[Show abstract][Hide abstract] ABSTRACT: Siraitia grosvenori extract has been used as a food additive. As a part of the safety assessment of the extracts, a 13-week repeated dose toxicity study was performed in Wistar Hannover (GALAS) rats. Male and female rats were divided into five groups consisting of eight animals each and given diet containing 0%, 0.04%, 0.2%, 1%, and 5% of S. grosvenori extract for 13 weeks. During the experiment, no deaths were observed in any groups, and there were no remarkable changes in general appearance, body weight, food and water consumption, hematological and serum biochemical parameters, organ weight and histopathological findings between the control and treated groups. On the basis of these data, the no-observed-adverse effect level (NOAEL) of S. grosvenori extract in Wistar Hannover rats was considered to be 5% (2520 mg/kg/day in males and 3200 mg/kg/day in females) or more.
Preview · Article · Aug 2007 · Food and Chemical Toxicology
[Show abstract][Hide abstract] ABSTRACT: Microarray and RT-PCR analyses were performed for the transgene and Ras-related genes in forestomach squamous cell carcinomas (SCCs) induced by 7,12-dimethylbenz[a]anthracene (DMBA) in rasH2 mice; these results were compared with our previous molecular data of N-ethyl-N-nitrosourea-induced forestomach SCCs and urethane-induced lung adenomas in rasH2 mice. Overexpression of the transgene was detected in the DMBA-induced SCCs, suggesting that the transgene plays an important role in enhanced carcinogenesis in rasH2 mice. In addition, the mouse endogenous ras genes were up-regulated in the DMBA-induced SCCs, and are probably involved in the tumorigenesis of forestomach SCCs. Genes such as osteopontin, Cks1b, Tpm1, Reck, gelsolin, and amphiregulin that were commonly altered in these three different carcinogen-induced tumors may contribute to the development of tumors in rasH2 mice.
[Show abstract][Hide abstract] ABSTRACT: Our previous study suggested the possibilities that dicyclanil (DC), a nongenotoxic carcinogen, produces oxidative stress in the liver of the two-stage hepatocarcinogenesis model of mice and the stress induced probably causes secondary oxidative DNA damage. However, clear evidences demonstrating the relationship between DC-induced hepatocarcinogenesis, oxidative stress, and oxidative DNA damage have not been obtained. To clarify the relationship, further investigations were performed in the liver of the partially hepatectomized (PH) mice maintained on diet containing 1,500 ppm of DC for 13 and 26 weeks after intraperitoneal injection of dimethylnitrosamine (DMN). Significant increases in mRNA expressions of some metabolism- and oxidative stress-related genes with a formation of gamma-glutamyltranspeptidase (GGT) positive foci were observed in the DMN + DC + PH group by the treatment of DC for 13 and 26 weeks. The levels of 8-hydroxy-deoxyguanosine (8-OHdG) in the liver DNA also significantly increased in mice of the DMN + DC + PH group at weeks 13 and 26 and mice given DC alone for 26 weeks. The in vitro measurement of reactive oxygen species (ROS) generation from the mouse liver microsomes showed a significant increase of ROS production in the presence of DC. These results suggest that DC induces oxidative stress which is probably derived from its metabolic pathway, partly, and support our previous speculation that oxidative stress plays one of the important roles in the DC-induced hepatocarcinogenesis in mice.
No preview · Article · Nov 2006 · Archive für Toxikologie
[Show abstract][Hide abstract] ABSTRACT: mRNA expression profiles in the liver from mice treated with flumequine (FL) were analyzed in order to elucidate the mechanism of its tumor-promoting effect. The liver from a C3H/He mouse that received a diet containing 4,000 ppm of FL for 4 weeks was examined by cDNA microarray in comparison with an untreated mouse. Furthermore, to obtain a more comprehensive sequence, time-course changes in selected genes were determined by real-time RT-PCR. Microarray analysis revealed 15 upregulated and 9 downregulated genes in an FL-treated mouse. The upregulated genes included signal transducers and cell cycle regulators. In addition, the levels of stress response genes, particularly glutathione S-transferase (GST) alpha and GSTmu, were very high, indicating the generation of oxidative stress. On the other hand, the downregulated genes included phase I metabolic enzymes, such as cytochrome P450 (CYPs) enzymes, and apoptosis-associated proteins. These changes were confirmed by quantitative RT-PCR and were generally consistent with each other. Time-course observations revealed consistent results, particularly with regard to GSTalpha, GSTmu, ERK5, and CYP2E1. In addition, the expression of 8-oxoguanine DNA glycosylase 1 (OGG1) was increased in a time-dependent manner. These results suggest the possibility that responses against oxidative stress may play a major role in hepatocarcinogenesis by FL in mice.
