for estrogen induced breast cancer include increased breast epithelial cell proliferation, the metabolism of estrogen to genotoxic metabolites, such as DNA-adducts, and the silencing of tumor suppressor genes (TSGs) that have been implicated in breast carcinogenesis by inducing gene promoter hypermethylation, which is potentially reversible. Animal studies suggest that physical activity decreases breast tumor growth by promoting changes in cellular proliferation and apoptosis. Human studies provide some support for exercise producing favorable changes in estrogen metabolism that may lead to reduced breast epithelial cell proliferation. No studies have been performed to determine whether exercise decreases the accumulation of estrogen metabolite DNA-adducts in breast tissue. However, research supports the hypothesis that physical activity reduces promoter hypermethylation of TSGs implicated in breast carcinogenesis by lowering circulating estrogen levels. Thus, further research is necessary to clarify the mechanisms that relate to physical activity as a negative modulator of breast cancer risk to develop meaningful guidelines for the use of physical activity in breast cancer prevention.

Genetic Reprogramming of Human Mammary Cells by Environmental Carcinogens Released into Breast Milk

Epidemiologic studies indicate that high serum levels of chlorinated pollutants such as dioxins or pesticides constitute a risk factor for breast cancer in postmenopausal women. This finding has been attributed to the ability of organochlorine compounds to induce the expression of cytochrome P450 (CYP) enzymes, which are thought to increase the burden of genotoxic metabolites. However, it was not clear whether the incriminated pollutants accumulate to sufficiently high levels within the human body to be able to promote significant transcriptional responses. Here, milk samples from nursing mothers were processed by gel permeation chromatography to isolate complex mixtures of residues containing both endogenous hormones and lipophilic contaminants of environmental origin. High-density oligonucleotide microarrays were used to monitor global transcriptional profiles in a human cell line (MCF7) that represents one of the most frequently used model systems to study breast cancer biology. We found that all breast milk extracts were able to reprogram the genome of MCF7 cells by imposing a typically estrogenic expression fingerprint. This estrogenic background was overlapped only by the induction of transcripts coding for CYP1A1 and, to a minor degree, CYP1B1. The magnitude of induction of these metabolic enzymes correlated with the respective organochlorine concentrations. Thus, in support of previous epidemiologic studies, we demonstrate that contaminants released from human adipose tissue trigger a potentially genotoxic pathway in cells from the mammary gland.

Genotoxic activation of 2-phenylbenzotriazole-type compounds by human cytochrome P4501A1 and N-acetyltransferase expressed in Salmonella typhimurium umu strains

Four 2-phenylbenzotriazole (PBTA)-type compounds (PBTA-4, PBTA-6, PBTA-7, and PBTA-8) were identified as major mutagens in blue cotton/rayon-adsorbed substances collected at sites below textile dyeing factories or municipal water treatment plants treating domestic waste and effluents from textile dyeing factories in several rivers in Japan. The main purpose of this study is to understand the basis of the roles of human cytochrome P450 (CYP) and N-acetyltransferases (NATs) in genotoxic activation of PBTA derivatives. We compared the induction of umuC gene expression as a measure of genotoxicity using Salmonella typhimurium TA1535/pSK1002 (parental strain), NM2009 (bacterial O-acetyltransferase-overexpressing strain) established in our laboratories. PBTA-4, PBTA-6, PBTA-7, and PBTA-8 induced the umuC gene expression more strongly in the bacterial O-acetyltransferase-overproducing strain than in the parental strain in the presence of rat S9 mix. We determined the activation of PBTA derivatives by cDNA-based recombinant ( Trichoplusia ni) systems expressing human or rat cytochrome P450 enzymes (P450 or CYP) and NADPH-P450 reductase using S. typhimurium NM2009. The results showed that human recombinant CYP1A1 enzyme was much more active than CYP1A2 and CYP3A4 in the genotoxic activation of PBTA-4, PBTA-6, PBTA-7, and PBTA-8. Similarly, rat recombinant CYP1A1 enzyme catalyzed the activation of these chemicals at high rates. α-Naphthoflavone, a known inhibitor of CYP1A1, was found to inhibit genotoxic activation caused by PBTA derivatives. We further determined the activation of PBTA derivatives using S. typhimurium NM6001 (human NAT1-expressing strain), S. typhimurium NM6002 (human NAT2-expressing strain), and S. typhimurium NM6000 ( O-AT-deficient parent strain) in the presence of S9 mix. PBTA-4 showed almost similar sensitivity in the NAT1-expressing strain and the NAT2-expressing strain, although NAT2-expressing strain exhibited relatively higher sensitivity to PBTA-6, PBTA-7, and PBTA-8 than NAT1-expressing strain. The results support the view that O-acetylation by human NAT1 and NAT2 enzymes is involved in the genotoxic activation of PBTA compounds. These results demonstrate for the first time that human P4501A1 and NATs (NAT1 and NAT2) contribute significantly to the activation of PBTA-type compounds to genotoxic metabolites that induce umuC gene expression in S. typhimurium tester strains.

