Inhibitory effects of organochlorine pesticides on intercellular transfer of Lucifer Yellow in cultured bovine oviductal cells

R. Pöhland and U. Tiemann

Research Institute for the Biology of the Farm Animals, Reproductive Biology, 18196 Dummerstorf, Germany

AIM: To investigate in which manner dichlorodiphenyltrichloroethane (DDT), methoxychlor (MXC), and g-hexachlorocyclohexane (gHCH, lindane) affect the gap junctional intercellular communication (GJIC) and the intracellular cAMP concentration [cAMP]i in cultured bovine oviductal cells. METHODS: Confluent grown oviductal cells were incubated with different concentrations of pesticides for 1 and 5 hrs at 37 °C. GJIC was evaluated by microinjection of fluorescent dye Lucifer Yellow and observing the inhibition of the spreading of dye into adjacent cells. The intracelluar cAMP level was indirectly measured by an increase in the 520 nm/580 nm fluorescence emission ratio of the protein kinase A fluorosensor (FICRhR) which was microinjected in single cells. RESULTS: After incubation for 1 h, a significant inhibition in GJIC begun at 32 µM DDT, MXC, or gHCH compared to non-exposed controls. After incubation for 5 h, a dose-dependent inhibition of GJIC was obtained in the concentration range from 8 to 64 µM of the pesticides. The first significant inhibitory effect on GJIC was caused by 8 µM DDT, 16 µM MXC and 32 µM gHCH. The 128 µM concentration of the pesticides was toxic. At pesticide concentration of 64 µM the decrease in dye-coupling observed was not due to lethal cell injury, as is indicated by the use of trypan blue dye exclusion. After removal of 64 µM DDT from the culture medium, intercellular communication was re-established within 3 h. Measurement of cytosolic free Ca2+ concentration [Ca2+]i in fura-2/AM-loaded oviductal cells showed that the inhibition of GJIC by addition of DDT, MXC or gHCH was not associated with a detectable increase in [Ca2+]i. Whereas, the addition of 16 or 32 µM DDT decreased the [cAMP]i significantly. Coincubation of the DDT with dibutyryl-cAMP prevented the 64 µM DDT-induced inhibition of intercellular communication in adherent oviduct cells. CONCLUSION: The inhibition of GJIC has been hypothesised to play a role in the promotion/progression phases of carcinogenesis and since the toxicants used in the study do inhibit GJIC in oviductal cells it can be speculated that they also can act as tumour promoters in cells responsible for reproduction as in rat liver epithelial cells.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Environment and Occupation.