Interleukin-6 selectively induces drug metabolism to potentiate the genotoxicity of dietary carcinogens in mammary cells.

Breast cancer is the most commonly diagnosed malignancy in females, the etiology being multifactorial and includes the role of lifestyle exposure to DNA-damaging chemicals such as dietary carcinogens benzo (a) pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP). Both compounds require cytochrome P450 (CYP)-mediated metabolic activation to DNA-damaging species, and both induce transcriptional responses through the nuclear receptors Aryl hydrocarbon receptor (AhR) and estrogen receptor α (ERα). BaP and PhIP are mammary carcinogens in rodents. Clinically, circulating IL-6 expression is linked with poor prognosis of cancer and 35% of the deaths in breast cancer are linked with inflammation. The objective of this work was to investigate the molecular toxicology and local activation of BaP and PhIP in the presence of IL-6. Our laboratory has previously reported that miR27b can regulate CYP1B1 expression in colorectal cells, here we have investigated if this mechanism is working in mammary cell models, MCF-7 and MDA-MB-231 cells. Treatment (24 h) of cells with BaP (10 nM-10 µM) and PhIP (100 nM-100 µM) significantly induced genetic damage (micronuclei formation) in a dose-dependent manner in both cell lines. This effect was potentiated in the presence of human IL-6 at concentrations reported to be expressed in clinical breast cancer. On its own, IL-6 treatment failed to induce micronuclei frequency above the control levels in these cells. Compared to BaP or PhIP treatment alone, IL-6 plus BaP or PhIP selectively induced CYP1B1 significantly in both cell lines. Additionally, miR27b expression was downregulated by IL-6 treatments and transfection with miR27b inhibitor confirmed that miR27b is a regulator of CYP1B1 in both cell lines. These data show that BaP- and PhIP-induced DNA damage in mammary cells is potentiated by the inflammatory cytokine IL-6 and that inflammation-induced CYP expression, specifically CYP1B1 via miR27b, is responsible for this effect.

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.

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.

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.

Viral Vectors: The Road to Reducing Genotoxicity.

Viral vector use in gene therapy has highlighted several safety concerns, including genotoxic events. Generally, vector-mediated genotoxicity results from upregulation of cellular proto-oncogenes via promoter insertion, promoter activation, or gene transcript truncation, with enhancer-mediated activation of nearby genes the primary mechanism reported in gene therapy trials. Vector-mediated genotoxicity can be influenced by virus type, integration target site, and target cell type; different vectors have distinct integration profiles which are cell-specific. Non-viral factors, including patient age, disease, and dose can also influence genotoxic potential, thus the choice of test models and clinical trial populations is important to ensure they are indicative of efficacy and safety. Efforts have been made to develop viral vectors with less risk of insertional mutagenesis, including self-inactivating (SIN) vectors, enhancer-blocking insulators, and microRNA targeting of vectors, although insertional mutagenesis is not completely abrogated. Here we provide an overview of the current understanding of viral vector-mediated genotoxicity risk from factors contributing to viral vector-mediated genotoxicity to efforts made to reduce genotoxicity, and testing strategies required to adequately assess the risk of insertional mutagenesis. It is clear that there is not a 'one size fits all' approach to vector modification for reducing genotoxicity, and addressing these challenges will be a key step in the development of therapies such as CRISPR-Cas9 and delivery of future gene-editing technologies.

Using 3D MCF-7 mammary spheroids to assess the genotoxicity of mixtures of the food-derived carcinogens benzo[a]pyrene and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

Genotoxic carcinogens are present in the human diet, and two important examples are benzo[a]pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). BaP is a polycyclic aromatic hydrocarbon generated by incomplete combustion of organic substances, thus contaminating numerous foodstuffs, and PhIP is a heterocyclic amine formed when meat is cooked. Genotoxicity testing of chemical carcinogens has focussed largely on individual chemicals, particularly in relation to diet, despite mixtures representing a more realistic exposure scenario. We have previously shown that exposure of MCL-5 cells to BaP-PhIP mixtures produces a TK mutation dose response that differs from the predicted additive response, using traditional regulatory-like two-dimensional (2D) cell culture. There is a large gap between 2D cell culture and the whole animal, and three-dimensional (3D) cell culture, shown to better represent in vivo tissue structure, may bridge the gap. The aim of the current study was to use 3D spheroids to characterise the DNA damage response following exposure to mixtures of the mammary carcinogens BaP and PhIP. Mammary MCF-7 cells were grown in 3D spheroids, exposed (24 h) to BaP (10-10 to 10-5 M) or PhIP (10-9 to 10-4 M) individually or in mixtures and DNA damage assessed by micronucleus (MN) formation. A dose-dependent increase in MN was observed for the individual chemicals in 3D cell culture. In line with our previous 2D TK mutation data, 3D mixture exposures gave a modified DNA damage profile compared to the individual chemicals, with a potent response at low dose combinations and a decrease in MN with higher concentrations of BaP in the mixture. Ethoxyresorufin-O-deethylase (CYP1A) activity increased with increasing concentration of BaP in the mixture, and for combinations with 10 µM BaP, CYP1A1 mRNA induction was sustained up to 48 h. These data suggest mixtures of genotoxic chemicals give DNA damage responses that differ considerably from those produced by the chemicals individually, and that 3D cell culture is an appropriate platform for DNA damage assays.

