Hepatocyte proliferation activity in untreated rats, measured by immunohistochemical detection of Ki-67: The effect of age on the repeated-dose liver micronucleus assay.

The repeated-dose liver micronucleus (RDLMN) assay is a widely accepted method for detecting genotoxic substances. We investigated the effect of animal age on this assay. Proliferation activity in the liver tissue of untreated rats at age = 3.5, 6, 8, 10, or 12 weeks was measured via immunohistochemical expression of Ki-67 protein. The percentage of Ki-67-positive hepatocytes decreased markedly with age, reaching very low levels after 10 weeks, indicating decline with age of proliferative capacities in the liver. We calculated the area under the curve (AUC) of the approximate curve generated from the percentage of Ki-67-positive cells, to estimate the hepatocyte proliferation activity over the dosing period in the two regimens of the 4-week RDLMN assay: dosing initiated at age = 6 or 8 weeks. Hepatocyte proliferation activity of the former regimen was approximately double that of the latter. We also calculated the AUC for the juvenile-rat method, in which rats are treated for two days at age = 3.5 weeks. The AUC calculated for that method was approximately half of that for the 4-week repeated-dosing regimen initiated at 6 weeks of age. These findings suggest that the 4-week RDLMN assay with dosing initiated at age = 6 weeks could be approximately twice as sensitive as the other two methods.

Increased bioavailability of diosmetin-7-glucoside-γ-cyclodextrin inclusion complex compared with diosmin in Sprague-Dawley rats.

Diosmin (DSN) is found mainly in citrus fruits, and has potent antioxidant effects. This study aimed to evaluate pharmacokinetics of diosmetin-7-glucoside-γ-cyclodextrin (DIOSG-CD) inclusion complex. The area under the curve values from AUC0-24 of DIOSG-CD, prepared by reacting DSN and naringinase with γ-CD, were approximately 800-fold higher than those of DSN following their administration in Sprague-Dawley rats.

Lack of hepatocarcinogenicity of 2,2'-[1,2-ethanediylbis(oxymethylene)]bis-oxirane, 3-hydroxy-2-naphthoic acid, and acetoacetanilide in a medium-term rat liver bioassay.

The carcinogenicity of 2,2'-[1,2-ethanediylbis(oxymethylene)]bis-oxirane (ethylene glycol diglycidyl ether; EGDE), 3-hydroxy-2-naphthoic acid (HNA), and acetoacetanilide (AAA) was investigated using a medium-term rat liver bioassay for an occupational safety assessment. F344 male rats were administered a single intraperitoneal injection of diethylnitrosamine (200 mg/kg body weight (bw)/day) and then starting 2 weeks later, they received EGDE at 6, 20, and 60 mg/kg bw/day, HNA at 20, 60, and 200 mg/kg bw/day, or AAA at 60, 200, and 600 mg/kg bw/day by oral gavage for 6 weeks. The animals in the positive control group received phenobarbital sodium solution (PB, 25 mg/kg bw/day) by oral gavage and those in the negative control group received a vehicle (water/corn oil) during the administration period of test substances in this model. All animals were subjected to two-thirds partial hepatectomy at week 3 and euthanized at week 8. Neither the number nor the area of hepatocellular foci positive for glutathione S-transferase placental form (GST-P) increased in any of the EGDE, HNA, or AAA treated groups. However, the number and area of GST-P-positive foci significantly increased in the positive control group treated with PB. The results indicate that EGDE, HNA, and AAA lack hepatocarcinogenicity in rats.

The effect of aging on the repeated-dose liver micronucleus assay using diethylnitrosamine.

BACKGROUND: The repeated-dose liver micronucleus (RDLMN) assay has been well-developed and applied because of its simplicity and the ease of integration into general toxicity studies which is the preferred method from the 3R's point of view. In this assay, we observed micronucleated hepatocytes which accumulated during a rather long-term dosing period. When considering integration into general toxicity studies, the effects of age of the animals used in the micronucleus assay becomes a major issue. The effect of age on the micronucleus induction rate has been reported in bone marrow micronucleus assays, and it is considered that the decrease in cell proliferation rate due to aging is the cause of the decrease in sensitivity. A decrease in sensitivity due to aging was also reported in a liver micronucleus assay using clofibrate and the cause is considered to be a decrease in hepatocyte proliferation activity due to aging. However, no actual decrease in hepatocyte proliferation rate due to aging has been reported. In addition, there are no reports, so far, on whether similar effects of aging appear when other substances were administered. To investigate the effects of aging in the RDLMN assay, this study focused on the effects of 14-day repeated administration of DEN, a well-known genotoxic hepatocarcinogen with the hepatocyte toxicity which should cause an elevation of cell proliferation rate as a reflective regeneration. RESULTS: The liver micronuclei induced by DEN were equivalent between the two age groups (i.e., six and eight weeks of age at the start of dosing). In the histopathological examination for the liver, single cell necrosis, karyomegaly, and increased mitosis were observed in the hepatocytes, and the frequency and severity were increased dose-dependently. Ki-67 immunohistochemical analysis which can detect all cells in the cell cycle other than those in the G0 phase revealed dose-dependent increase of cell proliferation activity, and the difference between ages was not observed. CONCLUSION: The effect of aging on the RDLMN assay could not be recognized when DEN was administered for 14 days in rats. Meanwhile, it was supported by the histopathological examination and Ki-67 immunohistochemical analysis that such an effect of aging was masked by the compensatory hepatocyte proliferation which was induced by the hepatocyte toxicity of DEN.

