Induction of dedifferentiated clones of LLC-PK 1 cells upon long-term exposure to dichlorovinylcysteine

Abstract

Dichlorovinylcysteine (DCVC), the key metabolite of the nephrotoxic and nephrocarcinogenic chemicals, trichloroethylene and dichloroacetylene, exerts potent acute cellular toxicity in LLC-PK 1 cells (Vamvakas S., Bittner D., Dekant W. and Anders M.W. (1992). Events that precede and that follow S-( l,2-dichlorovinyl)-L-cysteine-induced release of mitochondrial Ca + and their association with cytotoxicity to renal cells. Biochem. Pharmacol. 44, 1131–1138). In the present study we investigated whether long-term exposure of LLC-PK 1 cells to low, non-cytotoxic concentrations of DCVC results in stable morphological and biochemical dedifferentiation. After 7 weeks exposure to 1 and 5 μM DCVC, morphologically changed single cells were picked under the microscope and cultured in absence of DCVC for 4–8 weeks. In contrast to the physiological cuboidal shape of untreated LLC-PK, cells, the clones derived from long-term exposure to DCVC consisted of elongated, spindle-shaped cells tending to form irregular borders. Moreover, glucose uptake, pH-dependent ammonia production and dome formation, important indicators of the renal tubule origin of the LLC-PK 1 cells, were severely impaired in the clones. In addition to the loss of membrane polarity, the clones exhibited altered composition of the nuclear matrix and intermediate filament proteins by two-dimensional gel electrophoresis, increased poly(ADP-ribosyl)ation of nuclear proteins and enhanced expression of c- fos. The induction of dedifferentiated LLC-PK 1 clones with stable characteristics upon long-term exposure to the nephrocarcinogen DCVC may represent a useful in vitro model to study biochemical alterations involved in chronic renal toxicity and carcinogenicity.