Full-text · Article · Sep 2006 · Archive für Toxikologie
[Show abstract][Hide abstract] ABSTRACT: In order to evaluate the tumor-initiating activity of kojic acid (KA) in mouse liver, an in vivo initiation assay in liver was performed using partially hepatectomized mice. Male ICR mice were fed on a basal diet (BD) containing 0 or 3% KA for 4 weeks, followed by distilled water (DW) containing 0 or 500 ppm phenobarbital (PB) for 13 weeks. Two weeks after the treatment with PB, two-thirds partial hepatectomy was preformed in all mice in order to enhance the regeneration and proliferating activities of the hepatocytes. In microscopic examinations, no proliferative lesion was observed in any of the groups. There were no differences in the number of gamma-glutamyltransferase-positive cells, an expected marker for preneoplastic hepatocytes in mice, between the KA + DW and the KA + PB groups. In the immunohistochemical analyses of the proliferating activity of hepatocytes, significant increases in the labeling index of proliferating cell nuclear antigen (PCNA) were observed in the BD + PB and KA + PB groups as compared to the BD + DW group; however, no significant difference in the positivity of PCNA was observed between the BD + PB and the KA + PB groups. These results of the present study suggest the possibility that KA has no tumor-initiating activity in the liver of mice.
No preview · Article · Jun 2006 · Archive für Toxikologie
[Show abstract][Hide abstract] ABSTRACT: To evaluate the carcinogenic susceptibility of rasH2 mice to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 7-week-old rasH2 mice and their wild-type littermates (non-Tg mice) of both the sexes were fed a diet containing 0 or 300 ppm IQ for 26 weeks. Microscopical examinations revealed that the proliferative lesions of the forestomach, including squamous cell hyperplasias, papillomas, and carcinomas, were frequently encountered in male and female rasH2 mice fed with IQ. In non-Tg mice, no significant differences in the incidence of forestomach lesions were observed between the 0 ppm and 300 ppm groups. Histopathological changes such as periportal hepatocellular hypertrophy and oval cell proliferation in the liver were more apparent in female rasH2 and non-Tg mice than in males, and the incidence of hepatocellular altered foci significantly increased in female rasH2 mice in the 300 ppm group as compared to that in the 0 ppm group. These results suggest that the carcinogenic potential of IQ can be detected in rasH2 mice by a 26-week, short-term carcinogenicity test.
Preview · Article · Feb 2006 · Toxicologic Pathology
[Show abstract][Hide abstract] ABSTRACT: Our previous studies showed the possibility that oxidative stress, including oxidative DNA damage, is involved in the mechanism of dicyclanil (DC)-induced hepatocarcinogenesis at the preneoplastic stage in mice. In this study, the expression analyses of genes, including oxidative stress-related genes, were performed on the tissues of hepatocellular tumors in a two-stage liver carcinogenesis model in mice. After partial hepatectomy, male ICR mice were injected with N-diethylnitrosamine (DEN) and given a diet containing 0 or 1500 ppm of DC for 20 weeks. Histopathological examinations revealed that the incidence of hepatocellular tumors (adenomas and carcinomas) significantly increased in the DEN + DC group. Gene expression analysis on the microdissected liver tissues of the mice in the DEN + DC group showed the highest expression levels of oxidative stress-related genes, such as Cyp1a1 and Txnrd1, in the tumor areas. However, no remarkable up-regulation of Ogg1-an oxidative DNA damage repair gene-was observed in the tumor areas, but the expression of Trail-an apoptosis-signaling ligand gene-was significantly down-regulated in the tumor tissues. These results suggest the possibility that the inhibition of apoptosis and a failure in the ability to repair oxidative DNA damage occur in the hepatocellular DC-induced tumors in mice.
