Rhiannon M. David

Synergistic and Antagonistic Mutation Responses of Human MCL-5 Cells to Mixtures of Benzo[a]pyrene and 2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]pyridine: Dose-Related Variation in the Joint Effects of Common Dietary Carcinogens.

Background Chemical carcinogens such as benzo[a]pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) may contribute to the etiology of human diet-associated cancer. Individually, these compounds are genotoxic, but the consequences of exposure to mixtures of these chemicals have not been systematically examined. Objectives We determined the mutagenic response to mixtures of BaP and PhIP at concentrations relevant to human exposure (micromolar to subnanomolar). Methods Human MCL-5 cells (metabolically competent) were exposed to BaP or PhIP individually or in mixtures. Mutagenicity was assessed at the thymidine kinase (TK) locus, CYP1A activity was determined by ethoxyresorufin-O-deethylase (EROD) activity and qRT-PCR, and cell cycle was measured by flow cytometry. Results Mixtures of BaP and PhIP produced dose responses different from those of the individual chemicals; we observed remarkably increased mutant frequency (MF) at lower concentrations of the mixtures (not mutagenic individually), and decreased MF at higher concentrations of the mixtures, than the calculated predicted additive MF of the individual chemicals. EROD activity and CYP1A1 mRNA levels were correlated with TK MF, supporting involvement of the CYP1A family in mutation. Moreover, a cell cycle G2/M phase block was observed at high-dose combinations, consistent with DNA damage sensing and repair. Conclusions Mixtures of these genotoxic chemicals produced mutation responses that differed from those expected for the additive effects of the individual chemicals. The increase in MF for certain combinations of chemicals at low concentrations that were not genotoxic for the individual chemicals, as well as the nonmonotonic dose response, may be important for understanding the mutagenic potential of food and the etiology of diet-associated cancers. Citation David R, Ebbels T, Gooderham N. 2016. Synergistic and antagonistic mutation responses of human MCL-5 cells to mixtures of benzo[a]pyrene and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine: dose-related variation in the joint effects of common dietary carcinogens. Environ Health Perspect 124:88–96; http://dx.doi.org/10.1289/ehp.1409557

The selective cytotoxicity of the alkenyl glucosinolate hydrolysis products and their presence in Brassica vegetables.

Cruciferous vegetable consumption correlates with reduced risk of cancer. This chemopreventative activity may involve glucosinolates and their hydrolysis products. Glucosinolate-derived isothiocyanates have been studied for their toxicity and chemopreventative properties, but other hydrolysis products (epithionitriles and nitriles) have not been thoroughly examined. We report that these hydrolysis products differ in their cytotoxicity to human cells, with toxicity most strongly associated with isothiocyanates rather than epithionitriles and nitriles. We explored mechanisms of this differential cytotoxicity by examining the role of oxidative metabolism, oxidative stress, mitochondrial permeability, reduced glutathione levels, cell cycle arrest and apoptosis. 2-Propenylisothiocyanate and 3-butenylisothiocyanate both inhibited cytochome P450 1A (CYP1A) enzyme activity in CYP expressing MCL-5 cells at high cytotoxic doses. Incubation of MCL-5 cells with non-cytotoxic doses of 2-propenylisothiocyanate for 24h resulted in a dose-dependent inhibition of ethoxyresorufin O-deethylase, yet failed to affect CYP1A1 mRNA expression indicating interference with enzyme activity rather than inhibition of transcription. Increased reactive oxygen species (ROS) production was observed only for 2-propenylisothiocyanate treatment. 2-Propenylisothiocyanate treatment lowered reduced glutathione levels whereas no changes were noted with 3,4-epithiobutylnitrile. Cell cycle analysis showed that 2-propenylisothiocyanate induced a G2/M block whereas other hydrolysis products showed only marginal effects. We found that 2-propenylisothiocyanate and 3-butenylisothiocyanate induced cell death predominantly via necrosis whereas, 3,4-epithiobutylnitrile promoted both necrosis and apoptosis. Thus the activity of glucosinolate hydrolysis products includes cytotoxicity that is compound-class specific and may contribute to their putative chemoprotection properties.

The mutagenic effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine in Muta™Mouse colon is attenuated by resveratrol

Epidemiology studies show that consumption of certain naturally occurring chemicals (generally plant-derived) can protect against the development of cancer (chemoprevention). Resveratrol, a phytoalexin found in foods such as grapes and wine as a glucoside, is one such chemical. Our group has previously shown that treating mammalian cells with resveratrol aglycone can reduce the mutagenicity of the cooked meat-derived carcinogens 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP). However, the anti-mutagenic effect of resveratrol in vivo has not been previously reported. In this study, daily treatment of resveratrol up to 1000 μg kg−1 for 10 days was well tolerated in Muta™Mouse mice. Treating Muta™Mouse mice with the meat carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine induced mutation in the colon. Co-treatment of resveratrol and PhIP reduced the mutation frequency induced by PhIP in the colon by about 25% in a treatment group of mixed male and females. Analysing males and females separately revealed a sex difference in the response to PhIP and to the effect of resveratrol on PhIP-induced mutagenicity. In males compared to females, PhIP was a more potent colonic mutagen and resveratrol was more effective at attenuating the mutagenic response (∼35% in males but only 9% in females). The reason for this sex difference in both PhIP mutagenicity and response to resveratrol is not clear. However, resveratrol treatment was also shown to powerfully inhibit ethoxyresorufin-O-deethylase activity in vivo, indicating that the antimutagenic effects are likely linked to metabolic activation of PhIP. These data suggest that resveratrol has anti-mutagenic effects in vivo, supporting its potential to act as a chemopreventative.

