Emiko Asakawa

Induction of Renal Cell Tumors in Rats and Mice, and Enhancement of Hepatocellular Tumor Development in Mice after Long‐term Hydroquinone Treatment

Hydroquinone (HQ) was administered to F344 rats and B6C3F1 mice of both sexes at a level of 0.8% in the diet for two years. This treatment induced renal tubular hyperplasia as well as adenomas, predominantly in males of both species, and was associated with chronic nephropathy in rats. In addition, the occurrence of epithelial hyperplasia of the renal papilla was increased in male rats. Foci of cellular alteration of the liver were significantly reduced in number by HQ in rats, but in contrast, were increased in mice, where development of hepatocellular adenoma was also enhanced in males. The incidence of squamous cell hyperplasia of the forestomach epithelium was significantly higher in mice of both sexes given HQ than in the controls, but no corresponding increase in tumor development was observed. The present study strongly indicates potential renal carcinogenicity of HQ in male rats and hepatocarcinogenicity in male mice. Thus, it is possible that HQ, which is present in the human environment, may play a role in cancer development in man.

Stomach carcinogenicity of caffeic acid, sesamol and catechol in rats and mice

The carcinogenic potential of caffeic acid, sesamol and catechol was examined in male and female F344 rats and B6C3F1 mice, groups of 30 animals being treated with diets containing 2% caffeic acid, 2% sesamol or 0.8% catechol for 104 weeks (rats) or 96 weeks (mice). Histological examination revealed that caffeic acid induced forestomach squamous cell carcinoma in 57% (P<0.001 vs. controls) and 50% (P<0.001) of male and female rats, respectively, whereas sesamol was associated with squamous cell carcinoma at incidences of 31% (P<0.001) in male rats, and 38% (P<0.001) and 17% (P<0.05) in male and female mice, respectively. Catechol induced glandular stomach adenocarcinomas in 54% (P<0.001) and 43% (P<0.001) of male and female rats, respectively. The results thus clearly demonstrated that all three antioxidants are carcinogenic in rodent stomach epithelia.

The Modifying Effects of Indomethacin or Ascorbic Acid on Cell Proliferation Induced by Different Types of Bladder Tumor Promoters in Rat Urinary Bladder and Forestomach Mucosal Epithelium

The effects of indomethacin (IM) or L‐ascorbic acid (AsA) on cell proliferation induced by bladder tumor promoters such as butylated hydroxyanisole (BHA), sodium L‐ascorbate (Na‐AsA), sodium citrate (Na‐Cit), and diphenyl (DP) in rat bladder and forestomach epithelium were investigated. Treatment with IM in combination with BHA or Na‐AsA diminished DNA synthesis levels of bladder epithelium as compared to the BHA or Na‐AsA alone values. On the other hand, AsA further amplified the increase of bladder epithelial DNA synthesis caused by Na‐Cit treatment. Histopathologically, administration of Na‐AsA in combination with IM reduced the incidence of simple hyperplasia. In contrast, simultaneous treatment with Na‐Cit and AsA caused an increase of the hyperplasia development. No apparent combination effects were observed in the DP‐treated groups. In forestomach epithelium, AsA enhanced the BHA‐induced increase in DNA synthesis and epithelial hyperplasia, characterized by marked basal cell proliferation. The present results thus suggested that IM may exert inhibitory effects on promotion of bladder carcinogenesis by certain tumor promoter types, and AsA may enhance BHA forestomach carcinogenesis.

Forestomach Neoplasm Induction in F344/DuCrj Rats and B6C3F1 Mice Exposed to Sesamol

Sesamol was administered at a dietary level of 2% to groups of 30 male and female F344/DuCrj rats and B6C3F, mice for 104and 96 weeks, respectively. Squamous cell carcinomas in the forestomach were Induced in nine of 29 (31%) effective male rats, three of 30 (10%) female rats, eleven of 29 (38%) male mice and five of 30 (17%) female mice treated with sesamol. Papillomas developed in ten of 29 (34%) male rats and fourteen of 30 (47%) female rats, but not in any of the mice. Hyperplasias developed in almost all rats and mice of both sexes. Significant differences from control values were found for all three lesions in rats and for carcinoma and hyperplasia categories in mice. The incidences of other tumors in the 2% sesamol group were comparable with control values. In conclusion, sesamol induces squamous cell carcinomas in the forestomach of rats and mice, males being more susceptible than females.

Promoting Effect of the Peroxisome Proliferator, Clofibrate, but Not Di(2‐ethylhexyl)phthalate, on Urinary Bladder Carcinogenesis in F344 Rats Initiated by N‐Butyl‐N‐(4‐hydroxybutyl)nitrosamine

The modifying potential of clofibrate and di(2‐ethylhexyl)phthalate (DEHP) on second stage, N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN)‐initiated urinary bladder carcinogenesis was investigated in male F344 rats, using a uracil‐accelerated transitional cell proliferation model. Six‐week‐old animals received 0.05% BBN in their drinking water for 4 weeks and then clofibrate (1.0, 0.5, and 0.25%) and DEHP (1.2, 0.6, and 0.3%) were given during experimental weeks 5–8 and weeks 12–20. Uracil was administered during weeks 9–11 at a dietary level of 3.0%. Control rats were treated with BBN and uracil without peroxisome proliferator. Surviving animals were killed at the end of week 20 of the experiment, when the densities of putative preneoplastic, papillary or nodular (PN) hyperplasias (numbers per 10 cm of basement membrane) were significantly increased in all clofibrate‐treated, but not the DEHP groups. The incidences of PN hyperplasia were similar in both treated animals and controls. In a second experiment, rats fed diets containing 1.0% clofibrate or 1.2% DEHP were assessed for levels of DNA synthesis in urinary bladder epithelium by 5‐bromo‐2′‐deoxyuridine immunohistochemistry. Numbers of labeled nuclei remained within normal levels, and no proliferative changes were evident. Thus, the present experiments indicated that while clofibrate, but not DEHP, exerts weak enhancing effects on BBN‐initiated urinary bladder carcinogenesis in rats this is not associated with increased levels of DNA synthesis in the affected epithelium.

