Young-Man Cho

Evaluation of toxicity of green tea catechins with 90-day dietary administration to F344 rats

Green tea catechins (GTC), polyphenols extracted from the stalks and leaves of Camellia sinensis, are found in the different types of tea beverages and as antioxidant additives to many foods, snacks, fats and fatty oils. As a part of their safety assessment, subchronic toxicity was investigated in male and female F344 rats with dietary administration at concentrations of 0 (control), 0.3%, 1.25% and 5.0% for 90 days. The average daily intakes of GTC in each group were 180, 764 and 3525mg/kg body weight/day, respectively for males, and 189, 820 and 3542mg/kg body weight/day, respectively for females. No mortality or obvious clinical signs were observed throughout the experimental period but body weights were reduced from week 1 to the end of the experiment in 5.0% males. In serum biochemistry, alanine transaminase and alkaline phosphatase in 5.0% males and females and aspartate transaminase in 5.0% females were increased, together with the relative liver weights in both sexes receiving 5.0%. Although decreases were evident for total cholesterol in 0.3-5.0% males and triglycerides in 1.25% and 5.0% males and 5.0% females, these changes were not considered to be adverse. Hematology and histopathological observation revealed no GTC-related toxicological changes. Based on above findings, the no observed adverse effect level (NOAEL) of GTC was estimated to be 1.25% (764mg/kg body weight/day for males and 820mg/kg body weight/day for females).

Inhibition of conjugated fatty acids derived from safflower or perilla oil of induction and development of mammary tumors in rats induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

Chemopreventive effects of conjugated fatty acids derived from safflower oil (CFA-S), which contains large amounts of conjugated linoleic acid, and from perilla oil (CFA-P) with abundant conjugated alpha-linolenic acid were examined in a 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced rat mammary carcinogenesis model. Groups of 20-22 6-week-old female Sprague-Dawley (SD) rats were given eight intragastric injections of PhIP at a dose of 100 mg/kg b.w. during the initial 8 week period. Powdered basal diets containing 0.1% CFA-S or CFA-P were applied during or after PhIP treatment until week 40. In the rats receiving CFA-S or CFA-P together with PhIP treatment, retardation of mammary tumor emergence was observed until week 27. The groups given CFA-S or CFA-P after PhIP treatment, in contrast, demonstrated significant decrease in the final incidences of mammary adenocarcinomas. The indices of proliferating cell nuclear antigen positive cells in mammary adenocarcinomas were significantly reduced with both CFA-S and CFA-P in the post-initiation phase. Formation of aberrant crypt foci in the colon and basophilic foci of the pancreas due to the PhIP treatment group were not affected by CFA-S or CFA-P. In a second short-term experiment, female SD rats were maintained on powdered basal diet containing 0.03% PhIP alone or together with 0.1% CFA-S or CFA-P for 4 weeks. Immunohistochemically, CFA-S and CFA-P were revealed to suppress PhIP-DNA adduct formation in the epithelial cells of mammary gland (duct and alveolar cells), colon and pancreas. These results indicated that CFA-P and CFA-S may retard development of PhIP-induced mammary tumors with inhibition of PhIP-DNA adduct formation, and decreased mammary carcinogenesis in the post-initiation period with inhibition of cell proliferation.

Age-dependent histopathological findings in the prostate of probasin/SV40 T antigen transgenic rats: Lack of influence of carcinogen or testosterone treatment

Sequential changes in the phenotype of prostatic lesions and the impact of additional carcinogen treatment or castration on development and progression of prostate cancers were examined in probasin/simian virus 40 (SV40) T antigen transgenic (TG) rats. Non‐invasive prostate adenocarcinomas were evident in all lobes at 15 weeks of age. Invasive tumors were limited to the anterior lobe at this time point and were found in all lobes in an age‐dependent manner thereafter. No metastasis was apparent at any age. Additional carcinogen treatment or castration did not enhance progression or generate selective growth of hormone‐independent prostate cancer cells. These results suggest that our TG rats are suitable for clarification of mechanisms in early stages of prostate carcinogenesis, that is, from prostatic intraepithelial neo‐plasia (PIN) to non‐invasive and then invasive lesions. (Cancer Sci 2003; 94: 153–157)

Absence of in vivo genotoxicity of 3-monochloropropane-1,2-diol and associated fatty acid esters in a 4-week comprehensive toxicity study using F344 gpt delta rats