Dose-dependent synergistic and antagonistic mutation responses of binary mixtures of the environmental carcinogen benzo[a]pyrene with food-derived carcinogens

Cooking food at high temperatures produces genotoxic chemicals and there is concern about their impact on human health. DNA damage caused by individual chemicals has been investigated but few studies have examined the consequences of exposure to mixtures as found in food. The current study examined the mutagenic response to binary mixtures of benzo[a]pyrene (BaP) with glycidamide (GA), BaP with acrylamide (AC), or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) with GA at human-relevant concentrations (sub-nM). The metabolically competent human MCL-5 cells were exposed to these chemicals individually or in mixtures and mutagenicity was assessed at the thymidine kinase (TK) locus. Mixture exposures gave dose–responses that differed from those for the individual chemicals; for the BaP-containing mixtures, an increased mutation frequency (MF) at low concentration combinations that were not mutagenic individually, and decreased MF at higher concentration combinations, compared to the calculated predicted additive MF of the individual chemicals. In contrast, the mixture of PhIP with GA did not increase MF above background levels. These data suggest BaP is driving the mutation response and that metabolic activation plays a role; in mixtures with BaP the increased/decreased MF above/below the expected additive MF the order is PhIP > AC > GA. The increase in MF at some low concentration combinations that include BaP is interesting and supports our previous work showing a similar response for BaP with PhIP, confirming this response is not limited to the BaP/PhIP combination. Moreover, the lack of a mutation response for PhIP with GA relative to the response of the individual chemicals at equivalent doses is interesting and may represent a potential avenue for reducing the risk of exposure to environmental carcinogens; specifically, removal of BaP from the mixture may reduce the mutation effect, although in the context of food this would be significantly challenging.

Ethanol potentiates the genotoxicity of the food-derived mammary carcinogen PhIP in human estrogen receptor-positive mammary cells: mechanistic support for lifestyle factors (cooked red meat and ethanol) associated with mammary cancer

Consumption of cooked/processed meat and ethanol are lifestyle risk factors in the aetiology of breast cancer. Cooking meat generates heterocyclic amines such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Epidemiology, mechanistic and animal studies indicate that PhIP is a mammary carcinogen that could be causally linked to breast cancer incidence; PhIP is DNA damaging, mutagenic and oestrogenic. PhIP toxicity involves cytochrome P450 (CYP1 family)-mediated metabolic activation to DNA-damaging species, and transcriptional responses through Aryl hydrocarbon receptor (AhR) and estrogen-receptor-α (ER-α). Ethanol consumption is a modifiable lifestyle factor strongly associated with breast cancer risk. Ethanol toxicity involves alcohol dehydrogenase metabolism to reactive acetaldehyde, and is also a substrate for CYP2E1, which when uncoupled generates reactive oxygen species (ROS) and DNA damage. Here, using human mammary cells that differ in estrogen-receptor status, we explore genotoxicity of PhIP and ethanol and mechanisms behind this toxicity. Treatment with PhIP (10−7–10−4 M) significantly induced genotoxicity (micronuclei formation) preferentially in ER-α positive human mammary cell lines (MCF-7, ER-α+) compared to MDA-MB-231 (ER-α−) cells. PhIP-induced CYP1A2 in both cell lines but CYP1B1 was selectively induced in ER-α(+) cells. ER-α inhibition in MCF-7 cells attenuated PhIP-mediated micronuclei formation and CYP1B1 induction. PhIP-induced CYP2E1 and ROS via ER-α-STAT-3 pathway, but only in ER-α (+) MCF-7 cells. Importantly, simultaneous treatments of physiological concentrations ethanol (10−3–10−1 M) with PhIP (10−7–10−4 M) increased oxidative stress and genotoxicity in MCF-7 cells, compared to the individual chemicals. Collectively, these data offer a mechanistic basis for the increased risk of breast cancer associated with dietary cooked meat and ethanol lifestyle choices.

