Cytotoxic and genotoxic effect of inhibitor of vulcanisation N-cyclohexylthiophthalimide in a battery of in vitro assays

Mutagenicity of N-cyclohexylthiophthalimide (Duslin P) was tested first by the Ames test in the bacteria, Salmonella typhimurium. The negative results of the Ames test suggested that this compound does not induce mutations in the genome of S. typhimurium under the conditions used. To estimate the cy totoxicity of Duslin P to human cells, we measured cellular DNA and protein as well as cell proliferation, i.e., the mitotic index of treated and cont rol cells. The genotoxic effects were assayed by two biochemical methods de veloped for detection of single-strand breaks of DNA in mammalian cells, i. e., by the alkaline single cell gel electrophoresis (comet assay) and by th e DNA unwinding method, respectively. The DNA unwinding method showed that this compound did not induce DNA damage at concentrations < 7 mu g/ml. Alka line single cell gel electrophoresis revealed approximately double the leve l of DNA damage (in comparison to untreated control DNA) at a concentration of 2 mu g/ml, which reduced proliferation to approximately 30%, and triple the level of DNA damage at higher concentrations (6 and 7 mu g/ml), which inhibited completely both DNA synthesis and proteosynthesis. Cells with mod erately damaged DNA were more common than cells with heavily damaged DNA. P arallel experiments with the strong mutagen and carcinogen MNNG showed that MNNG induced in cells a high level of DNA damage at concentrations which d id not reduce the mitotic index or proteosynthesis, while DNA synthesis inh ibited only partially. After treatment with MNNG, cells with heavily damage d DNA were more common than cells with moderately damaged DNA. Duslin P-tre ated VH10 cells were also tested cytogenetically, confirming that Duslin P induced neither chromosomal aberrations nor aneuploidy. We conclude that Du slin P has no mutagenic effect on bacteria, does not induce chromosomal abe rrations and CREST positive or CREST negative micronuclei in human cells an d induces only a small increase of DNA damage in human cells which is consi stent with DNA fragmentation due to cell death. (C) 1999 Elsevier Science B .V. All rights reserved.