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Updated: Jul 8, 2026
An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry
Published on: May 13, 2019
Enhanced micronucleus formation and modulation of BCL-2:BAX in MCF-7 cells after exposure to binary mixtures
Rebecca Hewitt1, Albert Forero, Paz J Luncsford
1Biomedical Sciences Unit, Lancaster University, Lancaster, United Kingdom.
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.
Insights
Low-dose environmental toxins like benzo[a]pyrene and lindane can increase DNA damage and affect cell survival. These xenobiotic interactions highlight the role of endocrine activity in cellular responses to chemical mixtures.
Area of Science:
- Environmental toxicology
- Cellular biology
- Molecular toxicology
Background:
- Xenobiotic mixtures can cause additive, synergistic, or inhibitory effects in cells.
- The role of environmental concentrations of xenobiotics in human disease etiology is often unclear.
Purpose of the Study:
- To investigate the effects of benzo[a]pyrene (B[a]P) and PhIP, individually and in combination with lindane, on chromosomal aberrations in MCF-7 cells.
- To analyze intracellular responses, including gene expression and protein levels of key regulators of cell cycle and apoptosis.
Main Methods:
- Cytokinesis-block micronucleus assay to detect chromosomal aberrations.
- Quantitative gene expression analysis of CDKN1A, BCL-2, BAX, and CYP isoforms.
- Immunocytochemical analysis of p53, p21(Waf1/Cip1), Bcl-2, and Bax proteins.
Main Results:
- Binary mixtures of B[a]P/lindane or PhIP/lindane significantly increased micronucleus formation compared to individual agents.
- Lindane altered the Bcl-2:Bax ratio, mimicking effects of 17beta-estradiol (E(2)).
- B[a]P elevated P21(WAF1/CIP1) and CYP expression but reduced the Bcl-2:Bax mRNA ratio, effects partially reversed by lindane or E(2).
Conclusions:
- Low-dose xenobiotic mixtures, near environmental levels, can induce DNA damage and influence cell survival.
- The observed cellular effects are dependent on the endocrine activity of the tested agents.
- These findings underscore the potential health risks associated with complex environmental chemical exposures.