Estrogenic Phenol and Catechol Metabolites of PCBs Modulate Catechol-Omethyltransferase Expression Via the Estrogen Receptor: Potential Contribution to Cancer Risk

Commercial PCB mixtures have been shown to induce liver tumors in female rats and this effect has been attributed to the effects of PCBs on estrogen metabolism. Catechol metabolites of PCBs are potent inhibitors of COMT activity and are likely to contribute significantly to reduced clearance of genotoxic catechol metabolites of estrogen. The effect of PCB metabolites on COMT expression in cultured cells was investigated to explore potential mechanisms by which PCB exposure alters catechol estrogen clearance. We hypothesize that estrogenic PCB metabolites may contribute to reduction of COMT expression via interaction with the estrogen receptor. To test this hypothesis, human MCF-7 cells were exposed to PCB analogues and the expression of COMT determined. Western blot analysis demonstrated that COMT protein levels were statistically significantly reduced by both the phenolic and the catechol compounds, an effect which was abolished by the anti-estrogen, ICI182780. The above suggests that COMT levels may be reduced by estrogenic PCB metabolites, via interactions between PCB metabolites and the ER. It supports the hypothesis that both phenolic and catechol metabolites of PCBs may contribute to PCB-mediated carcinogenesis through reduction of COMT levels and activities and subsequent reduction in clearance of endogenous and xenobiotic catechols.

Quantifiable mRNA transcripts for tamoxifen-metabolising enzymes in human endometrium

Tamoxifen has been used in the management of receptor-positive breast cancer for >20 years. Usage confers an elevated risk of developing endometrial carcinoma. Its mechanism of carcinogenicity remains unresolved with controversy as to whether or not this is mediated through a genotoxic mechanism. Usage is not only associated with an elevated occurrence of endometrioid endometrial carcinoma, but also type 2 and mixed epithelial-stromal tumours (MESTs) that have a poorer prognosis. Following hysterectomy, endometrial tissues ( n = 18) classified as benign ( n = 6), non-tamoxifen-associated carcinoma ( n = 6) and tamoxifen-associated carcinoma ( n = 6) were obtained; quantitative gene expression was performed. Employing real-time RT-PCR, the relative gene expressions of phase I/II metabolic enzymes CYP1A2, CYP1B1 and CYP3A4, cathechol- O-methyltransferase ( COMT) and SULT2A1 were ascertained. Measurable mRNA transcripts, especially for those genes associated with tamoxifen bioactivation, were quantifiable in all the tissues examined. Whether this is evidence that generation of genotoxic tamoxifen metabolites may occur in human endometrial tissue remains to be ascertained.

