ABSTRACT
We have developed a simple liver micronucleus assay using young rats (up to 4 weeks old) to assess cytogenetic damage of chemicals in liver cells. Diethylnitrosamine (DEN) was used as a model rodent hepatocarcinogen in this study. Compared to the partial hepatectomy method most commonly used for the liver micronucleus assay, the present assay method showed equal or even higher practicability. The young rat liver micronucleus assay was also characterized for rat strain differences, sampling time after treatment, single treatment vs. split treatment, age of animals, administration route, and staining method. Although based on one model chemical, we propose an acceptable protocol for the micronucleus assay using young rat liver as follows: Up to 4-week-old rats should be used; oral or intraperitoneal administration can be used; single or repeated treatment protocols can be applied; sampling time is 3-5 days after the last treatment; hepatocytes are prepared by the collagenase perfusion method; and cells are stained with the AO-DAPI double staining method.
Subject(s)
Carcinogens/toxicity , Diethylnitrosamine/toxicity , Hepatocytes/drug effects , Micronucleus Tests/methods , Acridine Orange , Administration, Oral , Age Factors , Animals , Carcinogens/administration & dosage , Cell Separation/methods , DNA Damage , Diethylnitrosamine/administration & dosage , Dose-Response Relationship, Drug , Drug Administration Schedule , Fluorescent Dyes , Hepatectomy , Hepatocytes/ultrastructure , Indoles , Injections, Intraperitoneal , Liver/cytology , Liver/drug effects , Liver Regeneration , Male , Mitosis , Rats , Rats, Inbred F344 , Rats, Sprague-DawleyABSTRACT
1,4-Dioxane is a nongenotoxic hepatocarcinogen but in our previous replicative DNA synthesis (RDS) studies with the [3H]thymidine (TdR)-technique, it failed to increase hepatocyte RDS values when given by gavage to male F344 rats as a single 2000 mg/kg body weight dose. However, in a current series of trials with TdR, it showed equivocal responses 24 or 48 hr following treatment with 2000 mg/kg in time-course experiments, and positive responses 24 hr following 1000, 1500 and 2000 mg/kg in dose-response experiments. An increased RDS incidence was also observed at the dose of 2000 mg/kg with data for 5-bromo-2'-deoxyuridine (BrdU)-incorporation. These present findings thus support the hypothesis that a capacity to induce cell proliferation may play a key role in 1,4-dioxane hepatocarcinogenesis.
