title: Optimal dose selection of N-methyl-N-nitrosourea for the rat comet assay to evaluate DNA damage in organs with different susceptibility to cytotoxicity.
abstract: The in vivo rodent alkaline comet assay (comet assay) is a promising technique to evaluate DNA damage in vivo. However, there is no agreement on a method to evaluate DNA damage in organs where cytotoxicity is observed. As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the comet assay, we examined DNA damage in the liver, stomach, and bone marrow of rats given three oral doses of N-methyl-N-nitrosourea (MNU) up to the maximum tolerated dose based on systemic toxicity. MNU significantly increased the % tail DNA in all the organs. Histopathological analysis showed no cytotoxic effect on the liver, indicating clearly that MNU has a genotoxic potential in the liver. In the stomach, however, the cytotoxic effects were very severe at systemically non-toxic doses. Low-dose MNU significantly increased the % tail DNA even at a non-cytotoxic dose, indicating that MNU has a genotoxic potential also in the stomach. Part of the DNA damage at cytotoxic doses was considered to be a secondary effect of severe cell damage. In the bone marrow, both the % tail DNA and incidence of micronucleated polychromatic erythrocytes significantly increased at non-hematotoxic doses, which were different from the non-cytotoxic doses for liver and stomach. These findings indicate that an optimal dose for detecting DNA damage may vary among organs and that careful attention is required to select an optimum dose for the comet assay based on systemic toxicity such as mortality and clinical observations. The present study shows that when serious cytotoxicity is suggested by increased % hedgehogs in the comet assay, histopathological examination should be included for the evaluation of a positive response.
authors.affiliation: Environmental Health Science Laboratory, Sumitomo Chemical Co. Ltd. 3-1-98 Kasugadenaka, Konohana-ku, Osaka 554-8558, Japan. Electronic address: kitamotos3@sc.sumitomo-chem.co.jp.
authors.forename: Sachiko
authors.initials: S
authors.lastname: Kitamoto
authors.affiliation: Environmental Health Science Laboratory, Sumitomo Chemical Co. Ltd. 3-1-98 Kasugadenaka, Konohana-ku, Osaka 554-8558, Japan.
authors.forename: Ryoko
authors.initials: R
authors.lastname: Matsuyama
authors.affiliation: Preclinical Research Laboratories, Dainippon Sumitomo Pharma Co., Ltd. 3-1-98 Kasugadenaka, Konohana-ku, Osaka 554-8558, Japan.
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Uematsu
authors.affiliation: Environmental Health Science Laboratory, Sumitomo Chemical Co. Ltd. 3-1-98 Kasugadenaka, Konohana-ku, Osaka 554-8558, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Ogata
authors.affiliation: Environmental Health Science Laboratory, Sumitomo Chemical Co. Ltd. 3-1-98 Kasugadenaka, Konohana-ku, Osaka 554-8558, Japan.
authors.forename: Mika
authors.initials: M
authors.lastname: Ota
authors.affiliation: Preclinical Research Laboratories, Dainippon Sumitomo Pharma Co., Ltd. 3-1-98 Kasugadenaka, Konohana-ku, Osaka 554-8558, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Yamada
authors.affiliation: Environmental Health Science Laboratory, Sumitomo Chemical Co. Ltd. 3-1-98 Kasugadenaka, Konohana-ku, Osaka 554-8558, Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Miyata
authors.affiliation: Preclinical Research Laboratories, Dainippon Sumitomo Pharma Co., Ltd. 3-1-98 Kasugadenaka, Konohana-ku, Osaka 554-8558, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Funabashi
authors.affiliation: Environmental Health Science Laboratory, Sumitomo Chemical Co. Ltd. 3-1-98 Kasugadenaka, Konohana-ku, Osaka 554-8558, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Saito
mesh_terms: D000818:Animals; D001853:Bone Marrow / Q000187:drug effects; D002273:Carcinogens / Q000633:toxicity; D020552:Comet Assay / Q000379:methods*; D004249:DNA Damage / Q000187:drug effects*; D004198:Disease Susceptibility; D004305:Dose-Response Relationship, Drug; D008099:Liver / Q000187:drug effects; D008297:Male; D020714:Maximum Tolerated Dose; D008770:Methylnitrosourea / Q000633:toxicity*; D015162:Micronucleus Tests / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D013270:Stomach / Q000187:drug effects
chemical_list: D002273:Carcinogens; D008770:Methylnitrosourea
keywords: In vivo comet assay; In vivo rat micronucleus assay; JaCVAM validation study; N-methyl-N-nitrosourea