Induction of CYP1A1 and CYP1B1 and formation of carcinogen–DNA adducts in normal human mammary epithelial cells treated with benzo[a]pyrene
Introduction
Human exposure to mixtures of environmental pollutants that include polycyclic aromatic hydrocarbons (PAHs) is unavoidable [1]. Because many of these compounds are human carcinogens, elevated cancer rates may occur in individuals subjected to environmental and occupational PAH exposures [2]. Carcinogenic PAHs induce multiple metabolic pathways in human tissues and cells, as evidenced by DNA-microarray studies [3], [4]. In humans, carcinogenic PAHs are biotransformed through induction of multiple enzyme systems, particularly the cytochrome P450s [5], [6], and wide inter-individual variation in response to PAH exposures has been documented [4]. As breast cancer incidences appear to be increasing in recent years, several molecular epidemiologic studies have focused on normal breast tissue and breast cancer cells, and established associations between patterns of carcinogen–DNA adduct formation and cytochrome P450 polymorphisms [7], [8], [9]. These studies suggest that both environmental and genetic factors may contribute to the burden of human female breast cancer.
While CYP1A1 was cloned in 1985 and its expression had been studied long before that, CYP1B1 was not cloned until almost a decade later and has been less intensively investigated [10], [11]. Both CYP1A1 and CYP1B1 enzymes may be involved in the metabolic activation of exogenous carcinogens that reach human mammary tissues [12], [13], [14], [15], [16]. In addition, CYP1B1, known to metabolize steroids, may be involved in the metabolic activation of estrogens to carcinogenic intermediates; consequently, it has been identified as a target for inhibition in anticancer strategies [6], [17]. Both CYP1A1 and CYP1B1 are activating enzymes that can metabolize xenobiotics in concert with detoxifying enzymes. Glutathione-S-transferase M1 (GSTM1), a detoxication enzyme, contributes to the disposition of PAHs by effectively lowering the concentration of activated xenobiotic available for DNA-binding.
In these experiments, we have used normal human mammary epithelial cells (NHMECs), cultured from normal mammary tissues obtained at reduction mammoplasty from 22 individuals to study gene expression of the carcinogen-metabolizing enzymes CYP1A1 and CYP1B1. The data have been stratified by GSTM1 genotype. NHMEC strains were exposed to the carcinogenic PAH benzo[a]pyrene (BP) to explore inter-individual variability in BP–DNA adduct formation and to compare BP–DNA adduct formation with expression of the metabolic enzymes. An earlier study has documented the metabolism of PAHs in cells of similar derivation [18], but advanced methods to compare gene expression of the metabolic activation enzymes with DNA adduct formation (the toxicogenomic aspects of PAH metabolism) were not available at that time. In a previous study [4], we used oligonucleotide arrays to investigate the complete pattern of gene expression changes in BP-exposed cultured NHMECs obtained from four different donors, and those studies showed that some of the most consistently and extensively induced genes were CYP1A1 and CYP1B1. Here we compare the formation of BP–DNA adducts with the extent of CYP1A1 and CYP1B1 induction in cell strains from 22 individuals exposed to BP. Values for CYP1A1 and CYP1B1 induction determined by oligonucleotide arrays were confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and stratified by GSTM1 status for comparison with BP–DNA adduct levels.
Section snippets
Human tissues and cells
Reduction mammoplasty provides a valuable source of normal human mammary epithelial cells. Cells were isolated from discarded tissues of female donors by a process of mechanical and enzymatic disruption that has been well documented [19]. Cell strains were cultured for several passages, frozen and re-cultured in order to expose multiple strains simultaneously at the same passage. Tissues for this study were obtained through the Cooperative Human Tissue Network, which is sponsored by the
Origins of mammary epithelial cells
Normal human mammary epithelial cells were obtained from tissues salvaged at reduction mammoplasty. The 23 donors in this study were between 18 and 51 years old (28±7 years, mean±SD) and most were Caucasian (Table 1). There was no correlation between age and CYP1A1 or CYP1B1 induction or BP–DNA adduct levels (r=−0.032, −0.117 and 0.177, respectively).