Preview · Article · Feb 2006 · Toxicologic Pathology
[Show abstract][Hide abstract] ABSTRACT: Chronic toxicity and carcinogenicity studies of ammonium sulfate, used as a food additive in fermentation, were performed in male and female Fisher 344 rats at dietary concentrations of 0%, 0.1%, 0.6% and 3.0% in a 52-week toxicity study and 0%, 1.5% and 3.0% in a 104-week carcinogenicity study. Treatment with ammonium sulfate caused significant increase in kidney and/or liver weights in males and females of the 3.0% diet group, but no effects were found on survival rate, body weights, and hematological, serum biochemical or histopathological parameters at any dose levels in the chronic toxicity study. Regarding carcinogenicity, ammonium sulfate did not exert any significant influence on the incidences of tumors in any of the organs and tissues examined. It was concluded that the no observed adverse effect level of ammonium sulfate was the 0.6% diet, which is equivalent to 256 and 284 mg/kg b.w./day in males and females, respectively, and the compound is non-carcinogenic under the conditions of the study.
No preview · Article · Feb 2006 · Food and Chemical Toxicology
[Show abstract][Hide abstract] ABSTRACT: In our previous study, microarray analysis was performed on N-ethyl-N-nitrosourea (ENU)-induced forestomach tumors in transgenic mice carrying the human prototype c-Ha-ras gene (ras H2 mice). Ras-MAPK related genes, including the transgene and mouse endogenous ras genes, that are involved in enhanced carcinogenesis were up-regulated in these tumors. In the present study, ras H2 mice received five intraperitoneal injections of 1,000 mg/kg urethane at 2-day intervals. Subsequently, microarray and RT-PCR analyses for the transgene and some molecules involved in the Ras pathway were performed on the induced lung tumors. In the microarray analysis, gene expression profiles of normal lungs and adenomas showed a distinct pattern, and several genes related to the cell cycle and nucleotide metabolism were up-regulated in the adenomas. RT-PCR confirmed the overexpression of the transgene in lung tumors; however, the up-regulation of the mouse endogenous ras genes was not observed. Some genes showed a similar expression pattern in both ENU- and urethane-induced tumors. These results suggest that the overexpression of the transgene plays an important role in the carcinogenesis of both ENU- and urethane-induced tumors in ras H2 mice. The genes that showed a similar expression pattern in both tumors were considered to be the candidate genes responsible for enhanced carcinogenesis.
[Show abstract][Hide abstract] ABSTRACT: The present study was conducted to elucidate the possible molecular mechanism of germinal cell apoptosis induced by Sertoli cell damage after 1,3-dinitrobenzene (1,3-DNB), a testicular toxicant, was administered to laboratory male rats. In this study, male Sprague-Dawley rats were administered with a single oral dose of 1,3-DNB (25 mg/kg body weight). Histopathological examinations and TUNEL methods revealed a marked increase in the number of apoptotic pachytene spermatocytes in seminiferous tubules showing stages VII-VIII and IX-XI of the spermatogenic cycle at 24 h after 1,3-DNB treatment. In immunohistochemical analysis, the cytoplasm and nuclei of pachytene spermatocytes were sometimes stained with antibodies to Bax and cleaved caspase-3 at 24 h after treatment. RT-PCR analysis for apoptosis-related gene expression showed that the expression of Bax,Bcl-2, Bcl-xL, and Bcl-xs genes, which are implicated in mitochondrial pathway, was significantly upregulated in the testes of the treated rats. These results suggest that the mitochondrial pathway is mainly involved in the testicular germinal cell apoptosis in rats induced by 1,3-DNB.