Dose-Dependent Renal Tubular Toxicity of Harman and Norharman in Male F344 Rats*

The renal toxicity of harman and norharman, administered for 2 or 4 weeks at dietary levels of 1,000, 500, or 0 parts per million (ppm), was investigated in 6-week-old male F344/DuCrj rats. Although rats fed 1,000 ppm harman or norharman, but not the 500 ppm level, demonstrated marked body weight retardation from 1 week to termination, no mortalities occurred. Marked elevation of water consumption was evident in rats given harman or norharman at 1,000 ppm, but not at 500 ppm, together with large increases in urine of low specific gravity. Urinary lysosomal enzymes (N-acetyl-βT-D-glucosaminidase, NAG, and lactate dehydrogenase, LDH) and sugar levels were increased, and the brush border enzymes (γ-glutamyl transpeptidase, GGT, and alkaline phosphatase, ALP) decreased. Furthermore, serum biochemistry revealed clear elevation of parameters indicating renal toxicity in these rats. Histopathologically, rats fed 1,000 ppm harman or norharman, but not 500 ppm, demonstrated focal toxic renal degenerative/necrotic and regenerative lesions in proximal, distal, and collecting tubules. These changes were associated with a clearly increased labeling index (LI) of the nuclei of renal tubular epithelial cells on immunohistochemical staining for 5-bromo-2′-deoxyuridine (BrdU). Chemical specific crystal formation within tubular lumina was evident in rats fed 1,000 ppm, but not 500 ppm, this being considered the cause of the renal tubular lesions. It was concluded that harman and norharman exert renal toxicity at the dietary level of 1,000 ppm, but not 500 ppm, in male F344 rats.

Enhancing effects of harman and norharman on induction of preneoplastic and neoplastic kidney lesions in rats initiated with N-ethyl-N-hydroxyethylnitrosamine

The modifying potential of two nephrotoxic agents, harman and norharman, on N‐ethyl‐N‐hydroxyethylnitrosamine (EHEN)‐induced renal and hepatic carcinogenesis was investigated in male F344/DuCrj rats. Animals were given 0.1% EHEN in their drinking water for the first 2 weeks as an initiator. Subsequently, starting 3 weeks from the commencement, they were fed diet containing these compounds at concentrations of 1000, S00 or 0 ppm until week 26, and then killed for light microscopic examination. The mean numbers of renal tubular cell hyperplasias/cm2 and those of tumors/cm2 in rats given harman and norharman at 1000 ppm after initiation, but not at 500 ppm, were significantly increased as compared to the control values. However, neither compound modified liver carcinogenesis. It is concluded that harman and norharman show enhancing effects on rat kidney carcinogenesis, when ingested at dose levels which cause renal tubular damage.

No enhancing effects of calcium/magnesium salts of L-glutamate and L-ascorbate on tumor development in a rat medium-term multiorgan carcinogenesis bioassay

Calcium/magnesium salts of L-glutamate and L-ascorbate were tested for modification potential using a rat multiorgan carcinogenesis bioassay. Following sequential treatment with three different carcinogens (diethylnitrosamine, N-methylnitrosourea, and dihydroxydi-N-propylnitrosamine) over a 4-wk period, rats were given diet containing 5% monocalcium di-L-glutamate tetrahydrate (Ca-glutamate), 2.5% monomagnesium di-L-glutamate tetrahydrate (Mg-glutamate), 5% L-glutamic acid, 5% monocalcium di-L-ascorbate dihydrate (Ca-ascorbate), 2.5% monomagnesium di-L-ascorbate dihydrate (Mg-ascorbate), or 5% L-ascorbic acid for 16 wk. Body weight increase was slightly suppressed in the groups receiving Ca-ascorbate, Mg-ascorbate, and ascorbic acid supplementation after the carcinogen treatments. While administration of Ca-glutamate or Ca-ascorbate raised urinary pH, ascorbic acid values were decreased. Concentrations of calcium and magnesium ions in the urine increased after ingestion of Ca-glutamate or Ca-ascorbate, and Mg-glutamate or Mg-ascorbate, respectively, but phosphorus levels decreased in all groups given calcium and magnesium salts. No consistent treatment-related changes in the concentrations of sodium or potassium ions in the urine were detected. Histopathological investigation at wk 20 did not demonstrate any modification of tumorigenesis with regard to the incidence of frequency of lesions developing in the various target organs/tissues. The present results thus revealed no apparent enhancement of carcinogenesis at any site, including the urinary system, by calcium or magnesium salts using the present rat multiorgan carcinogenesis bioassay.