In vitro mechanistic studies of cell proliferation and cell death induced by the agrochemicals commonly used in the Red River Valley, Minnesota.

摘要

Epidemiologic studies showed hormonal alteration and increased frequency of birth defects correlated with the use of chlorophenoxy herbicides, adjuvants and fungicides in the Red River Valley of Minnesota. Based on epidemiologic outcome and known toxicity, eighteen most commonly used agrochemicals were selected for in vitro mechanistic studies of cell proliferation and cell death. MCF-7 human breast cancer cell line was used in order to define hormone-mediated mechanisms and development-related apoptosis that may participate in the reproductive and developmental effects observed in the population. The test chemicals were screened for cytotoxicity by measurement of cell number and viability. Based on the screening results, some adjuvants and commercial grade herbicides were selected for detailed cell proliferation and transcription activation assessment. The data demonstrated that the adjuvants X-77 and Activate Plus, both containing known estrogenic compounds nonyl- and octyl-phenols, induced significantly increased MCF-7 proliferation at 0.1 and 1 μg/ml. Commercial grade herbicides 2,4-D LV4 and 2,4-D Amine induced MCF-7 proliferation at 1 and 10 μg/ml. The reagent grade 2,4-D products failed to induce proliferation over the same concentration range. Ethylene thiourea (ETU), metabolite of the fungicide mancozeb, induced significant MCF-7 proliferation at 10-100 μg/ml. ETU also induced transcription activation in ERE-SV40-Luciferase Reporter Assay.; The fungicides triphenyltin (TPT) and mancozeb provoked apoptosis that peaked at 4.1μg/ml (10−5M) and 50μg/ml, respectively, in fluorescence-activated cell sorting (FACS) cell cycle analysis and terminal transferase dUTP nick-end labeling TUNEL assay. TPT and mancozeb also demonstrated different cell cycle specificity of apoptosis in two-color TUNEL assay: TPT induced apoptosis at S and G2/M phases, and mancozeb at G0/G1. ETU did not significantly increase apoptosis over the concentrations tested (10-1000 μg/ml. TPT demonstrated anti-estrogenic effect in ERE-SV40-luciferase reporter assay at 10−7 M, a non-cytotoxic concentration. This effect was abolished by 10−8 M estradiol.; These in vitro studies identified plausible biological mechanisms of reproductive effects observed in the epidemiologic studies. Further animal studies will be performed to complete the weight-of-evidence risk assessment of the pesticide related adverse health effects in the RRV.