Expression of CYP1A1 and CYP1B1 by oligonucleotide arrays and RT-PCR
RNA samples from 22 cells strains were examined for a battery of gene expression changes in BP-exposed cells (4 μM BP, 12 h), compared to unexposed
Discussion
This report shows that measurement of induction of cytochrome P450 genes, at the transcription level, by TaqMan™-based RT-PCR is highly correlated with DNA-oligonucleotide microarray data. However, RT-PCR is considerably more sensitive than DNA-microarrays for the determination of CYP1A1 and CYP1B1 transcription. These studies were originally designed to identify key gene expression biomarkers of BP exposure in normal human epithelial cells, and use those biomarkers to define inter-individual
Acknowledgements
The Cooperative Human Tissue Network, sponsored by the NCI and NDRI, is a critical source of human mammary tissue that is used for our on-going development of a large panel of normal human mammary epithelial cells. We are also indebted to Glory Johnson for invaluable editorial support.
References (51)
- et al.
Transcriptional signatures of environmentally relevant exposures in normal human mammary epithelial cells: benzo[a]pyrene
Cancer Lett
(2005) - et al.
Cytochrome P450 1B1: a target for inhibition in anticarcinogenesis strategies
Mutat. Res.
(2003) - et al.
Analyses of bulky DNA adduct levels in human breast tissue and genetic polymorphisms of cytochromes P450 (CYPs), myeloperoxidase (MPO), quinone oxidoreductase (NQO1), and glutathione S-transferases (GSTs)
Mutat. Res.
(2002) - et al.
Complete cDNA sequence of a human dioxin-inducible mRNA identifies a new gene subfamily of cytochrome P450 that maps to chromosome 2
J. Biol. Chem.
(1994) - et al.
Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method
Methods
(2001) - et al.
Comprehensive analysis of gene expression in rat and human hepatoma cells exposed to the peroxisome proliferator WY14,643
Toxicol. Appl. Pharmacol.
(2003) - et al.
A comparison of gene expression changes in response to diethylstilbestrol treatment in wild-type and p53+/− hemizygous knockout mice using focussed arrays
Toxicology
(2003) - et al.
Quantitative analysis of the Ah receptor/cytochrome P450 CYP1B1/CYP1A1 signalling pathway
Biochem. Pharmacol.
(2003) Range of environmentally responsive monooxygenase activities in human placental microsomes determined by direct fluorescence techniques
Biochem. Pharmacol.
(1981)- et al.
Tissue-specific induction of cytochromes P450 1A1 and 1B1 by polycyclic aromatic hydrocarbons and polychlorinated biphenyls in engineered C57BL/6J mice of arylhydrocarbon receptor gene
Toxicol. Appl. Pharmacol.
(2003)
Benzo(a)pyrene exposure induces CYP1A1 activity and expression in human endometrial cells
J. Steroid Biochem. Mol. Biol.
Certain polycyclic aromatic hydrocarbons and heterocyclic compounds
Chemical carcinogenesis
Applications of gene arrays in environmental toxicology: fingerprints of gene regulation associated with cadmium chloride, benzo(a)pyrene, and trichloroethylene
Environ. Health Perspect.
Mammary expression of xenobiotic metabolizing enzymes and their potential role in breast cancer
Cancer Res.
Genetic and environmental determinants on tissue response to in vitro carcinogen exposure and risk of breast cancer
Cancer Res.
Aromatic DNA adducts and polymorphisms of CYP1A1, NAT2, and GSTM1 in breast cancer
Carcinogenesis
Human dioxin-inducible cytochrome P1-450: complementary DNA and amino acid sequence
Science
A null association between active or passive cigarette smoking and breast cancer risk
Breast Cancer Res. Treat.
