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.
Hewitt
Rebecca
R
Biomedical Sciences Unit, Lancaster University, Lancaster, United Kingdom.
Forero
Albert
A
Luncsford
Paz J
PJ
Martin
Francis L
FL
D001564:Benzo(a)pyrene / Q000633:toxicity*; D001943:Breast Neoplasms / Q000378:metabolism; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000187:drug effects / Q000235:genetics; D004347:Drug Interactions; D004958:Estradiol / Q000633:toxicity; D005786:Gene Expression Regulation / Q000187:drug effects; D001556:Hexachlorocyclohexane / Q000633:toxicity*; D006801:Humans; D007093:Imidazoles / Q000633:toxicity*; D048629:Micronuclei, Chromosome-Defective / Q000187:drug effects*; D015162:Micronucleus Tests; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000187:drug effects / Q000378:metabolism; D015262:Xenobiotics / Q000633:toxicity; D051028:bcl-2-Associated X Protein / Q000187:drug effects / Q000378:metabolism
D050759:Cyclin-Dependent Kinase Inhibitor p21; D007093:Imidazoles; D019253:Proto-Oncogene Proteins c-bcl-2; D015262:Xenobiotics; D051028:bcl-2-Associated X Protein; D001564:Benzo(a)pyrene; D004958:Estradiol; D001556:Hexachlorocyclohexane; D003577:Cytochrome P-450 Enzyme System; C049584:2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine