Carcinogen risk assessment.

The National Academy of Sciences elected 12, not 6, new foreign associates (News and Comment, 3 June, p. 1028). The remaining six are Kimishige Ishizaka (Japan), medicine and microbiology, Johns Hopkins University School of Medicine; Ikuo Kushiro, petrology, University of Tokyo, Tokyo, Japan; Guido Pontecorvo (Italy). geneticist, Imperial Cancer Research Fund Laboratories, London, United Kingdom; Kai M. Siegbahn, University of Uppsala, Uppsala. Sweden; John R. Vane, research and development, Wellcome Research Laboratories, Kent, United Kingdom: Douglas F. Waterhouse (retired), entomology. CSIRO, Deakin, Australia.

Carcinogen testing: current problems and new approaches.

The classic procedures for testing potential carcinogens in animals have basically not changed in the past 50 years. Considerable knowledge of the mechanisms of carcinogenesis has accrued in the last 20 years, particularly concepts on the metabolic activation of chemicals to reactive electrophilic compounds that can interact with nucleophilic including DNA. These developments, in turn, have yielded a framework for integrating into carcinogen testing the determination of genetic effects of chemicals. A systematic decision point approach to carcinogen testing has been developed which entails a sequential decision-making process as specific tests are performed and evaluated prior to initiation of higher order, more complex tests. Compared to conventional bioassays in rodents, this approach provides knowledge based on mechanisms of carcinogenesis, yields a substantial amount of data at minimal cost, and forms a solid base for eventual heath risk assessment.

Mutagenic activity of nitrite-treated foods: human stomach cancer may be related to dietary factors.

By the Salmonella typhimurium test, extracts of Japanese raw fish treated in the laboratory with nitrite showed mutagenic activity which is prevented by addition of ascorbate. Extracts from similarly treated beef and hot dogs were nonmutagenic. The data conform to a working concept that the high stomach cancer incidence in Japanese and certain other populations may be due to specific dietary factors of an alkylnitrosamide type.

Fecal bacterial beta-glucuronidase: control by diet.

The effect of a mixed Western, high meat diet or a nonmeat diet on the intestinal bacterial beta-glucuronidase activity was studied in human volunteers. This enzyme was significantly higher in stools of subjects on a high meat diet as compared to the nonmeat regimen. Thus, intestinal flora of subjects on a high meat diet was more able to hydrolyze glucuronide conjugates than that of individuals on a nonmeat diet. This, in turn, may raise the amount of substances, such as carcinogens, within the colonic lumen.

Liver cancer: neonatal estrogen enhances induction by a carcinogen.

A single injection of 100 micrograms of estradiol benzoate into newborn rats was followed after weaning by dietary treatment with one of two dosages of the carcinogen N-hydroxy-N-2-fluorenylacetamide. Autopsies 26 weeks later showed a higher incidence of liver cancer in male and, particularly, female rats injected with hormone than in controls. The weights of livers were greater but gonads were smaller in size in the estradiol groups. Endocrine and possibly centralnervous-system factors may play roles in formation of liver tumors.

Effect of various levels of dietary butylated hydroxyanisole on methylazoxymethanol acetate-induced colon carcinogenesis in CF1 mice.

The effect of dietary butylated hydroxyanisole (BHA) on methylazoxymethanol (MAM) acetate-induced colon carcinogenesis was studied in female CF1 mice fed the NIH-07 open formula diet and the AIN-76 semipurified diet. BHA levels in the experimental diets were 0.6% in the AIN-76 diet and 0.03, 0.1, 0.3, and 0.6% in the NIH-07 diet. Starting at 5 weeks of age, groups of mice were fed diets with or without BHA. At 7 weeks of age, all animals except vehicle-treated controls were given ip injections of MAM acetate (15 mg/kg body wt), four times in 11 days (low dose) and eight times in 22 days (high dose). Animals were fed their experimental diets until 2 weeks after carcinogen treatment, when those receiving the BHA diets were fed their respective control diets without BHA until termination of the experiment. With a low dose of carcinogen, BHA in the NIH-07 diet inhibited lung tumor incidences in a dose-related manner; with a high dose of carcinogen the inhibition was apparent with 0.1-0.6% BHA. Lung tumor incidence was lower in the low carcinogen treated group fed the AIN-76 diet containing 0.6% BHA than in the animals fed the diet without BHA. Colon tumor incidence was lower in mice fed the NIH-07 diet containing 0.3 and 0.6% BHA and treated with a low dose of carcinogen than in the animals fed no BHA; colon tumor multiplicity (adenomas/animal and adenomas/tumor-bearing animal) was inhibited in mice fed the diets containing 0.03-0.6% BHA. In groups given a high dose of MAM acetate, the NIH-07 diet with 0.03-0.6% BHA and the AIN-76 diet with 0.6% BHA greatly inhibited colon tumor incidence and multiplicity.

