An evaluation of micronucleus induction in bone marrow and in hepatocytes isolated from collagenase perfused liver or from formalin-fixed liver using four-week-old rats treated with known clastogens.
The bone marrow (BM) micronucleus (MN) test is a sensitive assay for identifying clastogens. However, some clastogenic compounds and metabolites may never reach the BM. The liver has been suggested as an alternative tissue to BM but adult rat liver has a low mitotic index that increases the difficulty of evaluating hepatocytes (HEP) for MN induction. Chemical mitogens and partial hepatectomy have been used to increase HEP proliferation to improve the sensitivity for detection of clastogenic compounds, but these practices raise concerns for the evaluation of drug candidates. The use of 4-wk-old rats provides an alternative to mitogenic stimulation because livers from these animals have approximately 5.4% of their HEP in S-phase. HEP were isolated by collagenase perfusion, or from formalin-fixed tissue, from 4-wk-old treated rats. Six compounds were evaluated for the incidence of MN in HEP that were isolated by both methods. The results for MN induction by these compounds were similar for the two methods and confirmed that formalin-fixed tissue is an acceptable source of cells for evaluating MN induction in HEP. BM polychromatic erythrocytes (PCE) also were harvested at the end of the live phase for each study and then evaluated for the incidence of MN. Diethylnitrosamine and 2-nitrofluorene induced MN in HEP but had no effect in PCE. 2-Acetylaminofluorene, cyclophosphamide and 7,12-dimethylbenz[a]anthracene did not induce MN in HEP but were positive in PCE. The direct-acting clastogen, mitomycin C, was positive in both HEP and PCE. These results indicate that this modified liver micronucleus test, using 4-wk-old rats, offers an alternative to existing methods that use mitogens or partial hepatectomy to stimulate cell replication. Analysis of MN from formalin-fixed tissue provides additional flexibility by allowing the investigator to assess MN induction at a later time.
Parton
J W
JW
Toxicology Division, Lilly Research Laboratories, A Division of Eli Lilly and Company, Greenfield, Indiana 46140, USA.
Garriott
M L
ML
D000367:Age Factors; D000818:Animals; D001853:Bone Marrow / Q000187:drug effects*; D017364:Collagenases / Q000737:chemistry*; D004052:Diethylnitrosamine / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D005557:Formaldehyde; D008099:Liver / Q000166:cytology / Q000187:drug effects*; D008297:Male; D015162:Micronucleus Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity*; D009929:Organ Size; D010477:Perfusion; D051381:Rats; D011916:Rats, Inbred F344; D012680:Sensitivity and Specificity; D016707:Tissue Fixation / Q000379:methods
D009153:Mutagens; D005557:Formaldehyde; D004052:Diethylnitrosamine; D017364:Collagenases