Cigarette smoking and the risk of breast cancer in women: a review of the literature
Cancer Epidemiol. Biomarkers Prev.
Active and passive smoking and risk of breast cancer by age 50 years among German women
Am. J. Epidemiol.
Active and passive smoking in breast cancer: prospective results from the Nurses' Health Study
Epidemiology
Is there an association between passive smoking and breast cancer?
Eur. J. Epidemiol.
Elevated 4-hydroxylation of estradiol by hamster kidney microsomes: a potential pathway of metabolic activation of estrogens
Endocrinology
Polycyclic hydrocarbon activation and metabolism in epithelial cell aggregates prepared from human mammary tissue
Int. J. Cancer
Cited by (30)
Modification of adverse health effects of maternal active and passive smoking by genetic susceptibility: Dose-dependent association of plasma cotinine with infant birth size among Japanese women—The Hokkaido Study
2017, Reproductive ToxicologyCitation Excerpt :Tobacco smoke is a complex mixture of more than 4000 chemicals, such as polycyclic aromatic hydrocarbons (PAHs), N-nitrosamines, and nicotine, among others [12,13]. PAHs such as benzo[a]pyrene (BP) bind to the aromatic hydrocarbon receptor (AHR), and activate metabolic intermediates such as benzo[a]pyrene diolepoxide (BPDE) via xenobiotic-metabolizing enzymes (Phase I), including cytochrome P450 (CYP) 1A1, CYP1A2, and CYP1B1 [14,15]. The metabolic intermediates are then detoxified by xenobiotic-metabolizing enzymes (Phase II) such as glutathione S-transferase (GST) M1 and GSTT1, after which they are eliminated from the body [16].
Constitutive expression of the AHR signaling pathway in a bovine mammary epithelial cell line and modulation by dioxin-like PCB and other AHR ligands
2015, Toxicology LettersCitation Excerpt :At the same time, several evidences demonstrate the up-regulation of CYP1A1 and CYP1B1 in human and rodent mammary cell lines challenged with different DL-compounds (Chen et al., 2004), as well as in both mouse and rat mammary tissue following the in vivo treatment with TCDD or β-NAF, respectively (Collins et al., 2009; Larsen et al., 2004). As regards B[a]P, an increase in the levels of CYP1A1 and CYP1B1 mRNA has been reported in either human normal mammary epithelial and breast cancer cells after 12 and 6 h incubation, respectively (Kemp et al., 2006; Keshava et al., 2005a). Such data are consistent with the B[a]P-mediated time-course induction of CYP1A1 and CYP1B1 reported herein; however, the ligand concentrations employed in those studies were much higher than ours (5 μM vs 100 nM), suggesting that the bovine mammary cells might be more sensitive than their human counterparts to CYP1 family enzyme up-regulation upon B[a]P exposure.
Diallyl trisulfide as an inhibitor of benzo(a)pyrene-induced precancerous carcinogenesis in MCF-10A cells
2012, Food and Chemical ToxicologyCitation Excerpt :Studies vary as to BaP’s impact on cell proliferation in normal and cancerous breast cell lines. In normal primary breast tissue and several neoplastic breast cell lines (MCF-7, HCC 1806, T47-D, and MDA MB 231), BaP was not shown to significantly alter cell viability following a single exposure after 24 h (Keshava et al., 2005a,b; Sigounas et al., 2010; Tampio et al., 2009; Sadikovic and Rodenhiser, 2006). Our results were similar, as BaP did not significantly increase cell proliferation at 24 h, but increases were significant at 6 h, a time point that was not evaluated in other studies.
Transcriptional profiles of benzo(a)pyrene exposure in normal human mammary epithelial cells in the absence or presence of chlorophyllin
2008, Mutation Research - Fundamental and Molecular Mechanisms of MutagenesisBiomonitoring: Is body burden relevant to public health?
2006, Regulatory Toxicology and Pharmacology