No preview · Article · Jan 2006 · Archive für Toxikologie
[Show abstract][Hide abstract] ABSTRACT: In our previous study in which diets containing 0, 1.5 or 3% Kojic acid (KA) were administered to heterozygous p53-deficient mice of the CBA strain [p53 (+/-) mice] and their wild-type littermates [p53 (+/+) mice] for 26 weeks, the incidences of hepatocellular adenomas as well as altered hepatocellular foci were increased in p53 (+/-) and p53 (+/+) mice of the KA treated groups without initiation, as compared to those in the untreated control mice. In order to confirm the reproducibility of the induction of hepatocellular proliferative lesions in p53 (+/+) mice given KA, male CBA mice were given dietary administration of 0, 0.5, 1 or 2% KA for 26 weeks. Body weight gain in the 2% KA group was depressed after week 20 as compared to the corresponding control group. Significant increases of absolute and relative liver weights were observed in the groups treated with 1% and 2% KA. The incidences of hepatocellular adenomas in the control and 0.5, 1 and 2% KA groups were 5, 17, 10 and 21%, respectively, and those of hepatocellular foci in these groups were 15, 39, 45 and 47%, respectively, the difference between the control and 2% KA groups being statistically significant. The mean values for multiplicity of hepatocellular adenomas in the control and 0.5, 1 and 2% KA groups were 0.05, 0.2, 0.2 and 0.2, respectively, and those for hepatocellular foci were 0.2, 0.6, 0.8 and 0.6, respectively. The results of the present study suggest that KA has a hepatocarcinogenic potential in CBA mice. 2005
No preview · Article · Oct 2005 · Journal of Toxicologic Pathology
[Show abstract][Hide abstract] ABSTRACT: To investigate the mechanism of hepatocarcinogenesis due to dicyclanil (DC), an insect growth regulator for sheep, histopathological and molecular biological analyses were performed in the liver of male ICR mice fed on a diet containing 1500 ppm of DC for 2 weeks (Experiment I; Exp. I). In gene expression analyses using a large-scale cDNA microarray and RT-PCR, fluctuations of expressions of metabolism-/oxidation-/reduction-related genes, such as CYP1A, aldehyde dehydrogenase family 1 subfamily A1 (Aldh1a1), and thioredoxin reductase 1 (Txnrd1), were predominantly observed in the liver of the DC-treated group. In Experiment II (Exp. II), small-scale and metabolism/oxidative stress-specific cDNA microarray, real-time RT-PCR, and measurement of NF-kappaB protein were performed in the mice liver using a two-stage hepatocarcinogenesis model, in which the male ICR mice were fed on a diet containing 1500 ppm of DC for 7 weeks after a single injection of dimethylnitrosamine (DMN). These mice were subjected to two-thirds partial hepatectomy (PH) at week 3. During histopathological examinations, a remarkable increase in gamma-glutamyltransferase-positive cells was observed in the DMN+DC+PH group. During the microarray and PCR analyses, the metabolism and oxidative stress-related genes, such as Cyp1a, P450 oxidoreductase (Por), and thioredoxin reductase 1 (Txnrd1); a few DNA damage/repair genes, such as 8-oxoguanine DNA-glycosylase 1 (Ogg1); and growth arrest and DNA-damage-inducible 45 alpha (Gadd45a), were fluctuated in this group, together with a slight increase in the concentration of activated NF-kappaB. These results suggest that DNA damages due to oxidative stress may be involved in the mechanism of DC-induced hepatocarcinogenesis in mice.
[Show abstract][Hide abstract] ABSTRACT: In order to examine the modifying effects of co-administration of t-butylhydroquinone (TBHQ) and sodium nitrite (NaNO 2) on forestomach carcinogenesis, a total of 50 male transgenic mice carrying a human prototype c-Ha-ras gone (rasH2 mice) received a single intraperitoneal injection of 60 mg/kg body weight of N-ethyl-N-nitrosourea (ENU), and starting 1 week later, they were given diet containing 0 (control group) or 0.5% TBHQ (TBHQ alone group), drinking water containing 0.2% NaNO 2 (NaNO 2 alone group) or diet containing 0.5% TBHQ in combination with 0.2% NaNO 2 in drinking water (TBHQ+NaNO 2 group) for 20 weeks. Squamous cell hyperplasias, papillomas and carcinomas were induced in all of the ENU treated groups, and there were no significant differences in the incidence, multiplicity and PCNA labeling index of these forestomach proliferative lesions between the NaNO 2 alone, TBHQ alone, TBHQ+NaNO 2 and control groups. These results suggest that the co-administration of TBHQ and NaNO 2 does not cause any modifying effects on ENU-induced forestomach carcinogenesis in rasH2 mice. 2005
No preview · Article · Jan 2005 · Journal of Toxicologic Pathology
[Show abstract][Hide abstract] ABSTRACT: Kojic acid (KA) has been used as a food additive for preventing enzymatic browning of crustaceans and as a cosmetic agent for skin whitening. To date, it has been reported that female B6C3F1 mice receiving 3% KA in the diet were found to develop hepatocellular tumors, but the underlying mechanism of liver tumorigenicity is still not clear. In the present experiments, in order to investigate possible liver initiation activity, partially hepatectomized male F344 rats received a single oral dose of 0, 1000 and 2000 mg/kg body weight of KA followed by dietary administration of 0.015% of N-2-acetylaminofluorene (2-AAF) for 2 weeks and a single 0.8 mL/kg body weight dose of CCl4. Furthermore, male F344 rats were fed a diet containing 0 or 2% KA for 3, 7 and 28 days, and the 8-oxodeoxyguanosine (8-OxodG) levels in nuclear DNA were measured to examine the formation of oxidative DNA adduct and cell proliferating activities of hepatocytes in the liver. In the liver initiation assay, there were no significant differences in the number or area of glutathione S-transferase placental form (GST-P) positive foci, putative preneoplastic lesions, between the KA-treated and control groups. Cell proliferation of hepatocytes in rats given KA for 3 and 7 days was significantly increased as compared with the relevant control values, but no significant elevation in 8-OxodG levels was apparent. The results of the present study suggest that KA has neither liver initiation activity nor capability of 8-OxodG formation, but some evidences suggestive of liver tumor promoting effects in rats. 2005
No preview · Article · Jan 2005 · Journal of Toxicologic Pathology
[Show abstract][Hide abstract] ABSTRACT: To clarify the in vivo genotoxicity of cinnamaldehyde (CNMA) that is present in cigarette smoke, 8 organs and bone marrow of male mice given a single oral administration of 250 or 125 mg/kg body weight of CNMA were evaluated by an alkaline single-cell gel electrophoresis (comet) assay. The liver initiation activity of CNMA was also investigated by a short-term liver initiation assay. PH (Partially hepatectomized) male rats were given 1250 mg/kg body weight of CNMA by a single oral administration 12 hours after partial hepatectomy. In the comet assay, no significant differences of nuclear migration were observed in all tissues examined in mice treated with CNMA at 3 and 24 hours after administration. In the initiation assay, there were no significant increases in areas and numbers of glutathione S-transferase placental form.(GST-P) positive foci in the livers of rats treated with CNMA. Based on these results, the possibility that CNMA has in vivo genotoxicity or liver initiation activity appears to be extremely low. 2005
No preview · Article · Jan 2005 · Journal of Toxicologic Pathology
[Show abstract][Hide abstract] ABSTRACT: To clarify the mechanisms underlying enhancement of carcinogenesis in transgenic mice carrying a human prototype c-Ha- ras gene (rasH2 mouse), animals received a single intraperitoneal injection of 120 mg/kg N-ethyl-N-nitrosourea (ENU) and at 20 weeks thereafter expression profiles in three induced forestomach squamous cell carcinomas were assessed using high-density oligonucleotide microarrays. In addition, the reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to assess mRNA expression of human c-Ha- ras gene and some molecules involved in the Ras-regulated mitogen-activated protein kinase (MAPK) pathway. Compared with normal forestomach tissue from control mice, 416 and 368 genes, respectively, were found to be commonly up- and down-regulated by 2-fold or more in the three tumors. Many genes involved in tumor invasion and metastasis such as transforming growth factor beta1 and matrix metalloproteinases were up-regulated, reflecting tumor progression. RT-PCR analysis confirmed up-regulation of transgene, mouse endogenous Ha- ras, N- ras, raf, Mekk2, c- fos, junB, c- myc and cyclin D1. These results suggest that activation of the Ras-MAPK cascade following up-regulation of both human and mouse endogenous ras genes is involved in the enhanced tumorigenesis of ENU-induced forestomach squamous cell carcinomas in rasH2 mice.