Interference with xenobiotic metabolic activity by the commonly used vehicle solvents dimethylsulfoxide and methanol in zebrafish (Danio rerio) larvae but not Daphnia magna.

Organic solvents, such as dimethylsulfoxide (DMSO) and methanol are widely used as vehicles to solubilise lipophilic test compounds in toxicity testing. However, the effects of such solvents upon innate detoxification processes in aquatic organisms are poorly understood. This study assessed the effect of solvent exposure upon cytochrome P450 (CYP)-mediated xenobiotic metabolism in Daphnia magna and zebrafish larvae (4d post fertilisation). Adult D. magna were demonstrated to have a low, but detectable, metabolism of ethoxyresorufin in vivo and this activity was not modulated by pre-exposure to DMSO or methanol (24 h, up to 0.1% and 0.05% v/v, respectively). In contrast, the metabolism of ethoxyresorufin in zebrafish larvae was significantly reduced by both solvents (0.1% and 0.05% v/v, respectively) after 24 h of exposure. In zebrafish, these observed decreases in activity towards ethoxyresorufin were accompanied by decreased expression of a variety of genes coding for drug metabolising enzymes (corresponding to CYP1, CYP2, CYP3 and UDP-glucuronyl transferase [UGT] family enzymes), measured by quantitative PCR. Reduction of gene expression and CYP1 enzyme activities by methanol (0.05% v/v) in zebrafish larvae was partially reversed by co-exposure with Aroclor 1254 (100 µg L(-1)). Overall this study suggests that relatively low concentrations of organic solvents can impact upon the biotransformation of certain xenobiotics in zebrafish larvae, and that this warrants consideration when assessing compounds for metabolism and toxicity in this species.

Transcriptional responses in neonate and adult Daphnia magna in relation to relative susceptibility to genotoxicants.

Little information is available on the responses of lower animals to genotoxic chemicals or on their sensitivity for detecting genotoxic chemicals, especially at different life-stages, despite the established use of the water flea Daphnia magna in ecotoxicity testing. Comet assay methodology was developed and applied to daphnid cells but only limited, non-statistically significant responses to the genotoxicants sodium dichromate (0.2-1 µM), chrysoidine (0.1-2 µM), and mixtures of benzo-a-pyrene (BaP) and sodium dichromate were found (from 0.01 µM BaP & 0.1 µM sodium dichromate to 0.25 µM BaP & 0.75 µM sodium dichromate). Transcriptomic analyses using Agilent D. magna oligonucleotide microarrays were undertaken to assess the effect of a mixture of sodium dichromate and BaP (designed to produce both adducted and oxidised DNA) on gene transcription. Neonates (<24h) and adults (day 7) were exposed for 6h and 24h at two combination concentration levels (0.02 µM BaP & 0.15 µM sodium dichromate and 0.1 µM BaP & 0.75 µM sodium dichromate). The greatest differences in transcriptional profile occurred between adults and neonates. Subsets of the transcriptional profiles distinguished genotoxicant-exposed animals from controls, both for neonates and adults. Higher transcript levels of DNA repair genes were found in adults and adults also displayed significant induction of DNA repair gene transcripts in response to exposure whereas neonates did not. Transcriptional changes in response to genotoxicant exposure proved more sensitive than measurement of DNA strand breaks by the Comet assay and the extensive differences in transcription between adults and neonates emphasized the importance of life stage in toxicant testing with Daphnia.

Induction of DNA strand breaks by genotoxicants in the alga Chlamydomonas reinhardtii.

The use of unicellular algae in ecotoxicity testing is well established, particularly regarding whole-organism and population-level end points such as lethality and population growth. Conflicting information exists, however, on the potential for genetic toxicity to be incorporated into the safety studies in this test organism. In the present study, DNA strand breaks (Comet assay) and ethoxyresorufin-O-deethylase (EROD) activity were used as indicators of genetic toxicity and cytochrome P450 1A baseline xenobiotic metabolism, respectively, in the unicellular green alga Chlamydomonas reinhardtii. DNA strand breaks were quantified following exposure to the direct-acting genotoxic agents 4-nitroquinoline-1-oxide (NQO) and the N-hydroxy metabolite of 2-acetylaminofluorene (N-OH-2-AAF) and the indirect-acting genotoxin chrysoidine. Following compound exposure, chrysoidine and N-OH-2-AAF produced statistically significant increases in DNA strand breaks at both 0.1 and 10 microM and 0.05 and 5 microM, respectively (p < 0.05 and p < 0.01). Different light sources were also found to influence DNA strand breaks, the minimum response being observed using a source that omits the ultraviolet range. Compared to many mammalian cells, both DNA damage responses and EROD activity were relatively weak. EROD activity was 0.03 pmol/min/10(6) cells in control cells, and the maximum level of DNA strand breaks observed was 14.1% at a 5 nM concentration of NQO. The responses exhibited were not enhanced by the use of a cell wall-free mutant strain. In conclusion, C reinhardtii responded, albeit weakly, to selected direct- and indirect-acting genotoxicants and also exhibited measurable EROD activity.