Effect of a diet with high levels of protein and fat on colon carcinogenesis in F344 rats treated with 1,2-dimethylhydrazine.

Female inbred F344 rats fed diets containing high levels of beef protein and fat or high levels of soybean protein and corn oil and treated with 1,2-dimethylhydrazine had a greater incidence of colon tumours than did rats fed diets that had normal levels of such components and were treated similarly. The source of fat and protein had no major influence on the incidence of colon tumors.

Induction of carcinoma of the large intestine in guinea pigs by intratectal instillation of N-methyl-N-nitrosourea.

Intrarectal administration of 0.5 ml of a 0.25 percent solution of N-methyl-N-nitrosourea twice seekly for 42 weeks to female inbred strain-2 guinea pigs induced large-bowel adenocarcinomas in 9 of 10 animals in 38-56 weeks. Controls did not show cancer. The lesions were infiltrative or constrictive, which distinguished them from chemically induced large-bowel cancers of rats and mice.

In vitro binding of the food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline to dietary fibers.

The ability of three kinds of fiber to bind to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a potent bacterial mutagen formed during the cooking of meat, was investigated to determine interactions among important food components. At pH 6.5 corn bran, wheat bran, and alfalfa meal each bound approximately 50% of the available IQ after incubation for 1 hour. Binding was pH-dependent, occurred optimally between pH 4 and 6, and was probably due to a cation-exchange mechanism. The pH at which IQ is efficiently bound to fiber overlaps the range of pH in the human gastrointestinal tract.

Azoxymethane-induced liver hemangiosarcomas in inbred strain-2 guinea pigs.

Intrarectal administration of 0.5 ml of a 0.2% solution of azoxymethane twice weekly for 33 weeks to female inbred strain-2 guinea pigs specifically induced multiple liver hemangiosarcomas in 15 of 16 animals 32-54 weeks after the first treatment. No neoplasms and preneoplastic changes were found in the large intestine.

Effect of dietary alfalfa, pectin, and wheat bran on azoxymethane-or methylnitrosourea-induced colon carcinogenesis in F344 rats.

The effect of dietary alfalfa, pectin, and wheat bran on colon carcinogenesis was studied in female inbred F344 rats. Weanling rats were fed semipurified diets containing 0 or 15% alfalfa, pectin, or wheat bran. At 7 weeks of age, all animals except controls were given azoxymethane (AOM) sc at a dose rate of 8 mg/kg body weight/week for 10 weeks or methylnitrosourea (MNU) intrarectally at a dose rate of 2 mg/rat twice a week for 3 weeks. The AOM-treated group was autopsied 40 weeks and the MNU-treated group 30 weeks after the first injection of the carcinogen. No tumors were observed in the colon or other organs of untreated rats fed the various diets. The animals fed the alfalfa diet and treated with MNU had a higher incidence of colon tumors than did those fed the control diet or the diets containing pectin or wheat bran. The incidence of MNU-induced colon tumors did not differ between the animals fed the control diet or the diets containing pectin or wheat bran. However, the incidence of AOM-induced colon tumors in rats fed diets containing pectin or wheat bran was lower than that in rats fed the control diet or the alfalfa diet. These results thus indicate that the effect of fiber in colon carcinogenesis depends on the type of fiber and, possibly, the fiber's mode of action.

Effect of bile acids and neutral sterols on benzo[a]pyrene-induced tumorigenesis in skin of mice.

The effect of neutral sterols and bile acids were determined in the classic mouse skin tumor induction system by applying each sterol or acid with benzo[a]pyrene to the skin of Swiss mice three times per week for 60 weeks. The mice were killed 63 weeks after the experimenta was begun. Most of the chemicals had an inhibitory effect on skin tumor formation by benzo[a]pyrene. Unconjugated bile acids had higher inhibitory action than did conjugated bile acids. The inhibition decreased in the following order of acids: chenodeoxycholic, lithocholic, deoxycholic, and clinic. Within the group of conjugated acids, taurine conjugates were more effective inhibitors than were glycine conjugates. The neutral sterols cholesterol, coprostanol, and coprostanone showed relatively similar, moderate inhibitory effects.

Induction of cancer of the glandular stomach in rats by an extract of nitrite-treated fish.

Treatment of a homogenate of the mackerel fish Sanma hirakl with nitrite at pH 3 led to the development of direct-acting mutagenic activity for Salmonella typhlmurium TA-1535. Repeated gastric intubation three times/week for 6 months of an extract containing this mutagenic activity into noninbred Wistar rats led to the induction of tumors in 8 of 12 rats 12-18 months later. Adenomas and adenocarcinomas were found in the glandular stomach, squamous cell carcinoma was observed in the forestomach, and adenocarcinoma was found in the small intestine and pancreas. Furthermore, precancerous lesions (including intestinal metaplasia and glandular hyperplasia of the glandular stomach as well as squamous cell hyperplasia) were noted in virtually all of the animals at risk. No tumors were seen in 8 control rats given the untreated fish extract alone; 1 rat had glandular hyperplasia and intestinal metaplasia. Thus a mutagenic extract of nitrite-treated fish was demonstrated to induce, in the rat glandular stomach, cancers identical to gastric cancer observed in man. Preventive m:asures, including reduction of the intake of pickled foods and the year-round daily availability of foods containing vitamin C, are discussed.

Detection and estimation of azomethane in expired air of 1,2-dimethylhydrazine-treated rats.

Azomethane (AM) gas was identified as a major metabolite of 1,2-dimethylhydrazine (1,2-DMH) in the expired air of F344 rats. The compound was characterized by high-pressure liquid chromatography, gas chromatography, and mass spectrometry, in comparison to a synthetic standard. At a dose of 21 mg 1,2-DMH/kg sc, approximately 14 and 11% of the dose were exhaled as AM and CO2, respectively, in 24 hours. At 200 mg 1,2-DMH/kg, 23 and 4% of the dose appeared as AM and CO2, respectively, in the respired air within the same period. Most AM was seen in the first 6 hours, but the CO2 evolution was more progressive, especially after the higher dose of 1,2-DMH.

Carcinogenicity of 3-methyl-2-naphthylamine and 3,2'-dimethyl-4-aminobiphenyl to the bladder and gastrointestinal tract of the Syrian golden hamster with atypical proliferative enteritis.

3,2'-Dimethyl-4-aminobiphenyl (DMAB) and 3-methyl-2-naphthylamine (MeNA) were each administered to groups of 25 male noninbred Syrian golden hamster weanlings in doses of 100 mg/kg body weight by weekly sc injections for a total of 38 injections over 18--22 months. DMAB produced a high incidence of urinary bladder epithelial neoplasms and intestinal neoplasms. Other neoplasms included squamous papilloma of nonglandular stomach, lymphoma-leukemias, and squamous cell carcinomas of the ear duct and skin. With MeNA, urinary bladder epithelial neoplasms occurred but no intestinal tumors. Also present were soft-tissue sarcomas at the injection site, papilloma of forestomach, and lymphoma-leukemias. Most hamsters in both groups as well as the control group had a chronic form of atypical proliferative enteritis that affected the small or large intestine. The occurrence of intestinal tumors with DMAB could be related to sensitizing effect of increased intestinal mucosal proliferation. Apart from this effect, hamsters displayed a distinct susceptibility to bladder carcinogenesis by these aromatic amines.

Promoting effect of sodium deoxycholate on colon adenocarcinomas in germfree rats.

The promoting effect of sodium deoxycholate (DC) on colon carcinogenesis was studied in female F344 germfree rats. Animals received intrarectal (ir) instillations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for 4 weeks (total dose, 16 mg/rat), then weekly ir doses of DC (total dose, 3 g/rat); the rats were autopsied 52 weeks after the first injection. DC increased the number of MNNG-induced colon adenocarcinomas. No tumors were in the colons of germfree rats given DC alone. It was concluded that DC (present in high concentrations in human stools) had a promoting effect on colon carcinogenesis in rats.

Effect of dietary fat on X-ray-induced mammary cancer in Sprague-Dawley rats.

We studied the effect of dietary fat levels on the induction of mammary cancer by 350 rads total-body X-radiation given to noninbred albino Sprague-Dawley rats at 50 days of age. Compared to rats on a low-fat (LF) diet (5% lard), rats on a high-fat (HF) diet (20% lard) from 30 days of age had more tumors, with a higher multiplicity of carcinomas per rat. LF-fed groups exhibited a longer median tumor latency period thatn did HF-fed groups. A similar trend toward more tumors with an earlier time of death was seen in rats given single iv doses of 50 mg 1-methyl-1-nitrosourea/kg and fed an HF diet as compared to an LF diet.

Mechanisms of the inhibitory action of p-hydroxyacetanilide on carcinogenesis by N-2-fluorenylacetamide or N-hydroxy-N-2-fluorenylacetamide.

The metabolism of N-2-fluorenylacetamide (FAA) and N-hydroxy-2-fluorenylacetamide (N-OH-FAA) was studied in groups of rats that had been prefed the protective agent p-hydroxyacetanilide (p-OH-AA) alone or in combination with each of the carcinogens for 4 weeks. Compared with controls, pretreatment increased the percentage of metabolites in the urine, chiefly as glucuronic acid conjugates, whereas the fecal excretion of FAA metabolites was lowered. The levels of total and tissue-bound material in the liver and blood plasma were also lower after prefeeding. Liver aryl hydrocarbon hydroxylase and liver deacetylase were not affected by p-OH-AA pretreatment. However, liver glucuronyl transferase was increased by either prefeeding with p-OH-AA and/or the carcinogen. The protective effect of p-OH-AA against liver tumor induction with FAA or N-OH-FAA may in part result from a combination of the decreased binding of carcinogen to hepatic cellular macromolecules and the increased excretion as the glucuronide conjugates.

Aging changes in CD-1 HaM/ICR mice reared under standard laboratory conditions.

Three hundred CD-1 HaM/ICR mice were observed for 2 years, and useful necropsies were done on 99 males and 102 females. Mortality was 50% at 16 months in the males and 18 months in the females. Among mice that came to autopsy, total tumor incidence was 54% for males and 75% for females, with most neoplasms occurring after 18 months. Adenomas or adenocarcinomas of the lung were the most frequent, followed by lymphoreticular tumors, vascular tumors, hepatomas, subcutaneous fibrosarcomas, and adenocarcinomas of the mammary glands. Some degree of amyloidosis was seen in half the mice of both sexes, beginning at 8 months in males and 12 months in females. Variability in tumor incidence among small groups if mice emphasized the need for adequate samples.

Modification of diethylnitrosamine liver carcinogenesis with phenobarbital but not with immunosuppression.

Administration of 40 ppm diethylnitrosamine (DENA) in the drinking water for 10 weeks to male Fischer rats led to hepatocellular carcinoma in 100 percent with metastasis to the lung in 40 percent, of the animals living for the full experimental period of 20 weeks. Concurrent feeding of phenobarbital and DENA for 10 weeks produced cancer of the liver in 77 percent of the animals, but only 9 percent had metastases in the lung. A brief regimen of DENA for 4 weeks, followed by 16 weeks of observation, induced cancer of the liver in only 13 percent of the rats. Administration of phenobarbital, begun 1 week after cessation of DENA intake and terminated at week 20, led to liver cancer in 64 percent of the rodents. Hydroxyurea had no effect on this enhancement. Treatment with a purified gamma fraction of antilymphocytic serum after the DENA did not influence the outcome. Thus phenobarbital given together with DENA reduced the severity of the carcinogenic process, but when it was given after the hepatocarcinogen, it increased the effect.