3-Monochloropropane-1,2-diol (3-MCPD) is regarded as a rat renal and testicular carcinogen and has been classified as a possible human carcinogen (group 2B) by International Agency for Research on Cancer. This is potentially of great importance given that esters of this compound have recently found to be generated in many foods and food ingredients as a result of food processing. There have been a few reports about their toxicity, although we have recently found that the toxicity profile of 3-MCPD esters was similar to that of 3-MCPD in a rat 13-week repeated dose study, except for the acute renal toxicity seen in 3-MCPD-treated females. In the present study, to examine in vivo genotoxicity we administered equimolar doses of 3-MCPD or 3-MCPD fatty acid esters (palmitate diester, palmitate monoester and oleate diester) to 6-week-old male F344 gpt delta rats carrying a reporter transgene for 4 weeks by intragastric administration. In vivo micronucleus, Pig-a mutation and gpt assays were performed, as well as investigations of major toxicological parameters including histopathological features. As one result, the relative kidney weights of the 3-MCPD and all three ester groups were significantly increased compared with the vehicle control group. However, the frequency of micronucleated reticulocytes and Pig-a mutant red blood cells did not differ among groups. Moreover, no changes were observed in mutant frequencies of gpt and red/gam (Spi(-)) genes in the kidney and the testis of 3-MCPD and 3-MCPD-fatty-acid-esters-treated rats. In histopathological analyses, no treatment related changes were observed, except for decrease of eosinophilic bodies in the kidneys of all treated groups. These results suggest that 3-MCPD and its fatty acid esters are not in vivo genotoxins, although they may exert renal toxicity.

High susceptibility of the ACI and spontaneously hypertensive rat (SHR) strains to 2-amino-1-methyl-6-phenylimidazo[4,5.b]pyridine (PhIP) prostate carcinogenesis

Carcinogenic responses in the prostate to 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) were compared among seven rat strains (F344, ACI, Spontaneously Hypertensive Rat (SHR), Sprague‐Dawley (SD), Wistar, Lewis and Brown Norway (BN)). Ten‐week‐old animals of each strain were given PhIP at 400 ppm in the diet for 20 weeks then maintained until week 54. The final survival rates were 92, 92, 83, 75, 67, 42 and 42%, respectively, and the SHR strain showed the highest sensitivity with regard to development of prostatic intraepithelial neoplasias (PINs) in the ventral prostate. With regard to the induction of adenocarcinomas of the ventral prostate, the ACI strain was most sensitive, whereas Lewis and F344 rats were relatively resistant. No adenocarcinomas were found in the dorsolateral or anterior prostate or seminal vesicles in any of the strains. The levels of serum testosterone and estrogen, PhIP‐DNA adducts and cell kinetics did not correlate with the development of ventral prostatic lesions and thus other factors are presumably responsible for the variations in susceptibility. The present data indicate that ACI and SHR rats are appropriate strains for experimental investigation of PhIP‐induced prostate carcinogenesis.

Frequent c-Ha-ras gene mutations in rat mammary carcinomas induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant heterocyclic amine in cooked meat and fish, is carcinogenic to the mammary glands of rats. Mutations in the H-ras gene were here examined in PhIP-induced mammary tumors of female F344 rats by the polymerase chain reaction followed by single strand conformation polymorphism analysis (PCR-SSCP) and restriction enzyme length polymorphism analysis (RFLP). Mutations in codon 12 of the H-ras gene were detected in four out of 13 tumors by PCR-SSCP. Three of them were GGA to GAA, and one was GGA to GTA. However, by RFLP analysis, four additional mutations in codon 13 were also detected in the same samples. Two had a GGC to CGC mutation, and the other shifts were GGC to GAC and GGC to GTC. Therefore, overall eight out of 13 cases had H-ras gene mutations. These results indicate that changes in H-ras function may play important roles in PhIP-induced mammary carcinogenesis.

Development of invasive follicular cell carcinomas in a rat thyroid carcinogenesis model : Biological impact of capsular inflammation and reduced cyclooxygenase-2 expression