No preview · Article · Jan 2005 · Archive für Toxikologie
[Show abstract][Hide abstract] ABSTRACT: To evaluate the susceptibility of rasH2 mice to N-bis(2-hydroxypropyl)nitrosamine (DHPN), a potent carcinogen targeting the lung, liver, thyroid, and kidney, male, 6-week old, rasH2 mice and wild-type littermates (non-Tg mice) were given DHPN in drinking water at 0, 20 or 200 ppm, and 0 or 200 ppm, respectively, for 26 weeks. The experiment using rasH2 mice given 200 ppm DHPN and non-Tg mice given 200 and 0 ppm DHPN was completed at 20 weeks, since mortality in these groups was remarkably increased due to hemangiosarcomas of the liver. Histologically, tumors developed in the lung and liver in both rasH2 and non-Tg mice treated with DHPN. In addition, proliferative lesions were observed in the forestomach, urethra, and excretory duct of salivary glands in rasH2 mice given 200 ppm DHPN. RT-PCR analysis showed no marked difference in expression of mRNAs for the transgene and the endogenous mouse ras gene between the whole lung tissue containing a neoplasm and normal lung tissue. Our results suggest that rasH2 mice are highly susceptible to DHPN, the target organs including the forestomach, salivary gland and urethra, which have not been found to develop tumors in previous long-term carcinogenicity studies of DHPN in rats and mice.
No preview · Article · Jul 2004 · Toxicologic Pathology
[Show abstract][Hide abstract] ABSTRACT: Rice bran glycosphingolipid (RBGSL), one of the glycosphingolipids (GSLs), has been widely used as a food additive, a base of cosmetics, and so on. As a part of the safety assessment of RBGSL, a 13-week repeated dose toxicity study was performed in Wistar Hannover (GALAS) rats. Male and female rats were divided into 4 groups consisting of 8 animals and were given 0, 60, 250, and 1000 mg/kg BW of RBGSL orally 5 times weekly for 13 weeks. During the experiment, no deaths were observed in any groups, and there were no remarkable changes in general appearance, body weight, food and water consumption, hematological and serum biochemical parameters, organ weight and histopathological findings between the control and treated groups. On the basis of these data, the no-observed-adverse effect level (NOAEL) of RBGSL in Wistar Hannover rats was considered to be 1000 mg/kg BW/ day or more.
No preview · Article · Mar 2004 · The Journal of Toxicological Sciences
[Show abstract][Hide abstract] ABSTRACT: To clarify the mechanisms underlying the testicular toxicity of oxfendazole (OX), adult Wistar rats were orally administered a dose of 100 mg/kg/day for 3, 7, or 14 days. Assays of sex-related hormones showed a significant decrease in only the estradiol serum level at days 3 and 7, as compared with the control group. Histopathologically, marked degeneration of meiotic spermatocytes was observed in stage XIV-I seminiferous tubules from day 3 onwards, and these spermatocytes gave positive results on terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL). Abnormalities of spermiogenesis such as megakaryospermatids and binucleated spermatids were also observed in the testes of OX-treated rats. Under the electron microscope, lipid accumulation and dilatation of the endoplasmic reticulum were frequently found in the cytoplasm of the Sertoli cells on day 3. These results strongly suggest that OX induces both apoptosis of meiotic spermatocytes, most probably due to disruption of the microtubules, and degeneration of the Sertoli cells, characterized by distended endoplasmic reticulum and prominent cytosolic lipid accumulation.
Full-text · Article · Jan 2004 · Toxicologic Pathology
[Show abstract][Hide abstract] ABSTRACT: Wormwood, Artemisia absinthium, is a very bitter plant, and its extract has been used as food additives such as seasonings for food and drinks. A 13-week repeated dose toxicity study of wormwood extract was performed in both sexes of Wistar Hannover (GALAS) rats. Rats were divided into 4 groups consisting of 10 males and 10 females each, and were given water containing 0, 0.125, 0.5, or 2% wormwood extract. All rats had survived at the end of the study, and no changes indicating obvious toxicities that are attributable to the treatment of wormwood extract were observed in the body weights, hematological and serum biochemical examinations, organ weights, and histopathological examinations. Based on the results of the present study, the NOAEL (no-observed-adverse-effect-level) of wormwood extract of Wistar Hannover rats was estimated to be 2% (equivalent to 1.27 g/kg/day in males and 2.06 g/kg/day in females) or more.
No preview · Article · Jan 2004 · The Journal of Toxicological Sciences