Mechanistic evidence that benzo[a]pyrene promotes an inflammatory microenvironment that drives the metastatic potential of human mammary cells

Benzo[a]pyrene (B(a)P) is a major cancer-causing contaminant present in food such as cooked meats and cereals, and is ubiquitous in the environment in smoke derived from the combustion of organic material. Exposure to B(a)P is epidemiologically linked with the incidence of breast cancer. Although B(a)P is recognized as a complete genotoxic carcinogen, thought to act primarily via CYP-mediated metabolic activation to DNA-damaging species, there is also evidence that B(a)P exposure elicits other biological responses that promote development of the cancer phenotype. Here in mechanistic studies using human mammary cells MCF-7 and MDA-MB-231, we have explored mechanisms whereby B(a)P (10− 8 to 10− 5M) promotes inflammation pathways via TNF-α and NFκB leading to IL-6 upregulation, microRNA (Let7a, miR21 and miR29b) dysregulation and activation of VEGF. The miRNA dysregulation is associated with altered expression of inflammation mediators and increased migration and invasive potential of human mammary cancer cells. Our data suggest that mammary cell exposure to B(a)P results in perturbation of inflammation mediators and dysregulation of tumorigenic miRNAs, leading to an inflammation microenvironment that facilitates migration and invasion of mammary epithelial cells. These properties of B(a)P, together with its well-established metabolic activation to DNA-damaging species, offer mechanistic insights into its carcinogenic mode of action.

Abstract 5454: Genotoxic consequences of exposure to mixtures of food-derived chemical carcinogens

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL It has been shown that mutagenic chemicals can form naturally in foods during cooking. Examples include Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon generated by incomplete combustion of organic substances such as lipids, leading to contamination of numerous foodstuffs (Lijinski and Shubik, Science 1964, 145, 53), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine that is formed during the cooking of meat (Murray, et al. J Chromatog 1993, 616, 211), and glycidamide (GA) is the ultimate genotoxic metabolite of acrylamide (AC), which forms when foods, such as potatoes and cereals, are cooked at temperatures exceeding 100°C, in a reaction between the amino acid asparagine and reducing sugars that are naturally present in these foods (Tornqvist, Adv Exp Biol Med 2005, 561, 1). These chemicals have individually been shown to be genotoxic but the biological consequences of exposure to mixtures of these chemicals have not been systematically examined. The aim of the current study was to examine the biological response of MCL-5 cells (a metabolically competent human lymphoblastoid cell line) to mixtures of these genotoxic chemicals at concentrations that are relevant to human exposure (mM to sub-nM). Cells were exposed to the chemicals individually or in mixtures for 24h and mutagenicity was assessed through resistance to trifluorothymidine at the thymidine kinase (TK) locus and 6-thioguanine at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus. The mixture exposures gave modified dose-response curves from the individual chemicals, with increased mutation frequency (MF) at some low concentration combinations that were not mutagenic individually, and decreased MF at some combinations of higher concentrations that did produce a mutation response individually. Responses also differed between the TK and HPRT loci, with a higher MF observed for high concentration combinations at HPRT compared to TK. Ethoxyresorufin-O-deethylase (EROD) activity was measured and was found to correspond to the MF at the TK locus suggesting the involvement of CYP1A family in the mutation response. These data suggest that mixtures of genotoxic chemicals produce different mutation responses to the individual chemicals. The increase in MF at some low concentration combinations where the individual chemicals are not genotoxic is of significance when considering the carcinogenic potential of food. 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 5454. doi:1538-7445.AM2012-5454

Abstract 4630: Cruciferous vegetable glucosinolate hydrolysis products protect against Benzo[a]pyrene-induced genotoxicity

Recent epidemiology studies have indicated that cruciferous vegetable intake is inversely correlated with lung cancer risk among smokers (Tang et al., BMC Cancer 2010, 10, 162). Benzo[a]pyrene (BaP) is a prominent contaminant of cigarette smoke, a model genotoxic carcinogen and powerful mutagen. Glucosinolates found in cruciferous vegetables are known to be hydrolysed to compounds that have been shown to be chemopreventive in both animal and cell-based systems, while, paradoxically, evidence is gathering that high concentrations of some of these hydrolysis products are genotoxic. The aim of the current study was to investigate whether pre-treatment of MCL-5 cells (a metabolically competent human lymphoblastoid cell line) with glucosinolate hydrolysis products conferred protection against the cytotoxicity and genotoxicity induced by a subsequent exposure to BaP. In the case of AITC (allyl isothiocyanate), a model glucosinolate hydrolysis product, toxicity and mutagenicity to MCL-5 cells was established for a range of concentrations. Mutagenicity was assessed through resistance to trifluorothymidine at the thymidine kinase (TK) locus and 6-thioguanine at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus. The chemical treatments revealed a consistent trend for reduced cytotoxicity in cells pre-treated with AITC prior to BaP compared to those treated with BaP alone. Mutation frequency at the TK locus was consistently lower in the cells pre-treated with AITC compared to those treated with BaP alone. High concentrations of AITC induced a prolonged, dose-dependent cytotoxicity with cells only returning to normal growth rate after 7 days. These data suggest that pre-treatment of MCL-5 cells with glucosinolate hydrolysis products such as AITC affords protection against the genotoxic insult induced by mutagens such as BaP. 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 4630. doi:10.1158/1538-7445.AM2011-4630