Enhanced micronucleus formation and modulation of BCL-2:BAX in MCF-7 cells after exposure to binary mixtures

Background: Within mixtures, interactions between different xenobiotics may occur to give rise to additive, synergistic, inhibitory and/or stimulatory effects in target cells. The role that xenobiotics individually or in mixtures, and at environmental concentrations, play in the etiology of common human diseases often remains obscure.

Methods: In the presence or absence of lindane, chromosomal aberrations were detected in MCF-7 cells after 24-hr treatment with benzo[a]pyrene (B[a]P) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) using the cytokinesis-block micronucleus assay. Micronuclei were scored in 1,000 binucleate cells/treatment. We investigated intracellular responses using quantitative gene expression analyses of cyclin-dependent kinase inhibitor 1A [CDKN1A (P21(WAF1/CIP1))], B-cell leukemia/lymphoma 2 (BCL-2), BCL-2-associated X (BAX), and isoforms of cytochrome P450 (CYP), CYP1A1, CYP1A2, and CYP1B1. Immunocytochemical analyses of p53, p21(Waf1/Cip1), Bcl-2 and Bax protein expression in MCF-7 cells were also carried out.

Results: After exposure to binary mixtures of B[a]P plus lindane or PhIP plus lindane, a 10-fold increase in micronucleus formation resulted; these test agents individually induced 2- to 5-fold increases. Lindane increased the ratio of Bcl-2:Bax, as did 17beta-estradiol (E(2)). Although treatment with B[a]P alone was found to elevate expression of P21(WAF1/CIP1)and CYP isoenzymes, it reduced the ratio of BCL-2:BAX mRNA transcripts. Treatment with a binary mixture of 10(-8) M B[a]P plus 10(-12) M lindane or 10(-10) M E(2) reversed B[a]P-induced reductions in the ratio of Bcl-2- to Bax-positive cells. In contrast, treatments with PhIP (known to possess hormonelike properties) plus lindane or E(2) resulted in profound reductions in Bcl-2:Bax ratio.

Conclusions: Our results suggest that low-dose treatments (i.e., close to environmental levels) may increase DNA damage while influencing survival in exposed cells and that these effects may depend on the endocrine activity of test agents.