We have previously reported that thyroid capsular inflammation induced by sulfadimethoxine (SDM), a goitrogen, might play a role in development of invasive follicular cell adenocarcinomas in rats initiated with N‐bis(2‐hydroxypropyl)nitrosamine (DHPN). The present study was designed to examine the role of cyclooxygenase (COX)‐2, widely known to be up‐regulated in inflammatory states, during chemically induced rat thyroid carcinogenesis. Male F344 rats received a subcutaneous DHPN (2800 mg/kg) injection, and 1 week later were allowed free access to drinking water containing antithyroidal propylthiouracil (PTU, 0.003%) or SDM (0.1%) for 4 or 10 weeks. Control groups receiving goitrogen alone and no treatment were also included. At week 4, diffuse follicular cell hyperplasia was induced in all PTU‐ and SDM‐treated groups, along with fibrous capsular thickening and capsular thickening with inflammation, respectively. Additionally, multiple focal follicular cell hyperplasias and adenomas were observed in the DHPN + PTU and DHPN + SDM cases. At week 10, adenocarcinomas invasive to the capsule and restricted to the capsular adjacent region, were frequent in the DHPN + SDM group, but not observed in the animals given DHPN + PTU. Western blots and immunohistochemistry revealed constitutive COX‐2 expression in non‐neoplastic follicular cells of the control and all of the PTU‐ and SDM‐treated rats. However, COX‐2 reactivity was significantly reduced or negative in the preneoplastic/neoplastic lesions in the DHPN‐treated groups. In fibrous or inflamed thickened capsules, only a few component cells with inflammatory elements were positive for COX‐2, and there was no significant difference in this regard between the PTU and SDM treatments. The present results suggest that capsular inflammation could play a role in development of invasive carcinomas, but COX‐2 expression does not make a major contribution. (Cancer Sci 2005; 96: 31–38)

Anti‐Inflammatory Effects of Capsaicin and Piperine on Helicobacter pylori‐Induced Chronic Gastritis in Mongolian Gerbils

Spices have been used for thousands of years, and recent studies suggest that certain spices confer beneficial effects on gastric disorders. The purpose of this study was to evaluate possible chemopreventive effects of spice‐derived compounds on Helicobacter pylori (H. pylori)‐induced gastritis.

Modification of an in vivo lung metastasis model of hepatocellular carcinoma by low dose N-nitrosomorpholine and diethylnitrosamine.

Previously, we established the in vivo lung metastasis model of rat HCC induced by two hepatocarcinogens, diethylnitrosamine (DEN) and N-nitrosomorpholine (NMOR) at a dose of 120 ppm. This model allows us to investigate modifying factors leading to the inhibition of metastasis formation. However, low survival rates made the evaluation of metastasis formation difficult. The current experiments were conducted to modify the experimental protocol to improve survival and to establish a better animal metastasis model. Lower doses of NMOR (80 or 40 ppm in drinking water) were given to F344 rats for 14 weeks after DEN treatment. Survival rates in the 80 ppm group and in the 40 ppm group were 57% and 81%, respectively and these values were significantly higher than that in 120 ppm. Incidences of lung metastasis in the 40 ppm group steadily increased up to 67% by week 36 while that in the 80 ppm increased sharply up to 86% by week 24. Severity of lung metastases in the 40 ppm group at week 36 was mild compared with the 80 ppm group at week 24. In the second experiment, in order to characterize HCC development and lung metastasis in the 40 ppm group, rats given DEN and then followed with 40 ppm NMOR were killed sequentially. Development of HCC was observed at week 14 and reached 100% incidence at week 20. First lung metastatic lesions were evident at week 22, and incidence of lung metastasis reached 100%. Tumor cells were identified in the blood at week 20 by RT-PCR. The current study revealed that 40 ppm NMOR for 14 weeks after DEN treatment developed HCC without lung metastases at week 22, then HCC with a frequent lung metastasis at week 40. Thus, it can be said that this system is a more appropriate model for elucidation of mechanisms of metastasis and also for analysis of factors to inhibit natural metastasis.

Down-regulation of Lsm1 is involved in human prostate cancer progression

Elucidation of genetic alterations is an approach to understanding the underlying molecular mechanisms of progression of human prostate cancers. We have searched for genes differentially expressed in advanced prostate cancers using cDNA-representational difference analysis, and thereby isolated the Lsm1 as one of down-regulated gene. An Lsm1 expression vector was transfected into PC3 cells, normally featuring down-regulated Lsm1, and four transfectants were established. No differences in morphology or cell proliferation were evident in comparison with parent PC3 or PC3/mock-transfectants. In contrast, significant suppression of invasive potential or metastatic ability of Lsm1 transfectants was observed in the Matrigel chemoinvasion assay and in nude mice, respectively. With human prostate cancers, almost all of informative prostatectomised cases without neoadjuvant therapy showed allelic retention in the Lsm1 region, whereas refractory cancers frequently showed allelic loss in this region. No critical gene mutations were found in open reading frame of Lsm1 in prostate cancers examined by PCR–SSCP analysis, including localised and refractory cancers. These results suggest that Lsm1 is deeply involved in prostate cancer progression through its down-regulation, independent of any gene mutation.