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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 tests of certain environmental and industrial chemicals.

Fourteen chemicals of varied uses were tested for carcinogenicity by oral administration in male and female Charles River CD rats. Under the conditions of the tests, propane sultone, propylene imine, and ethylenethiourea, in addition to the positive control N-2-fluorenylacetamide, were carcinogenic. Avadex, bis(2-chloroethyl) ether, the potassium salt of bis(2-hydroxyethyl) dithiocarbamic acid, ethylene carbonate, and semicarbazide hydrochloride were not carcinogenic under the test conditions. Dithiooxamide, glycerol alpha-monochlorohydrin, and thiosemicarbazide gave somewhat ambiguous results, though administered at high enough dose levels to be toxic. An inadequate number of animals survived treatments with sodium azide, sodium bisulfide, and vinylene carbonate, or the animals may not have received sufficiently high doses of the test chemicals to provide maximum test sensitivity. However, there were no indications that these three chemicals were carcinogenic under the test conditions.

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.

Inhibition of chemically induced mammary carcinogenesis in rats by long-term exposure to butylated hydroxytoluene (BHT): interrelations among BHT concentration, carcinogen dose, and diet.

In outbred female Sprague-Dawley rats long-term exposure to dietary butylated hydroxytoluene [3,5-di-tert-butyl-4-hydroxytoluene (BHT); CAS: 128-37-0] 1 week before carcinogen administration to termination resulted in a dose-related inhibition of mammary tumorigenesis and adrenocortical nodulogenesis. In animals fed the cereal-based NIH-07 diet and receiving a low dose (5 mg/rat) of 7,12-dimethylbenz [a] anthracene [(DBMA) CAS: 57-97-6], there was a significant overall inhibitory trend in tumor incidence observed among those receiving 300, 1,000, 3,000, and 6,000 ppm BHT. Maximal inhibition was approximately 50% at the highest concentration of BHT (6,000 ppm). The inhibitory effect of BHT on mammary tumor incidence was less pronounced when BHT was administered to rats initiated with a high carcinogen dose: At 15 mg DMBA/rat maximal inhibition was only 20% at the highest concentration of BHT (6,000 ppm). In contrast, when tumor yield was assessed in terms of latency or tumor multiplicity, the inhibitory effect of BHT was more pronounced in the groups given a high dose of DMBA than in the groups given a low dose. In animals given a low dose of DMBA (5 mg) and fed 6,000 ppm BHT in the casein-based AIN-76A diet, tumor incidence was inhibited by 50% of that of the controls; in contrast, when initiation was with a high dose of DMBA (15 mg), tumor incidence was decreased by only 28% of that of the controls. In animals fed the NIH-07 diet, DMBA-induced adrenocortical nodule formation was also inhibited in a dose-dependent fashion by BHT. At 5 mg DMBA maximal inhibition was 86% of control levels (6,000 ppm BHT); at 15 mg DMBA maximal inhibition was 66% of control levels (6,000 ppm BHT). However, when BHT was incorporated into the AIN-76A diet, its inhibitory effects on adrenocortical nodulogenesis were unexpectedly feeble and unrelated to carcinogen dose: In animals initiated with 5 mg DMBA and administered 6,000 ppm BHT, nodule incidence was decreased by only 25%, whereas in animals initiated with 15 mg DMBA, nodule incidence was decreased by 30% of that of the controls. These results indicate that while chronic exposure to dietary BHT suppressed the development of DMBA-induced mammary tumors and adrenocortical nodules, the degree of suppression depended on the dose of carcinogen administered, the level of BHT in the diet, and the parameter being measured. Diet-dependent differences in BHT action were observed with regard to DMBA-induced adrenocortical nodulogenesis but not with regard to mammary tumorigenesis.

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.