Paul M. Newberne

Carcinogenic effects of low dietary levels of aflatoxin B1 in rats.

Abstract Aflatoxin B1 was added to a semi-synthetic diet at levels of 1, 5, 15, 50 and 100 μg/kg (ppb). These diets were fed to male Fischer rats, beginning when their body weights were 80 g and continuing for up to 105 wk. A control group received the same diet without aflatoxin. Hepatocellular carcinomas were induced in a high incidence by levels of 50 and 100 ppb, and in a lower incidence by the other dietary levels. Tumours were induced in two of 22 animals fed 1 ppb, and pathological lesions were present in the livers of seven of the remaining survivors. These results suggest that, under these experimental conditions, the sensitivity of the Fischer rat to aflatoxin carcinogenesis is approximately the same as that previously reported for the rainbow trout.

Preventive role of vitamin A in colon carcinogenesis in rats.

Recent literature reviews (Cancer Res. 35:3295, Cancer Res. 35:3427, 1975) have re‐emphasized the importance of vitamin A in the differentiation and maintenance of integrity of epithelium and increasing resistance to some forms of cancer. We have observed an increased incidence of colon carcinoma (100% vs. 60%) in rats deficient in vitamin A and exposed to dimethylhydrazine (Cancer Res. 33:1003, 1973). Other studies have shown that a marginal dietary level of vitamin A not only enhanced liver cancer in rats exposed to aflatoxin B1 but resulted in a 29% incidence of colon cancer as well (J. Natl. Cancer Inst. 50:439, 1973). Recent unpublished results with DMH‐induced colon tumors indicated a significant protection from increased dietary levels of vitamin A analog, 13‐Cis retinoic acid (20/20 vs. 8/20 animals with tumors, 3.1 vs. 2.3 average tumors/animal). These data correlate with depressed glycopeptide synthesis by the gut epithelium (J. Biol. Chem. 245:4551, 1970). Results of these studies indicate that a deficiency of dietary vitamin A can increase susceptibility to DMH‐induced colon tumors in rats, resulting in colon tumors as well as liver tumors in rats exposed to aflatoxin B1 and that that the retinoid, 13 Cis‐retinoic acid can inhibit colon tumor induction by DMH. These findings further substantiate the importance of adequate vitamin A and the potential for the therapeutic use of 13‐Cis retinoic acid.

Choline Deficiency, Partial Hepatectomy, and Liver Tumors in Rats and Mice

In standard two year tumorigenicity studies some gestagens, alone or in combination with an estrogen have been shown to produce proliferative liver lesions (PLL) in rats. Estrogens in general have not produced PLLs; for ethinyl estradiol the situation is equivocal. The steroids which increase the incidence of PLLs have not been shown to have the characteristics usually associated with classical hepatocarcinogens such as diethylnitrosamine (e.g. negative mutagenicity and cytoxicity, failure to bind covalently to DNA). Therefore, another mechanism must be assumed. A possible explanation is given by the ability demonstrated for a few steroids (mestranol alone or in combination with norethindrone, ethinylestradiol) to promote preneoplastic islands induced by diethylnitrosamine, resulting in an increase in tumor incidence over rats given only the carcinogen. Spontaneous preneoplastic islands in rats occur in various incidence in different strains and we have recently shown that their reaction to steroids—enhanced proliferation—is the same as the effect on preneoplastic islands produced by classical carcinogens. Sex steroids also enhance the growth or normal hepatocytes. It, therefore, seems reasonable to assume that sex steroids produce PLLs by acting on spontaneous preneoplastic lesions and not by the mechanisms usually associated with classical carcinogens. Extrapolating the incidence of PLLs in rats to a risk in humans may be highly misleading because of different pharmacokinetic patterns. Even in rats treated orally for two years with sex steroids dosed as multiples of the human contraceptive dose, resorption and biovailability may vary by a factor of 10 or more. It is, therefore, inappropriate to state that one sex steroid is more active in the production of PLLs than another, unless the data from pharmacokinetic studies are taken into account. Available evidence shows that sex steroids act as tumor promotors and enhance the growth of normal hepatocytes. It is extremely doubtful whether a sex steroid producing PLLs in the rat after two years of oral treatment can be classified as an initiating tumorigen for the rat. Substantial risk for humans cannot be inferred, because of the high incidence of spontaneous preneoplastic lesions in rats, and because of the very high sensitivity of the rat liver to a variety of chemically unrelated compounds including sex steroids.

Acute Toxicity of Aflatoxin B1 in the Dog

This work is a study of the acute effects of aflatoxin B1 in dogs. The dog has a remarkable susceptibility to aflatoxin B1 given by oral and intraperitoneal routes. Intraperitoneal administration resulted in the shortest survival time and the most profound pathological changes. However, massive single oral doses produced lesions of similar nature and intensity. Oral administration of aflatoxin in small, divided doses, over extended periods proved to be less toxic. The experimentally induced disease resembled hepatitis X in many respects.

Lipotrope deficiency in experimental carcinogenesis

Abstract Diets deficient in lipotropes (methionine, choline, and folate) and high in fat increase hepatocarcinogenesis by many chemicals, including aflatoxin B1 (AFB1) and N‐2‐fluor‐enylacetamide (AAF). The increase can be corrected in most cases by lipotrope supplementation, but the degree of correction appears to be influenced by the type of fat in the diet. A lipotrope‐deficient, high‐fat diet also increases dimethylhydrazine carcinogenesis in the colon, an effect due to the dietary fat content, not to lipotrope deficiency. In contrast, mammary carcinogenesis by dimethylbenzanthrene or AAF is decreased or unchanged in rats fed the deficient diet. Hepatic microsomal oxidase activity, cytochrome P450 and conversion of AFB 1 to a bacterial mutagen all are decreased in assays in vitro using tissues from lipotrope‐deficient rats. However, urine mutagen content is increased after AFB1 treatment, as is urine content of activated AAF. AFB1 binding to hepatic DNA in vivo is unchanged or is slightly decreased. T...

