Hisaharu Yamada

Evaluation of the repeated-dose liver, bone marrow and peripheral blood micronucleus and comet assays using kojic acid.

The repeated-dose liver micronucleus assay has the potential to detect liver carcinogens and could be integrated into general toxicological studies. To assess the performance of this assay, kojic acid was tested in 14-day and 28-day liver micronucleus assays. We evaluated the incidence of micronucleated cells in liver, bone marrow and peripheral blood and performed comet assays in both the liver and peripheral blood (comet assay was performed only for 14-days). Kojic acid, a skin-whitening agent used in cosmetic products, was orally dosed in six-week-old male rats at 250, 500 and 1000mg/kg/day for 14 days, and at 125, 250 and 500mg/kg/day for 28 days. Organ weight and histopathology were examined at the end of the experiment. Neither a clear, positive response in micronucleus (MN) incidence nor changes in the percent of tail DNA in the comet assays was noted in liver and bone marrow. An increase of relative liver weight was observed in 1000mg/kg/day for 14 days. In histopathology, minimal hypertrophy of hepatocytes was found at 1000mg/kg/day for 14 days. The results of both the micronucleus assay and the comet assay indicate that 14-day and 28-day repeated dosing of kojic acid are non-genotoxic in the liver and bone marrow. Kojic acid has been known to act as a tumor-promoter in thyroid carcinogenesis but has not been shown to have initiation activities in liver carcinogenesis. Findings in this study are consistent with the evidence that kojic acid is not an apparent initiator of liver carcinogenesis. Therefore, the liver micronucleus assay is simple and sensitive to detect genotoxic liver carcinogens.

Sex differences in the excretion levels of traditional and novel urinary biomarkers of nephrotoxicity in rats

Urinary biomarkers have been used widely in preclinical toxicity studies to detect dysfunctions and injuries of the kidney caused by drugs under development. While they have been well studied for evaluating nephrotoxicity, knowledge of sex differences in excretion levels of urinary biomarkers remains inadequate. We conducted experiments focused on effects of endogenous sex hormones on urinary biomarkers using intact and castrated male and female rats. Comparisons of the urinary biomarker excretion levels between intact male and female rats at 5, 7, 9 and 12 weeks of age revealed higher excretion levels of leucine aminopeptidase (LAP), γ-glutamyl transpeptidase (γGTP), total protein, liver-type fatty acid-binding protein (L-FABP), cystatin C (Cys-C) and β2-microglobulin (β2-MG), and lower excretion level of kidney injury molecule 1 (Kim-1), in male rats as compared to female rats. Orchidectomized male rats showed lower urinary excretion levels of alkaline phosphatase (ALP), LAP, γGTP, N-acetyl-β-D-glucosaminidase (NAG), glucose, total protein, L-FABP, Cys-C, β2-MG and neutrophil gelatinase-associated lipocalin (NGAL), and higher urinary excretion levels of clusterin (CLU) and Kim-1, than sham-operated male rats. On the other hand, no significant differences in the urinary biomarker excretion levels excluding ALP were observed between ovariectomized and sham-operated female rats. In the present study, we demonstrated the existence of sex differences in excretion levels of urinary biomarkers that are universally used in preclinical toxicity studies, and also that these differences, especially in relation to the urinary excretions of ALP, LAP, γGTP, total protein, L-FABP, Cys-C, and β2-MG, may closely relate to the endogenous testosterone.