Brinda Mahadevan

Altered Gene Expression Patterns in MCF-7 Cells Induced by the Urban Dust Particulate Complex Mixture Standard Reference Material 1649a

Human exposures to polycyclic aromatic hydrocarbon (PAH) occur in complex mixtures. Here, gene expression patterns were investigated using standard reference material (SRM) 1649a (urban dust). MCF-7 cells were exposed to SRM 1649a alone or SRM 1649a with either benzo[a]pyrene (BP) or dibenzo[a,l]pyrene (DBP) for 24 hours. Global analyses of the gene expression data revealed alterations of 41 RNA transcripts with at least 2-fold change (signal log ratio </= -1 or >/= 1) in response to SRM 1649a exposure. Increase in expression of cytochrome P450 (CYP) genes was observed in response to BP exposure (CYP1A1 and CYP1B1; signal log ratio of 4.7 and 2.5, respectively). An additive induction of CYP1A1 and CYP1B1 was observed with cotreatment of SRM 1649a and BP. On the contrary, no change in gene expression of CYP1A1 and CYP1B1 was observed when the cells were exposed to DBP. Furthermore, to study the effect of complex PAH mixtures on the metabolic activation of carcinogenic PAH to DNA-binding derivatives and to relate this with gene expression studies, PAH-DNA adduct formation was determined. SRM 1649a decreased the total level of BP-DNA adducts in comparison with BP alone. No significant difference in adduct levels was observed in response to either DBP alone or in combination with SRM 1649a. These results provide a transcriptional signature for chemical carcinogen exposure; in addition, they suggest a major factor in carcinogenic activity of PAH within complex mixtures is their ability to promote or inhibit the activation of carcinogenic PAH by the induction of CYP enzymes.

Activation of the aryl hydrocarbon receptor is the major toxic mode of action of an organic extract of a reference urban dust particulate matter mixture: The role of polycyclic aromatic hydrocarbons

Many of the toxic and carcinogenic effects of urban air pollution have been linked to polycyclic aromatic hydrocarbons (PAHs) adsorbed to airborne particulate matter (PM). The carcinogenic properties of PAHs in complex organic mixtures derived from PM have been chiefly attributed to their mutagenicity. Nevertheless, PAHs are also potent activators of the aryl hydrocarbon receptor (AhR), which may contribute to their nongenotoxic effects, including tumor promotion. As the genotoxicity of carcinogenic PAHs in complex mixtures derived from urban PM is often inhibited by other mixture constituents, the AhR-mediated activity of urban PM extracts might significantly contribute to the carcinogenic activity of such mixtures. In the present study, we used an organic extract of the urban dust standard reference material, SRM1649a, as a model mixture to study a range of toxic effects related to DNA damage and AhR activation. Both the organic extract and its neutral aromatic fraction formed a low number of DNA adducts per nucleotide in the liver epithelial WB-F344 cells model, without inducing DNA damage response, such as tumor suppressor p53 activation and apoptosis. In contrast, we found that this extract, as well as its neutral and polar fractions, were potent inducers of a range of AhR-mediated responses, including induction of the AhR-mediated transcription, such as cytochrome P450 1A1/1B1 expression, and the AhR-dependent cell proliferation. Importantly, these toxic events occurred at doses one order of magnitude lower than DNA damage. The AhR-mediated activity of the neutral fraction was linked to PAHs and their derivatives, as polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls were only minor contributors to the overall AhR-mediated activity. Taken together, our data suggest that more attention should be paid to the AhR-dependent nongenotoxic events elicited by urban PM constituents, especially PAHs and their derivatives.

Competitive inhibition of carcinogen‐activating CYP1A1 and CYP1B1 enzymes by a standardized complex mixture of PAH extracted from coal tar

