S-(1,2-dichlorovinyl)-L-cysteine-induced dedifferentiation and p53 gene mutations in LLC-PK1 cells: a comparative investigation with S-(2-chloroethyl)cysteine, potassium bromate, cis-platinum and styrene oxide.
Exposure of cultured renal (LLC-PK1) cells for 7 weeks to non-cytotoxic concentrations of S-(1,2-dichlorovinyl)-L-cysteine had resulted in the induction of morphologically and biochemically dedifferentiated clones, which retained their altered properties after removal of the chemical. In this study we investigated by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis and direct sequencing if S-(1,2-dichlorovinyl)-L-cysteine-induced LLC-PK1 clones display mutations in the p53 gene in comparison with wild-type clones. In addition, the characteristics of S-(1,2-dichlorovinyl)-L-cysteine-induced clones were compared with clones induced by carcinogens/metabolites of carcinogens with different mechanisms of action: (i) The potent alkylating agent and bacterial mutagen chloroethylcysteine, the key metabolite of the carcinogen dichloroethane; (ii) potassium bromate, a nephrocarcinogen inducing reactive oxygen species, which give rise to the formation of 8OHdG and DNA strand-breaks; (iii) cis-platinum, a bifunctional cross-linking agent and strand-break inducer and (iv) styrene oxide, the main intermediate metabolite of styrene, an epoxide whose carcinogenicity is thought to be based on cytotoxicity. Three essential markers of the physiological integrity and renal tubule origin of the wild-type LLC-PK1 cells were disrupted in all chemical-derived clones: (i) the polarisation of the plasma membrane into a luminal and basolateral part; (ii) the sodium-dependent glucose uptake and (iii) the pH-dependent ammonia production. Compared with the wild-type clones, poly(ADP-ribosyl)ation, a posttranslational modification of nuclear proteins, was clearly increased in clones induced by S-(1,2-dichlorovinyl)-L-cysteine, potassium bromate and cis-platinum. These clones displayed also band shifts of p53 exon 7, indicating mutations, which were confirmed by sequencing: a double mutation consisting of a base substitution followed by one base insertion in the case of S-(1,2-dichlorovinyl)-L-cysteine and potassium bromate and a base substitution in the case of cis-platinum. The base insertions both lead to the formation of the stop codon UGA resulting in loss of protein function.
Richter
H
H
Institut für Toxikologie der Universität Würzburg, Germany.
Vamvakas
S
S
D000641:Ammonia / Q000378:metabolism; D000818:Animals; D001959:Bromates / Q000633:toxicity; D002273:Carcinogens / Q000633:toxicity*; D002478:Cells, Cultured; D002945:Cisplatin / Q000633:toxicity; D003545:Cysteine / Q000031:analogs & derivatives* / Q000633:toxicity; D004852:Epoxy Compounds / Q000633:toxicity; D016158:Genes, p53; D006863:Hydrogen-Ion Concentration; D018374:LLC-PK1 Cells; D008757:Methylglucosides / Q000378:metabolism; D017354:Point Mutation; D011064:Poly Adenosine Diphosphate Ribose / Q000096:biosynthesis; D018807:Polymorphism, Single-Stranded Conformational; D014176:Protein Biosynthesis; D011506:Proteins; D013552:Swine; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D001959:Bromates; D002273:Carcinogens; D004852:Epoxy Compounds; D008757:Methylglucosides; D011506:Proteins; D016159:Tumor Suppressor Protein p53; C024857:poly(ADP)-ribosylated proteins; C019536:potassium bromate; D011064:Poly Adenosine Diphosphate Ribose; C045285:S-(2-chloroethyl)cysteine; C027020:methylglucoside; C039961:S-(1,2-dichlorovinyl)cysteine; D000641:Ammonia; C013690:styrene oxide; D003545:Cysteine; D002945:Cisplatin