Effectiveness of the liver micronucleus assay using juvenile mice

Abstract

This study investigated the effectiveness of the liver micronucleus (MN) assay using juvenile mice. Therefore, we analyzed various hepatic cytochrome P450 (CYP)- mediated activities of ethoxyresorufin O-deethylation, pentoxyresorufin O-dealkylation, tolbutamide hydroxylation, bufuralol 1'-hydroxylation, aniline hydroxylation and midazolam 4-hydroxylation by CYP1A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A, respectively, in non-treated male ICR mice aged between 3 and 8 weeks. The enzyme efficiency levels in 3- and 4-week-old mice were approximately similar to or higher than those in 8-week-old mice, except for CYP1A and CYP2E in 3- and 4-week-old mice, respectively. Since these results suggest that juvenile mice have sufficient activities for most CYP enzymes, we also conducted a liver MN assay using diethylnitrosamine (DEN), a rodent hepatocarcinogen, on male ICR mice aged between 3 and 6 weeks. A peripheral blood (PB) MN assay was performed simultaneously in 4-week-old mice. Assays incorporating DEN produced positive results in 3- and 4-week-old mice and showed a dose-dependent increase in the micronucleated hepatocyte frequencies at 4 weeks. Both the liver MN assay in 5- and 6-week-old mice and the PB MN assay had negative results when using DEN. These results suggest that 3- and 4-week-old mice have micronuclei-inducing potential in the liver to detect genotoxic compounds using the liver MN assay.

Keywords: CYP; genotoxicity assay; juvenile mouse; liver micronucleus assay.

Fig. 1.

Frequencies of micronucleated hepatocytes (MNHEPs, evaluated from 2,000 hepatocytes) and metaphase (M-phase) cells (evaluated from 2,000 hepatocytes) after administration of physiological saline and DEN (12.5, 25 and 50 mg/kg/day) to 4-week-old male mice. Data are the mean of four animals with standard deviation. *P<0.01, significantly different from solvent control (Fisher’s exact test). ^#P<0.01, significantly different from solvent control (Cochran-Armitage trend test).

Fig. 2.

Frequencies of micronucleated reticulocytes (MNRETs, evaluated from 2,000 reticulocytes) and reticulocytes (RETs, evaluated from 1,000 erythrocytes) after administration of physiological saline and DEN (12.5, 25 and 50 mg/kg/day) to male mice aged at 4 weeks. Data are the mean of four animals with standard deviation.