Development of a highthroughput yeast-based assay for detection of metabolically activated genotoxins

The potential genotoxicity of drug candidates is a serious concern during drug development. Therefore, it is important to assess the potential genotoxicity and mutagenicity of a compound early in the discovery phase of drug development. AMES Salmonella assay is the most widely used assay for the assessment of mutagenicity and genotoxicity. However, the AMES assay is not readily adaptable to highthroughput screening and several strains of Salmonella must be employed to ensure that different types of DNA damage can be studied. Therefore, an additional robust highthroughput genotoxicity screen would be of significant value in the early detection and elimination of genotoxicity. The complexity of DNA damage requires numerous cellular pathways, thus using single model organism to predict genotoxicity in early stage is challenging. Another critical component of such screens is that they incorporate the capability of metabolic activation to ensure that no genotoxic metabolites are generated. We have developed a novel highthroughput reporter assay for DNA repair that detects genotoxicity, and which incorporates metabolic activation. The assay has a low compound requirement as compared to Ames, and relies upon two different reporter genes cotransfected into a yeast strain. The gene encoding Renilla luciferase is fused to the constitutive 3-phosphoglycerate kinase ( PGK1) promoter and integrated into the yeast genome to provide a control for cell numbers. The firefly luciferase gene is fused to the RAD51 (bacterial RecA homolog) promoter and used to report an increase in DNA repair activity. A dual luciferase assay is performed by measuring the firefly and Renilla luciferase activities in the same sample. The result is expressed as the ratio of the two luciferase activities; changes from the base level (control) are interpreted as induction of the RAD51 promoter and evidence of DNA repair activity in eukaryote cells due to DNA damage. The yeast dual luciferase reporter has been characterized with and without S-9 activation using positive and negative control agents. This assay is efficient, requires little time and low amounts of compound. The assay is compatible with metabolic activation, adaptable to a highthroughput platform, and yields data that accurately and reproducibly detects DNA damage. Whereas the normal yeast cell wall, plasma membrane composition and the presence of active transporters can prevent the entry or persistence of some compounds internally in yeast cells, our assay did show concordance with regulatory mutagenicity assays, many of which require metabolic activation and are poorly detected by bacterial mutagenicity assays. Although there were false negative results, in our hands this assay performs as well as or better than other commercially available genetox assays. Furthermore, the RAD51 gene is strongly inducible by homologous intrachromosomal recombination; thus this assay may provide a means to detect clastogens. The RAD51 promoter fused dual luciferase assay represents a valuable addition to the armamentarium for the early detection of genotoxic compounds.

Acrylamide exposure and incidence of breast cancer among postmenopausal women in the Danish Diet, Cancer and Health Study

Acrylamide, a probable human carcinogen, is formed in several foods during high-temperature processing. So far, epidemiological studies have not shown any association between human cancer risk and dietary exposure to acrylamide. The purpose of this study was to conduct a nested case control study within a prospective cohort study on the association between breast cancer and exposure to acrylamide using biomarkers. N-terminal hemoglobin adduct levels of acrylamide and its genotoxic metabolite, glycidamide in red blood cells were analyzed (by LC/MS/MS) as biomarkers of exposure on 374 breast cancer cases and 374 controls from a cohort of postmenopausal women. The adduct levels of acrylamide and glycidamide were similar in cases and controls, with smokers having much higher levels (approximately 3 times) than nonsmokers. No association was seen between acrylamide-hemoglobin levels and breast cancer risk neither unadjusted nor adjusted for the potential confounders HRT duration, parity, BMI, alcohol intake and education. After adjustment for smoking behavior, however, a positive association was seen between acrylamide-hemoglobin levels and estrogen receptor positive breast cancer with an estimated incidence rate ratio (95% CI) of 2.7 (1.1-6.6) per 10-fold increase in acrylamide-hemoglobin level. A weak association between glycidamide hemoglobin levels and incidence of estrogen receptor positive breast cancer was also found, this association, however, entirely disappeared when acrylamide and glycidamide hemoglobin levels were mutually adjusted.