A complex mixture of polycyclic aromatic hydrocarbons (PAH) extracted from coal tar, the Standard Reference Material (SRM) 1597, was recently shown to decrease the levels of DNA binding of the 2 strong carcinogens benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) in the human mammary carcinoma‐derived cell line MCF‐7 (Mahadevan et al., Chem Res Toxicol 2005;18:224–231). The present study was designed to further elucidate the biochemical mechanisms involved in this inhibition process. We examined the effects of SRM 1597 on the metabolic activation of BP and DBP toward DNA‐binding derivatives in Chinese hamster cells expressing either human cytochrome P450 (CYP) 1A1 or CYP1B1. SRM 1597 inhibited BP‐DNA adduct formation through the entire exposure time in cells expressing human CYP1A1, while it significantly inhibited adduct formation only up to 48 hr when co‐treated with DBP. Conversely, human CYP1B1‐expressing cells were unable to catalyze PAH‐DNA adduct formation on treatment with SRM 1597 alone, and on co‐treatment with BP or DBP. The data obtained from biochemical experiments revealed that SRM 1597 competitively inhibited the activity of both human enzymes as analyzed by 7‐ethoxyresorufin O‐deethylation assays. While the Michaelis‐Menten constant (KM) was <0.4 μM in the absence of SRM 1597, this value increased up to 1.12 (CYP1A1) or 4.45 μM (CYP1B1) in the presence of 0.1 μg/ml SRM 1597. Hence the inhibitory effects of the complex mixture on human CYP1B1 were much stronger when compared to human CYP1A1. Taken together, the decreases in PAH‐DNA adduct formation on co‐treatment with SRM 1597 revealed inhibitory effects on the CYP enzymes that convert carcinogenic PAH into DNA‐binding metabolites. The implications for the tumorigenicity of complex environmental PAH mixtures are discussed.

Reduction in Tamoxifen-Induced CYP3A2 Expression and DNA Adducts Using Antisense Technology

Tamoxifen (TAM) is widely used in the treatment and prevention of breast cancer. There is clear evidence that cytochrome P450 (CYP) 3A enzymes play an important role in TAM metabolism, resulting in metabolites that lead to formation of TAM–DNA adducts. We have investigated the effect of CYP3A2 antisense (AVI‐4472) exposure on CYP3A2 transcription, enzyme activity, translation, and TAM–DNA adducts, in livers of rats administered TAM (50 mg/kg body weight [bw]/day) for 7 days. The study design included administration of 0, 0.5, 2.5, or 12.5 mg AVI‐4472/kg bw/day for 8 days, beginning 1 day before TAM exposure. The specific activity of CYP3A2 was increased after TAM administration, and decreased significantly (∼70%) in the presence of 12.5 mg AVI‐4472. CYP3A2 protein levels, determined by immunoblot analysis, showed a similar pattern. Hepatic TAM–DNA adduct levels were measurable in all TAM‐exposed groups. However, when rats were co‐treated with 2.5 and 12.5 mg AVI‐4472/kg bw/day, statistically significant (∼50%) reductions in TAM–DNA adduct levels (2.0–2.8 adducts/108 nucleotides) were observed compared to rats treated with TAM alone (5.1 adducts/108 nucleotides). Rat toxicology U34 arrays (Affymetrix) were used to investigate the modulation of gene expression patterns on co‐administration of TAM with AVI‐4472. Results indicated that several CYP genes were down regulated although no significant induction of CYP3A2 was observed in the TAM‐exposed rats co‐treated with AVI‐4472. Overall the data suggest the utility of antisense technology in the redirection of TAM metabolism thereby lowering TAM genotoxicity in rat liver. Published 2005 Wiley‐Liss, Inc.

The Role of Cytochrome P450 1B1 in Dibenzo[ a,l ]pyrene-induced Carcinogenesis

In human cells, the most carcinogenic polycyclic aromatic hydrocarbon dibenzo[ a,l ]pyrene (DB[ a,l ]P) forms high levels of DNA adducts through formation of the ( m )- anti -(11 R ,12 S )-diol (13 S ,14 R )-epoxide (DB[ a,l ]PDE) and its metabolic precursor, the ( m )-(11 R ,12 R )-diol. Generation of these adducts results from the catalytic activity of cytochrome P450 (P450) 1A1 and 1B1. Additional adducts such as (+)- syn -DB[ a,l ]PDE-DNA or more polar DNA adducts were detected only after increasing exposure doses of the parent compound or in cells that express P450 1A1. At low concentrations (·;100 nM) exclusively ( m )- anti -DB[ a,l ]PDE-DNA adducts were formed by P450 1B1, which is constitutively expressed in many mammalian tissues. Measurement of DNA binding and mutagenicity of DB[ a,l ]P in V79 cells expressing human P450 enzymes revealed a higher activity of P450 1B1 compared to 1A1 at low concentrations. Treatment of P450 1B1 knockout mice and DNA binding studies with fibroblasts isolated from these animals provided further evidence for the central role of P450 1B1-catalyzed formation of ( m )- anti -DB[ a,l ]PDE-DNA adducts in DB[ a,l ]P-induced carcinogenesis.

