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Inhibition of gap junctional intercellular communication in heptachlor- and heptachlor epoxide-treated normal human
K Nomata1, K S Kang, T Hayashi
1Department of Pediatrics/Human Development and Institute of Environmental Toxicology, Michigan State University, East Lansing 48824, USA.
Abstract:
Based on the concern of organochlorides in the environment and in human tissue, this study was designed to determine whether various noncytotoxic levels of heptachlor and heptachlor epoxide could inhibit, reversibly, gap junctional intercellular communication in human breast epithelial cells (HBEC). Cytotoxicity and gap junctional intercellular communication (GJIC) were evaluated by lactate dehydrogenase assay and fluorescence redistribution after photobleaching analysis, respectively. Both heptachlor and heptachlor epoxide were noncytotoxic up to 10 microg/ml. At this concentration, heptachlor and heptachlor epoxide inhibited GJIC of normal human breast epithelial cells after 1 h treatment. Within a 24 h treatment with heptachlor and heptachlor epoxide at 10 microg/ml, recovery of GJIC had not returned. GJIC completely recovered after a 12 h treatment of 1 microg/ml heptachlor epoxide, but it did not recover after a 24 h treatment of 1 microg/ml heptachlor. RT-PCR and Western blots were analyzed to determine whether the heptachlor or heptachlor epoxide might have altered the steady-state levels of gap junction mRNA and/or connexin protein levels or phosphorylation state. No significant difference in the level of connexin 43 (Cx43) message between control and heptachlor-treated cells was observed. Western blot analyses showed hypophosphorylation patterns in cells treated with 10 microg/ml heptachlor and heptachlor epoxide for 1 h with no recovery within 24 h. Immunostaining of Cx43 protein in normal HBEC indicated that heptachlor and heptachlor epoxide caused a loss of Cx43 from the cell membranes at noncytotoxic dose levels. Taken together, these results suggest that heptachlor and heptachlor epoxide can alter GJIC at the post-translational level, and that, under the conditions of exceeding a threshold concentration in the breast tissue containing 'initiated' cells for a long time and not being counteracted by anti-tumor-promoting chemicals, they could act as breast tumor promoters.
Insights
Organochlorines like heptachlor and heptachlor epoxide disrupt gap junction intercellular communication (GJIC) in human breast cells at non-toxic levels. These chemicals may promote breast tumors by altering cell communication pathways.
Area of Science:
- Environmental toxicology
- Cell biology
- Cancer research
Background:
- Organochlorines are environmental contaminants found in human tissues.
- Disruption of gap junction intercellular communication (GJIC) is implicated in cancer promotion.
Purpose of the Study:
- To investigate the effects of non-cytotoxic levels of heptachlor and heptachlor epoxide on GJIC in human breast epithelial cells (HBEC).
- To determine if these effects are reversible and the underlying molecular mechanisms.
Main Methods:
- Cytotoxicity assessed via lactate dehydrogenase assay.
- GJIC evaluated using fluorescence redistribution after photobleaching.
- Gene and protein expression analyzed by RT-PCR and Western blots.
- Connexin 43 (Cx43) localization studied by immunofluorescence.
Main Results:
- Heptachlor and heptachlor epoxide inhibited GJIC at 10 microg/ml without cytotoxicity.
- Inhibition of GJIC persisted for 24 hours at 10 microg/ml, with partial recovery at 1 microg/ml for heptachlor epoxide.
- Hypophosphorylation of Cx43 and loss of membrane-associated Cx43 were observed.
- No significant changes in Cx43 mRNA levels were detected.
Conclusions:
- Heptachlor and heptachlor epoxide impair GJIC in HBEC at the post-translational level.
- These organochlorines may act as breast tumor promoters by disrupting cell communication.
- Findings highlight potential risks of environmental contaminants in breast cancer development.