Modulatory activity of a Lactobacillus casei strain on 1,2‐dimethylhydrazine‐induced genotoxicity in rats

The present study was designed to investigate the putative antigenotoxic effects of supplementing the diet of rats treated with the colon carcinogen 1,2-dimethylhydrazine hydrochloride (DMH) with a Lactobacillus casei strain using an in vivo approach. The antigenotoxic response was evaluated in colon and liver cells using the alkaline comet assay. Since the balance between the bioactivation and detoxification metabolic pathways is crucial for the formation of toxic and genotoxic metabolites, alterations in the level of some xenobiotic metabolizing enzymes (XME) were studied in liver preparations. In the challenge group (L. casei + DMH), lactobacilli-supplemented diet, there was a decrease in the extent of DMH-induced DNA damage, especially in colon cells. Compared with control rats, there was less basal DNA damage in colon cells of rats fed on a lactobacilli-supplemented diet. These findings are the first to give clear evidence of DNA-protective effects of lactobacilli against basal DNA damage. Moreover, the chemopreventive effects were accompanied by changes in the activities of several XME. The observed decrease in the concentration of nonenzymatic antioxidants (i.e. GSH) and the reduced activity of enzymatic antioxidants (i.e., GST, GPx, and SOD) in liver could reflect an overall reduction in the level of oxidative stress in rats on a diet supplemented with the L. casei suspension compared with control rats (basal state). Thus, the concentrations of GSH and the activities of GST, GPx, and SOD could be downregulated by supplementing the diet with L. casei as a response to an improved antioxidant status.

Phosphatidylethanol Accumulation Promotes Intestinal Hyperplasia by Inducing ZONAB-Mediated Cell Density Increase in Response to Chronic Ethanol Exposure

Chronic alcohol consumption is associated with increased risk of gastrointestinal cancer. High concentrations of ethanol trigger mucosal hyperregeneration, disrupt cell adhesion, and increase the sensitivity to carcinogens. Most of these effects are thought to be mediated by acetaldehyde, a genotoxic metabolite produced from ethanol by alcohol dehydrogenases. Here, we studied the role of low ethanol concentrations, more likely to mimic those found in the intestine in vivo, and used intestinal cells lacking alcohol dehydrogenase to identify the acetaldehyde-independent biological effects of ethanol. Under these conditions, ethanol did not stimulate the proliferation of nonconfluent cells, but significantly increased maximal cell density. Incorporation of phosphatidylethanol, produced from ethanol by phospholipase D, was instrumental to this effect. Phosphatidylethanol accumulation induced claudin-1 endocytosis and disrupted the claudin-1/ZO-1 association. The resulting nuclear translocation of ZONAB was shown to mediate the cell density increase in ethanol-treated cells. In vivo, incorporation of phosphatidylethanol and nuclear translocation of ZONAB correlated with increased proliferation in the colonic epithelium of ethanol-fed mice and in adenomas of chronic alcoholics. Our results show that phosphatidylethanol accumulation after chronic ethanol exposure disrupts signals that normally restrict proliferation in highly confluent intestinal cells, thus facilitating abnormal intestinal cell proliferation.

Carboxylic Acid Drug-Induced DNA Nicking in HEK293 Cells Expressing Human UDP-Glucuronosyltransferases: Role of Acyl Glucuronide Metabolites and Glycation Pathways