Responses of Human Cells to PAH-Induced DNA Damage

Benzo[ a ]pyrene (B[ a ]P) and dibenzo[ a,l ]pyrene (DB[ a,l ]P) induce cytochrome P450 (CYP) CYP1A1 and CYP1B1, which metabolize these polycyclic aromatic hydrocarbons (PAHs) into DNA-binding species. In order to detail roles of CYP1A1 and CYP1B1 in activation of DB[ a,l ]P to the diol epoxide, we here report the inhibition of CYP1A1 in human MCF-7 cells with phosphorodiamidate morpholino antisense oligomers (morpholinos). PAH-DNA adduct formation was also determined after treatment with morpholinos and B[ a ]P or DB[ a,l ]P. p53 is involved in DNA repair, cell cycle arrest, and apoptosis. Cells with normal p53 protein arrest in the G1 phase of the cell cycle on exposure to DNA-damaging agents (presumably allowing the cell sufficient time to repair damaged DNA prior to replication). Previous studies in human MCF-7 cells indicate that cells with PAH-DNA adducts escape cell cycle arrest and accumulate in the S phase. In the present study the effect of PAH-DNA adducts on the cell cycle were observed in human diploid fibroblasts (HDF). We found that treatment of HDF with the diol epoxide of DB[ a,l ]P causes cell cycle arrest in G 1 . An increase in DNA adduct formation with increase in concentration of dibenzo[ a,l ]pyrene diol epoxide {( m )- anti -DB[ a,l ]PDE} was also observed.

Genotoxicity Assessment of Nanomaterials: Recommendations on Best Practices, Assays, and Methods

Nanomaterials (NMs) present unique challenges in safety evaluation. An international working group, the Genetic Toxicology Technical Committee of the International Life Sciences Institutes Health and Environmental Sciences Institute, has addressed issues related to the genotoxicity assessment of NMs. A critical review of published data has been followed by recommendations on methods alterations and best practices for the standard genotoxicity assays: bacterial reverse mutation (Ames); in vitro mammalian assays for mutations, chromosomal aberrations, micronucleus induction, or DNA strand breaks (comet); and in vivo assays for genetic damage (micronucleus, comet and transgenic mutation assays). The analysis found a great diversity of tests and systems used for in vitro assays; many did not meet criteria for a valid test, and/or did not use validated cells and methods in the Organization for Economic Co-operation and Development Test Guidelines, and so these results could not be interpreted. In vivo assays were less common but better performed. It was not possible to develop conclusions on test system agreement, NM activity, or mechanism of action. However, the limited responses observed for most NMs were consistent with indirect genotoxic effects, rather than direct interaction of NMs with DNA. We propose a revised genotoxicity test battery for NMs that includes in vitro mammalian cell mutagenicity and clastogenicity assessments; in vivo assessments would be added only if warranted by information on specific organ exposure or sequestration of NMs. The bacterial assays are generally uninformative for NMs due to limited particle uptake and possible lack of mechanistic relevance, and are thus omitted in our recommended test battery for NM assessment. Recommendations include NM characterization in the test medium, verification of uptake into target cells, and limited assay-specific methods alterations to avoid interference with uptake or endpoint analysis. These recommendations are summarized in a Roadmap guideline for testing.

UNIT 4.38 CYP1B1 Detection

This unit describes procedures for measuring CYP1B1 gene expression by reverse transcription real‐time PCR (qRT‐PCR), CYP1B1 protein levels by western blotting, and CYP1B1 enzyme activity through conversion of 7‐ethoxyresorufin substrate. To achieve specific measurement of CYP1B1 activity in the presence of CYP1A1 and CYP1A2, CYP1B1 inhibition and a subtractive approach have been adopted. 2,4,3′,5′‐Tetramethoxystilbene (TMS) is a potent and selective competitive inhibitor of CYP1B1 with an IC50 of 3 nM for EROD and ∼90 nM for E2 4‐hydroxylation. Binding studies with purified CYP1B1 suggests that TMS interferes in the proximity of the heme region of CYP1B1 with high affinity. Compared to other potent inhibitors such as α‐naphthoflavone, which is a known CYP1 family inhibitor with no selectivity between CYP1B1 and CYP1A2, TMS is ∼50‐ and 520‐fold selective for inhibition of CYP1B1 when compared to CYP1A1 and CYP1A2, respectively. Thus, TMS can serve as a helpful chemical scalpel for dissecting CYP1B1 activity from the overall activity of CYP1 family members against ethoxyresorufin. Curr. Protoc. Toxicol. 51:4.38.1‐4.38.26.