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Rebecca Hewitt, Albert Forero, Paz J. Luncsford, and Francis L. Martin

Biomedical Sciences Unit, Lancaster University, Lancaster, United Kingdom

Abstract
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 17β-estradiol (E₂) . Although treatment with B[a]P alone was found to elevate expression of P21^WAF1/CIP1and 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 E₂ 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 E₂ 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.

Key words: 17β-estradiol, BAX, Bcl-2, benzo[a]pyrene, binary mixture, clonogenic assay, cytokinesis-block micronucleus assay, lindane, micronucleus, PhIP. Environ Health Perspect 115(suppl 1) :129–136 (2007) . doi:10.1289/ehp.9361 available via http://dx.doi.org/ [Online 8 June 2007]

Address correspondence to F.L. Martin, Biomedical Sciences Unit, Department of Biological Sciences, Lancaster University, Lancaster LA1 4YQ, UK. Telephone: 44 1524 594505. Fax: 44 1524 593192. E-mail: f.martin@lancaster.ac.uk

This article is part of the monograph "Endocrine Disruptors—Exposure Assessment, Novel End Points, and Low-Dose and Mixture Effects."

We gratefully acknowledge grant support from the North West Cancer Research Fund (R.H.) and Rosemere Cancer Foundation (F.L.M.) . A.F. and P.J.L. were both recipients of a Fogarty Minority International Research Training Fellowship Program grant from the National Cancer Institute.

The author declares he has no competing financial interests.

Received 22 May 2006 ; accepted 5 September 2006.