Glucuronidation of carboxylic-acid-containing drugs can yield reactive acyl (ester-linked) glucuronide metabolites that are able to modify endogenous macromolecules. Previous research has shown that several carboxylic acid drugs are genotoxic in isolated mouse hepatocytes, and that DNA damage is prevented by the glucuronidation inhibitor, borneol. Whether these species induce comparable genetic damage in human cells is unknown. In this study, we investigated the mechanisms of clofibric acid-induced genotoxicity in HEK293 cells expressing the human UDP-glucuronosyltransferases UGT1A3, UGT1A9, or UGT2B7, and screened three other carboxylic acid drugs for UGT-dependent genotoxicity. DNA damage was detected using the alkaline version of the comet assay. HEK293 cells were incubated for 18 h with vehicle (2.5 mM NaOH), 0.1-2.5 mM clofibric acid or 0.1-1.0 mM benoxaprofen, bezafibrate, or probenecid. To identify mechanisms underlying any observed genotoxicity, we treated UGT2B7 transfectants with 10 mM aminoguanidine, 1 mM borneol, or 2 mM desferrioxamine mesylate prior to co-incubation with 1 mM clofibric acid for 18 h. Compared to vehicle, clofibric acid, benoxaprofen, and probenecid produced significant DNA damage in all three UGT-transfected HEK293 cell lines, detectable from the lowest concentration tested. Bezafibrate caused DNA damage only at higher concentrations (1.0 mM) in UGT2B7- and UGT1A9-, but not UGT1A3-transfected cells. No drug-induced DNA damage was detected in untransfected cells, consistent with the limited glucuronidation capacity of these cells. The glycation/glycoxidation inhibitor aminoguanidine and the glucuronidation inhibitor borneol significantly decreased clofibric-acid-mediated DNA damage in UGT2B7 transfected cells by 73.5 and 94.8%, respectively. The inhibitor of transition-metal-catalyzed oxidation, desferrioxamine mesylate, had no significant effect on DNA damage. This study demonstrates the substrate-dependent role of human UGTs in the bioactivation of carboxylic acid drugs to genotoxic acyl glucuronide metabolites that are able to damage nuclear DNA via glycation and/or glycoxidation mechanisms.

DNA adducts formation and induction of apoptosis in rat liver epithelial ‘stem-like’ cells exposed to carcinogenic polycyclic aromatic hydrocarbons

The bipotent liver progenitor cells, so called oval cells, may participate at the early stages of hepatocarcinogenesis induced by chemical carcinogens. Unlike in mature parenchymal cells, little is known about formation of DNA adducts and other genotoxic events in oval cells. In the present study, we employed spontaneously immortalized rat liver WB-F344 cell line, which is an established in vitro model of oval cells, in order to study genotoxic effects of selected carcinogenic polycyclic aromatic hydrocarbons (PAHs). With exception of dibenzo[ a, l]pyrene, and partly also benzo[ g]chrysene and benz[ a]anthracene, all other PAHs under the study induced high levels of CYP1A1 and CYP1B1 mRNA. In contrast, we observed distinct genotoxic and cytotoxic potencies of PAHs. Dibenzo[ a, l]pyrene, and to a lesser extent also benzo[ a]pyrene, benzo[ g]chrysene and dibenzo[ a, e]pyrene, formed high levels of DNA adducts. This was accompanied with accumulation of Ser-15 phosphorylated form of p53 protein and induction of apoptosis. Contrary to that, benz[ a]anthracene, chrysene, benzo[ b]fluoranthene and dibenzo[ a, h]anthracene induced only low amounts of DNA adducts formation and minimal apoptosis, without exerting significant effects on p53 phosphorylation. Finally, we studied effects of 2,4,3′,5′-tetramethoxystilbene and fluoranthene, inhibitors of CYP1B1 activity, which plays a central role in metabolic activation of dibenzo[ a, l]pyrene. In a dose-dependent manner, both compounds inhibited apoptosis induced by dibenzo[ a, l]pyrene, suggesting that it interferes with the metabolic activation of the latter one. The present data show that in model cell line sharing phenotypic properties with oval cells, PAHs can be efficiently metabolized to form ultimate genotoxic metabolites. Liver progenitor cells could be thus susceptible to this type of genotoxic insult, which makes WB-F344 cell line a useful tool for studies of genotoxic effects of organic contaminants in liver cells. Our results also suggest that, unlike in mature hepatocytes, CYP1B1 might be a primary enzyme responsible for formation of DNA adducts in liver progenitor cells.

Comparative mutational profiles of the environmental mammary carcinogen, 6-nitrochrysene and its metabolites in a lacI mammary epithelial cell line

The dietary and environmental agent, 6-nitrochrysene (6-NC) is a powerful mammary carcinogen and mutagen in rats. It is known to be metabolized by ring-oxidation, nitro-reduction and a combination of the two pathways. In order to determine the ultimate mutagenic metabolites, we have compared the previously determined mutational profile of 6-NC in rat mammary gland [T. Boyiri, et al. (2004) Carcinogenesis, 25, 637-643] with that of five of its known metabolites in the cII gene of lacI mammary epithelial cells in vitro. In vivo, 6-NC gives rise to three major mutations, AT > GC, AT > TA and GC > TA (in decreasing order) which comprise >70% of the mutations. The metabolite whose mutational profile was most similar to that of 6-NC in vivo was trans-1,2-dihydroxy-1,2-dihydro-N-hydroxy-6-aminochrysene (1,2-DHD-6-NHOH-C) which arises from both ring-oxidation and nitro-reduction. However, metabolites arising from either ring-oxidation or nitro-reduction alone exhibited some similarities to mutational profile of 6-NC. These results, taken in conjunction with previous data showing that the major DNA adducts in mammary tissue of rats treated with 6-NC are products of the reaction of 1,2-DHD-6-NHOH-C with guanine and adenine, make a strong case that 1,2-DHD-6-NHOH-C is the ultimate genotoxic metabolite from 6-NC.

Risk assessment of workers exposed to fumes of bitumen: genotoxic effects and urinary metabolites of naphthalene, phenanthrene, and pyrene

Risk assessment of workers exposed to fumes of bitumen: genotoxic effects and urinary metabolites of naphthalene, phenanthrene, and pyrene

P2-117: Increase of carcinogenic risk via enhancement of cyclooxygenase-2 expression and

Animal studies demonstrated that females are more susceptible than males to benzo[a]pyrene (BaP)-induced toxicities, including lung carcinogenesis. Elevation of cyclooxygenase-2 (COX-2) ex-pression has been shown to increase the risk of cancer development. BaP induces COX-2 expression, and an interaction between BaP and estrogen in relation to COX-2 expression is suspected. In the present study, 10 mMBaP alone only slightly increased COX-2 mRNA expression and 10 nM 17-beta estradiol (E2) alone slightly increased prostaglandin E2 (PGE2) secretion in human bronchial epithelial cells. However, co-treatment with BaP and E2 potentiated COX-2 mRNA expression and significantly elevated PGE2 secretion. Utilizing specific inhibitors and reporter assays, we further investigated the potentiation mechanisms of E2 on BaP-induced COX-2 expression. First, E2 activated estrogen receptor to increase PGE2 secretion, which directly increased COX-2 expression. Second, E2 potentiated BaP-induced nuclear factor-kB (NF-kB) activation, which regulates COX-2 expression. Third, although the aryl hydrocarbon receptor (AhR) did not play a role in BaP-induced COX-2 expression, the potentiation effect of E2 itself was AhR dependent. We further demonstrated that BaP induced the production of genotoxic E2 metabolites (2- and 4- hydroxyestradiols) via AhR-up-regulated cytochromes P450 1A1 and 1B1. These metabolites could directly activate NF-kB to further promote COX-2 mRNA expression in human lung epithelial cells. These findings were further supported by increased PGE2 secretion in rat lung slice cultures. Our findings that the BaP-VE2 interaction enhanced COX-2 expression and hydroxyestradiol accumulation in the media of cultivated lung cells and tissues provide the needed scientific basis for higher risk of BaP-associated lung cancer in females.

Guanine Alkylation by the Potent Carcinogen Aflatoxin B1: Quantum Chemical Calculations