Diet and aflatoxin B1 toxicity in rats

Abstract The effects of aflatoxin B 1 (AFB 1 ) in rats are markedly influenced by diet. Fischer and Sprague-Dawley male rats were fed either a complete synthetic diet or a synthetic diet marginally deficient in lipotropes, which had enhanced AFB 1 carcinogenesis in earlier studies. Deficient rats of both strains were resistant to the toxicity of a single dose of AFB 1 (7 or 9 mg/kg), which killed 60–100% of rats fed the complete diet. In contrast, the deficient rats were more sensitive to the acute toxicity of repeated small doses of AFB 1 . The dietary influence may be explained by the low resting levels of the hepatic drug-metabolizing enzymes and by the failure of the enzyme levels to respond to repeated doses of AFB 1 in the livers of deficient rats.

Acute and chronic toxicity of rubratoxin B.

Abstract Rubratoxin B was found to have the following LD50 values after a single ip dose, dissolved in dimethyl sulfoxide: mice, 0.27 mg/kg; rats 0.35 mg/kg; guinea pigs, 0.48 mg/kg; and cats, 0.2 mg/kg. Comparable values for propylene glycol solutions in dogs and chickens were greater than 5 and 4 mg/kg, respectively. Administration by stomach tube increased the LD50 value to 400–450 mg/kg in rats, possibly due to decomposition of the toxin in the acidic conditions of the stomach. Histologic lesions in animals poisoned by the toxin consisted of congestive, hemorrhagic, and degenerative lesions of the liver and spleen. Among rats treated by stomach tube 3 times/wk for 60 wk, 5 8 animals survived total doses of 458 or 616 mg per rat, and 11 18 survived a total dose of 307 mg/rat. No evidence of preneoplastic or neoplastic lesions was observed in survivors killed after 82–87 wk. Evidence of potentiation of the lethal action of rubratoxin B, but not of the carcinogenic action of aflatoxin B1, was found in rats treated with rubratoxin B (25 mg/kg, 3 times/wk for 5 wk) and fed over the same period a diet containing 0.2 ppm aflatoxin B1.

Dose-response study of the carcinogenicity of dietary sodium nitrite and morpholine in rats and hamsters.

Abstract Long-term feeding studies were conducted in Sprague-Dawley rats and Syrian golden hamsters using various dietary concentrations of nitrite and morpholine (up to 1000 ppm of each) or N -nitrosomorpholine (5 or 50 ppm). Most combinations of the two chemicals induced a high incidence of hepatocellular carcinoma in rats and a lower incidence in hamsters. The highest level of nitrite and morpholine (1000 ppm of each) had a stronger potential for carcinogenesis in both rats and hamsters than did a dietary level of 50 ppm of preformed N -nitrosomorpholine. Nitrite and morpholine also induced angiosarcomas in both species, most frequently in the liver, with the lung as the next most common site. The nitrite concentration in the diet seemed to have a greater effect on the incidence of hepatocellular carcinoma and angiosarcoma in the rat than did the concentration of morpholine. Dietary concentrations of 5 ppm each of nitrite and morpholine induced hepatocellular carcinoma and angiosarcoma in some rats. High concentrations of sodium nitrite alone were associated with a relatively high incidence of lymphoreticular tumours.

Effects of Bowman-Birk inhibitor on rat colon carcinogenesis.

The present study was undertaken to determine whether the Bowman-Birk inhibitor (BBI) could prevent colon carcinogenesis in rats treated with dimethylhydrazine (DMH) and whether there were adverse side effects associated with treatment with BBI for cancer prevention. BBI was evaluated in the forms of purified BBI (PBBI) or an extract of soybeans enriched in BBI, termed BBI concentrate (BBIC). The results demonstrate that PBBI and BBIC reduced the incidence and frequency of tumors in DMH-treated rats compared with animals treated with DMH alone. Autoclaved BBIC, in which the protease inhibitor activity of BBI was destroyed, had a weak and statistically insignificant, suppressive effect on DMH-induced colon carcinogenesis in rats, suggesting that the protease inhibitor activity of BBI is likely to be responsible for the anticarcinogenic activity of BBIC. Soy molasses, which contains soy isoflavones, did not have an effect on colon cancer carcinogenesis in DMH-treated rats. Similar to results from previous studies (Nauss et al. JNCI 73, 915-924, 1984), the most aggressive, malignant colon adenocarcinomas developed within or in association with gut-associated lymphoid tissue aggregates. No adverse side effects on the pancreas or animal growth were observed in rats treated with PBBI or BBIC. These results demonstrate that PBBI and BBIC may be used to prevent colon cancer without significant adverse side effects.

Lipid Peroxidation and atherosclerosis

Atherosclerosis and its clinical sequelae are among the most important of human cardiovascular diseases. Abnormal amounts of lipid are usually present in the thickened intima of atherosclerotic arteries. The notion that lipid peroxidation and polymerization occur in these lipid deposits is based largely on the fact that ceroid pigments have been demonstrated in atherosclerotic plaques. The detection of cholesteryl hydroxyoctadecadienoic acids in lipids of atheromas is suggestive evidence of lipoperoxidation; detection of lipoperoxides as such is technically difficult and the results to date are inconclusive. Studies of arteries from man and animals indicate that ceroid pigments occur late in the disease process and are not likely to be initiators of atherosclerosis. It is possible that hydroperoxides or intermediate free radiclals participate in the early stages of atherogenesis, but this theory is without conclusive support. Ceroid has been implicated as an inflammatory or sclerosing agent in arteries, p...