Cytotoxic evaluation of organochlorine insecticides and metals in vitro on CHO-K1 cell line using four different alternative methods

Abstract

The cytotoxicity of twelve organochlorine insecticides and seven metals has bene evaluated by four different in vitro alternative methods using the established CHO-K1 cell line as a mammalian model. The assessed techniques represent distant endpoints, neutral red (NR) assay, methyl tetrazolium test (MTT) assay, total cell protein content assay and lactate dehydrogenase (LDH) leakage test. Results of the first three in vitro methods showed that, the sequence of potency for the tested insecticides was p,p'-TDE > p,p'-DDT > p,p'-DDE and/or methoxychlor as a diclorodiphenyethanes derivatives. In case of the hexachlorocyclohexane isomers and cyclodiene derivatives, the results of both NR and total cell protein content assays showed that the mix of isomers of HCH were more toxic than the gamma isomer (lindane), and the derivatives of cyclodiene, displayed the following order of toxicity ranking chlordane > heptachlor > aldrin > endosulfan > toxaphene. The MTT test showed different results in the sequence of potency of the hexachloryclohexane isomers and cyclodiene derivatives. According to NR and total cell protein content assays the toxicity of the tested metals was in the following rank order: Hg²⁺ > CD²⁺ > Cu²⁺ > Zn²⁺ > Co²⁺ > Ni²⁺ > Pb²⁺, while MTT test exhibited the toxicity ranking as Hg²⁺ > Cd²⁺ > Zn²⁺ > Cu²⁺ > Ni²⁺ > Co²⁺ > Pb²⁺. Furthermore, the total cell protein content assay had the highest sensitivity, for p,p'-TDE, p,p'-DDT, p,p'-DDE, methoxychlor, HCH, lindane and toxaphene. Conversely, MTT test was more sensitive than the other tests against aldrin, dieldrin and endosulfan, while the NR assay had higher sensitivity than the other tests against chlordane and heptachlor. In case of the tested metals, the sequence of sensitivity between the followed methods was: MTT > NR > protein content assay. According to the LDH leakage test, it could not be reach to the midpoint cytotoxicity value to the majority of the tested agents.