We report a series of ab initio and density functional theory simulations of guanine alkylation by aflatoxin B 1 exo-8,9-epoxide. This reaction represents an initial step of carcinogenesis associated with aflatoxin B 1, a potent genotoxic fungal metabolite. Effects of hydration were considered in the framework of the Langevin dipoles solvation model and the solvent reaction field method of Tomasi and co-workers. In silico-calculated activation free energies are in good agreement with the experimental value of 15.1 kcal/mol. This agreement presents strong evidence in favor of the validity of the proposed S(N)2 reaction mechanism and points to the applicability of quantum chemical methods in studies of reactions associated with carcinogenesis. In addition, we predict that the preference of aflatoxin B 1 exo-8,9-epoxide over the endo stereoisomer for the reaction with guanine exists in the aqueous solution and is only further amplified in the DNA duplex. Finally, through comparison with an analogous reaction between 3a,6a-dihydrofuro[2,3- b]furan exo-4,5-epoxide and guanine, we show that the large planar body of aflatoxin B 1 does not enhance its reactivity and related carcinogenicity. This explains why the planar region of related mycotoxins sterigmatocystin and aflatoxin G 1 could have been evolutionarily optimized in a different way.

Effects of the environmental mammary carcinogen 6-nitrochrysene on p53 and p21 Cip1 protein expression and cell cycle regulation in MCF-7 and MCF-10A cells

The environmental pollutant 6-nitrochrysene (6-NC) is a potent mammary carcinogen in rats; it is more potent than numerous classical mammary carcinogens such as benzo[ a]pyrene (BaP). The mechanisms that account for the remarkable carcinogenicity of 6-NC remain elusive. Similar to BaP, 6-NC is also known to induce DNA damage in rodents and in human breast tissues. As an initial investigation, we reasoned that DNA damage induced by 6-NC may alter the expression of p53 protein in a manner that differs from other DNA damaging carcinogens (e.g. BaP). Using human breast adenocarcinoma MCF-7 cells and immortalized human mammary epithelial MCF-10A cells, we determined the effects of 6-NC on the expression of p53 protein and its direct downstream target cyclin-dependent kinase inhibitor p21 Cip1 as well as on the cell cycle progression. Western blot analysis demonstrated that treatments of MCF-7 and MCF-10A cells with 6-NC for 12, 24 or 48 h did not increase the level of total p53 protein; however, an increase of p21 Cip1 protein and a commitment increase of G 1 phase were observed in MCF-10A cells but not in MCF-7 cells. Further studies using 1,2-dihydroxy-1,2-dihydro-6-hydroxylaminochrysene (1,2-DHD-6-NHOH-C), the putative ultimate genotoxic metabolite of 6-NC, was conducted and showed a significant induction of p53 ( p < 0.05) in MCF-7 cells; however, this effect was not evident in MCF-10A cells, indicating the varied DNA damage responses between the two cell lines. By contrast to numerous DNA damaging agents such as BaP which is known to stimulate p53 expression, the lack of p53 response by 6-NC imply the lack of protective functions mediated by p53 (e.g. DNA repair machinery) after exposure to 6-NC and this may, in part, account for its remarkable carcinogenicity in the mammary tissue.

Comparison of Estimated Dietary Intake of Acrylamide with Hemoglobin Adducts of Acrylamide and Glycidamide

In a study comprising 50 subjects, we investigated the relationship between acrylamide (AA) intake from food using food frequency questionnaires and the concentration of hemoglobin (Hb) adducts of AA and its genotoxic metabolite glycidamide (GA) as a measure of the internal exposure. A method using solid-phase extraction and liquid chromatography with negative electrospray tandem mass spectrometric (MS/MS) detection for the determination of the Hb adducts as phenylthiohydantoin derivatives in human blood was developed. The limit of quantification for AA- and GA-Hb adducts were 2 and 6 pmol/g globin, respectively, and the between-assay precision was below 25%. The estimated dietary intake of AA was (median and range) 13.5 microg/day (4.1-72.6) in nonsmokers and 18.3 microg/day (7.8-32.0) in smokers. In nonsmokers, males had a higher intake than females, 16.6 microg/day (18.6-72.6) and 12.8 microg/day (4.1-30.2), respectively. Nonsmokers had a median AA and GA adduct concentration of 36.8 (range 17.9-65.5) and 18.2 (range 6.7-45.6) pmol/g globin, respectively. In smokers, the values were 165.8 (98.8-211) and 83.2 (29.1-99.0) pmol/g globin, respectively. Using multiple linear regression analysis, a significant positive correlation was found between the AA-Hb adduct concentration and the intake of chips/snacks and crisp bread. GA-Hb adduct did not correlate with consumption of any of the main food groups. Neither AA-Hb nor GA-Hb adduct concentration correlated with total dietary intake of AA as calculated from the reported food intake. Adduct concentrations did not correlate with 24 h urinary excretion of mercapturic acid metabolites of AA and GA in the same subjects reported previously.

