Both CYP1A1 and CYP1B1 are expressed extra-hepatic and are under regulatory control of the Ah receptor (Hankinson 2016). only in ER- (+) MCF-7 cells. Importantly, simultaneous treatments of physiological concentrations ethanol (10?3C10?1 M) with PhIP (10?7C10?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. two receptors, estrogen-receptor (ER-) and ER- with ER- being more abundantly expressed (Hewitt and Korach 2003) in approximately two-thirds of breast tumors and its presence determines the responsiveness towards hormone therapy (Williams et al. 2008). Interestingly, PhIP exhibits its estrogenic activity exclusively ER- (Lauber et al. 2004). The estrogenic behavior of PhIP has been shown to increase the invasiveness of breast cancer cells (Lauber and Gooderham 2011) but the role of ER in the genotoxicity and metabolic activation of PhIP has not been explored. A number of cytochrome P450 enzymes (CYPs) are known to be involved in metabolism of steroid hormones, particularly CYP1A1, 1A2 and 1B1 (Go et al. 2015). Additionally, CYP2E1 is usually reported to be differentially expressed in hormone-responsive MCF-7 cells compared to non-responsive MDA-MB-231 cells (Leung et al. 2013). Moreover, female steroid hormones (estrogen and progesterone) are known to regulate CYP2E1 expression (Konstandi et al. 2013). In view of the regulation of CYP2E1 estrogen and the hormone-like activity (estrogen) of PhIP (Lauber and Gooderham 2007), the possibility exists that PhIP might GHRP-6 Acetate regulate CYP2E1 expression. Epidemiology shows that consumption of ethanol is usually associated with breast cancer (Hamajima et al. 2002; Singletary and Gapstur 2001; Smith-Warner et al. 1998), with an intake of 10?g ethanol per day (approximately 1.25 units) increasing the risk of breast cancer between 6C10% (IARC 2012 https://monographs.iarc.fr/ENG/Monographs/vol96/mono96.pdf). Social consumption of ethanol readily achieves mM plasma concentrations. The risk is usually dose-dependent and the evidence that alcoholic drinks are a cause of pre- and post-menopausal breast cancer is usually sufficiently convincing that IARC have classed ethanol as a class 1 GHRP-6 Acetate carcinogen (carcinogenic in humans) (https://monographs.iarc.fr/ENG/Monographs/vol96/mono96.pdf). Although ethanol can be metabolised to acetaldehyde, which forms adducts with DNA (Abraham et al. 2011), overall the case for ethanol being a genotoxic carcinogen is usually weak (https://www.gov.uk/government/publications/consumption-of-alcoholic-beverages-and-risk-of-cancer), and a non-genotoxic mode of action is likely to contribute. Rabbit polyclonal to PRKCH Thus, although epidemiological evidence supports a positive association between alcohol intake and the risk for breast cancer, a mechanistic understanding of this association is usually lacking. In the present work, we describe mechanistic studies that explore the toxicity of PhIP and ethanol and their respective abilities to damage DNA. We further show the involvement of ER- and that ethanol can potentiate the genotoxicity of the mammary carcinogen PhIP through mutually interactive biochemistry. Methods Cell culture and treatment The human breast adenocarcinoma MCF-7 (ER-+) and MDA-MB-231 (ER-?) cell lines were purchased from ATCC (LGC Prochem, Middlesex,UK) and were grown in minimum essential medium (MEM) (GIBO, Life technologies, Paisley, UK) supplemented with 10% fetal bovine serum (FBS), 100 units/ml of penicillin and streptomycin 100?g and 2?mM L-glutamine. Cells were cultured routinely in 75-cm2 flasks in a humidified incubator at 37?C, 5% CO2. Prior to treatment, cells (MCF-7 and MDA-MB-231) at a density of 25,000 cells/well in 24-well plates, were cultured in MEM supplemented with 5% dextran-coated charcoal-stripped FBS (Stripped media) for 72?h. Cells were treated with PhIP (0C100?M, Toronto Research Chemicals Inc., Toronto, Canada) and Estradiol (E2) dissolved in dimethyl sulphoxide (DMSO). For treatment with estrogen-receptor inhibitor, cells were co-treated with PhIP and selective estrogen inhibitor Fulvestrant ICI 182,780 (ICI) (Sigma-Aldrich) for 24?h. PhIP, E2 and ICI were dissolved in DMSO. For STAT3 inhibition, cells were co-treated for 24?h with PhIP and 25?M STAT3 inhibitor (STAT3 inhibitor VIII 5, 15 diphenylporphyrin, Millipore, Feltham, UK). STAT3 inhibitor was dissolved in DMSO. For ethanol treatment, GHRP-6 Acetate media was supplemented with different concentrations of ethanol (10?mM-100?mM, Sigma-Aldrich) and was added to the cells. In some experiments, (10?min 2C8?C). The upper aqueous phase was transferred to a fresh tube and 5?g of RNase-free GHRP-6 Acetate glycogen (as carrier to aqueous phase) and 0.5?ml of isopropyl alcohol was added to precipitate RNA.