The genotoxic potential of methapyrilene using the alkaline Comet assay in vitro and in vivo.
The genotoxicity of methapyrilne (MP) has been evaluated in a number of assays since it was found to be a rat hepatocarcinogen with subsequent withdrawal as an over-the-counter antihistamine. Whilst it has not been classified as a genotoxin, there are reports of positive findings from mammalian cell gene mutation and transformation assays. To investigate further the genotoxic potential of MP, the alkaline Comet assay was used to evaluate DNA damage both in primary hepatocytes in culture and in vivo in the rat. To confirm bioactivation was required to induce the hepatotoxic mechanism, aminobenzotriazole, a broad spectrum cytochrome P450 enzyme inhibitor was used as a pre-treatment. The levels of glutathione and glutathione disulfide were determined in both hepatocytes in culture and in the liver following in vivo exposure. MP showed significant increases in DNA damage in freshly isolated male rat hepatocyte suspensions that could be significantly reduced by pre-incubation of aminobenzotriazole (ABT). DNA damage showed a marked sex difference, with male hepatocytes being more susceptible, and showing a concurrent depletion of glutathione (GSH) compared with female hepatocytes. Modulation of the GSH levels by diethylmaleate and γ-glutamylcysteinylethyl ester, elevated and reduced the levels of DNA damage, respectively. In the in vivo Comet assay, there was no evidence of DNA damage following MP (150mg/kg p.o) treatment for three consecutive days, although histological and liver enzyme changes were seen. Total protein GSH content was elevated in MP-treated animals and superoxide dismutase levels were increased specifically in periportal regions. Taken together, these data support the potential for MP to induce oxidative stress. The differences in DNA damage detected by the Comet assay in vitro, and in rat liver in vivo, could be attributed to differences in metabolism and response to oxidant insult or the inability of the assay to discriminate damage in a small number of individual cells in the whole liver.
Safety Assessment, AstraZeneca R&D, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK. catherine.priestley@astrazeneca.com
Catherine C
CC
Priestley
Sophie
S
Regan
B
B
Kevin Park
Dominic P
DP
Williams
D000818:Animals; D020552:Comet Assay; D004249:DNA Damage / Q000187:drug effects*; D005260:Female; D005978:Glutathione / Q000187:drug effects / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects* / Q000473:pathology; D006634:Histamine H1 Antagonists / Q000633:toxicity*; D008099:Liver / Q000187:drug effects / Q000473:pathology; D008297:Male; D008701:Methapyrilene / Q000633:toxicity*; D009152:Mutagenicity Tests; D018384:Oxidative Stress / Q000187:drug effects*; D051381:Rats; D017208:Rats, Wistar; D012737:Sex Factors; D013482:Superoxide Dismutase / Q000187:drug effects / Q000378:metabolism; D014230:Triazoles / Q000494:pharmacology
D006634:Histamine H1 Antagonists; D014230:Triazoles; C033020:1-aminobenzotriazole; D008701:Methapyrilene; D013482:Superoxide Dismutase; D005978:Glutathione