Carcinogenesis, Vol. 21, No. 2, 289-294,
February 2000
© 2000 Oxford University Press
Carcinogenesis |
Kinetics of DNA adduct formation and removal in mouse hepatocytes following in vivo exposure to 5,9-dimethyldibenzo[c,g]carbazole
Rhône-Poulenc Rorer, Nonclinical Safety Assessment, 13 Quai Jules Guesde, BP 14, 94403 Vitry sur Seine Cedex and
1 Institut Curie-Biologie, 91405 Orsay Cedex, France
5,9-Dimethyldibenzo[c,g]carbazole (DMDBC), a potent mouse hepatocarcinogen, has been shown to induce a non-linear increase in mutant frequency in the liver of the transgenic MutaTMMouse. To gain insight into the mechanisms underlying the mutagenicity of DMDBC in vivo, DNA damage formation and removal were monitored in mouse hepatocytes over 4–144 h after a single skin application of 10 or 90 mg/kg DMDBC. DNA adducts were measured by 32P-post-labeling. DNA repair was assessed by: (i) the unscheduled DNA synthesis (UDS) assay, which measures [3H]thymidine incorporation into hepatocyte DNA undergoing excision repair; (ii) the Comet assay, which detects DNA strand breaks transiently produced between the incision and rejoining steps of the excision repair process. A plateau of ~400 DNA adducts/108 nucleotides was reached 24 h after treatment with 10 mg/kg and remained unchanged until 144 h. UDS activity was significantly induced at 15 and 24 h, while no DNA strand breaks were observed at any sampling time. These results suggest that DNA repair mechanisms were efficiently induced and the formation of a high degree of DNA damage was avoided at this dose level. Following exposure to 90 mg/kg DMDBC, the number of DNA adducts increased sharply to a maximum at 24 h (~8000/108 nucleotides) and then declined to ~500/108 nucleotides at 144 h. UDS activity was markedly induced from 15 to 72 h. Low levels of DNA strand breaks were observed at 24 and 48 h. The formation of large numbers of DNA adducts and the emergence of DNA strand breaks despite a strong initial induction of UDS activity suggested that DNA repair mechanisms were saturated at this dose level. This phenomenon could partly account for the non-linear induction of gene mutations previously reported in the liver of the transgenic MutaTMMouse.
Abbreviations: DBC, 7H-dibenzo[c,g]carbazole; DMDBC, 5,9-dimethyldibenzo[c,g]carbazole; DMN, dimethylnitrosamine; FBS, fetal bovine serum; MF, mutant frequency; NER, nucleotide excision repair; NNG, net nuclear grain; TM, tail moment; UDS, unscheduled DNA synthesis; WME, William's medium E.