Genotoxicity and mutagenicity evaluation of isoquercitrin-γ-cyclodextrin molecular inclusion complex using Ames test and a combined micronucleus and comet assay in rats.

Flavonoids such as quercetin and its glucosides, especially isoquercitrin are well known as anti-inflammatory, anti-allergic, and anti-carcinogenic, etc. The safety of isoquercitrin formulations needs to be established prior to their use in functional food applications. The mutagenicity and genotoxicity of the IQC-γCD inclusion complex were assessed with three standard assays of the bacterial reverse mutation assay (Ames test) and using a combined in-vivo micronucleus and comet assay under the Organisation for Economic Co-operation and Development (OECD) guidelines. In combined rat bone marrow micronucleus and rat liver comet assay performed in male Sprague Dawley (SD) rats, the various doses of IQC-γCD inclusion complex (max. 2000 mg/kg bw) and positive controls ethyl methanesulfonate (EMS) and mitomycin C (MMC), respectively, and negative control (vehicle) were administrated. The results of the Salmonella typhimurium mutagenicity assay (strains TA100, TA1535, WP2uvrA, TA98, and TA1537) after exposure to the IQC-γCD inclusion complex with the absence and presence of the metabolic activation system (S9 fraction from rat liver) revealed a weakly positive response but with no biologically relevant mutagenicity at the conditions examined according to recommended regulatory guidelines. The combined micronucleus and comet assay results reveal that the IQC-γCD inclusion complex did not induce in-vivo genotoxic potential or indication of any oxidative DNA damage in rat liver tissues. Altogether, considering the results of the study, it is unlikely that the consumption of IQC-γCD inclusion complex as food or supplement would present any concern for humans regarding the mutagenicity and genotoxicity.

Detection of hepatocarcinogens by combination of liver micronucleus assay and histopathological examination in 2-week or 4-week repeated dose studies.

BACKGROUND: Currently, revisions to the ICH S1 guidance on rodent carcinogenicity testing are being proposed. Application of this approach would reduce the use of animals in accordance with the 3Rs principles (reduce/refine/replace). The method would also shift resources to focus on more scientific mechanism-based carcinogenicity assessments and promote safe and ethical development of new small molecule pharmaceuticals. In the revised draft, findings such as cellular hypertrophy, diffuse and/or focal cellular hyperplasia, persistent tissue injury and/or chronic inflammation, preneoplastic changes, and tumors are listed as histopathology findings of particular interest for identifying carcinogenic potential. In order to predict hepatocarcinogenicity of test chemicals based on the results from 2- or 4-week repeated dose studies, we retrospectively reanalyzed the results of a previous collaborative study on the liver micronucleus assay. We focused on liver micronucleus induction in combination with histopathological changes including hypertrophy, proliferation of oval cells or bile duct epithelial cells, tissue injuries, regenerative changes, and inflammatory changes as the early responses of hepatocarcinogenesis. For these early responses, A total of 20 carcinogens, including 14 genotoxic hepatocarcinogens (Group A) and 6 non-liver-targeted genotoxic carcinogens (Group B) were evaluated. RESULTS: In the Group A chemicals, 5 chemicals (NPYR, MDA, NDPA, 2,6-DNT, and NMOR) showed all of the 6 early responses in hepatocarcinogenesis. Five chemicals (DMN, 2,4-DNT, QUN, 2-AAF, and TAA) showed 4 responses, and 4 chemicals (DAB, 2-NP, MCT, and Sudan I) showed 3 responses. All chemicals exhibited at least 3 early responses. Contrarily, in the Group B chemicals (6 chemicals), 3 of the 6 early responses were observed in 1 chemical (MNNG). No more than two responses were observed in 3 chemicals (MMC, MMS, and KA), and no responses were observed in 2 chemicals (CP and KBrO3). CONCLUSION: Evaluation of liver micronucleus induction in combination with histopathological examination is useful for detecting hepatocarcinogens. This assay takes much less time than routine long-term carcinogenicity studies.

Crystallization and preliminary X-ray analysis of CooA from Carboxydothermus hydrogenoformans.

CooA, a homodimeric haem-containing protein, is responsible for transcriptional regulation in response to carbon monoxide (CO). It has a b-type haem as a CO sensor. Upon binding CO to the haem, CooA binds promoter DNA and activates expression of genes for CO metabolism. CooA from Carboxydothermus hydrogenoformans has been overexpressed in Escherichia coli, purified and crystallized by the vapour-diffusion method. The crystal belongs to space group P2(1), with unit-cell parameters a = 61.8, b = 94.7, c = 92.8 angstroms, beta = 104.8 degrees. The native and anomalous difference Patterson maps indicated that two CooA dimers are contained in the asymmetric unit and are related by a translational symmetry almost parallel to the c axis.