The stereochemistry of benzo[a]pyrene‐2′‐deoxyguanosine adducts affects DNA methylation by SssI and HhaI DNA methyltransferases

The biologically most significant genotoxic metabolite of the environmental pollutant benzo[a]pyrene (B[a]P), (+)-7R,8S-diol 9S,10R-epoxide, reacts chemically with guanine in DNA, resulting in the predominant formation of (+)-trans-B[a]P-N(2)-dG and, to a lesser extent, (+)-cis-B[a]P-N(2)-dG adducts. Here, we compare the effects of the adduct stereochemistry and conformation on the methylation of cytosine catalyzed by two purified prokaryotic DNA methyltransferases (MTases), SssI and HhaI, with the lesions positioned within or adjacent to their CG and GCGC recognition sites, respectively. The fluorescence properties of the pyrenyl residues of the (+)-cis-B[a]P-N(2)-dG and (+)-trans-B[a]P-N(2)-dG adducts in complexes with MTases are enhanced, but to different extents, indicating that aromatic B[a]P residues are positioned in different microenvironments in the DNA-protein complexes. We have previously shown that the (+)-trans-isomeric adduct inhibits both the binding and methylating efficiencies (k(cat)) of both MTases [Subach OM, Baskunov VB, Darii MV, Maltseva DV, Alexandrov DA, Kirsanova OV, Kolbanovskiy A, Kolbanovskiy M, Johnson F, Bonala R, et al. (2006) Biochemistry45, 6142-6159]. Here we show that the stereoisomeric (+)-cis-B[a]P-N(2)-dG lesion has only a minimal effect on the binding of these MTases and on k(cat). The minor-groove (+)-trans adduct interferes with the formation of the normal DNA minor-groove contacts with the catalytic loop of the MTases. However, the intercalated base-displaced (+)-cis adduct does not interfere with the minor-groove DNA-catalytic loop contacts, allowing near-normal binding of the MTases and undiminished k(cat) values.

An alternatively spliced cytochrome P4501A1 in human brain fails to bioactivate polycyclic aromatic hydrocarbons to DNA‐reactive metabolites

CYP1A1, a cytochrome P450 enzyme, metabolizes polycyclic aromatic hydrocarbons to genotoxic metabolite(s) that bind to DNA and initiate carcinogenesis. RT-PCR amplification of the complete open reading frame of CYP1A1 generated an amplicon of 1593 bp having deletion of 87 bp of exon-6 that translated into functional P450 enzyme. Unlike wild type CYP1A1, exon 6 del CYP1A1 did not metabolize polycyclic aromatic hydrocarbons such as, benzo(a)pyrene to genotoxic, ultimate carcinogens that form DNA adducts. Exon 6 del CYP1A1 metabolized ethoxyresorufin (the classical substrate for CYP1A1) less efficiently compared with wild type CYP1A1 while pentoxy and benzyloxyresorufin (classical substrates for CYP2B) were dealkylated more efficiently. In silico docking showed alteration of the substrate access channel in exon 6 del CYP1A1 such that benzo(a)pyrene does not bind in any orientation that would permit the formation of carcinogenic metabolites. Genotyping revealed that the splice variant was not generated due to differences in genomic DNA sequence and the variant was present only in brain but not in liver, kidney, lung, or heart from the same individual. We provide evidence that unique P450 enzymes, generated by alternate splicing in a histiospecific manner can modify genotoxic potential of carcinogens such as benzo(a)pyrene by altering their biotransformation pathway.