Safety assessment of gamma-cyclodextrin.

Gamma-cyclodextrin (gamma-CD) is a cyclic alpha-(1,4)-linked oligosaccharide consisting of eight glucose molecules. Like other cyclodextrins, gamma-CD can form inclusion complexes with a variety of organic molecules because the inner side of the torus-like molecule is less polar than the outer side. In foods, gamma-CD may be used as a carrier for flavors, vitamins, polyunsaturated fatty acids, and other ingredients. It also has useful properties as a stabilizer in different food systems. The daily intake from all its intended uses in food at highest feasible concentrations has been estimated at 4.1g/person/day for consumers of gamma-CD containing foods. The present review summarizes the safety data of gamma-CD. The toxicity studies consist of standard genotoxicity tests, subchronic rat studies with oral and intravenous administration of gamma-CD for up to 3 months, a subchronic (3-month) toxicity study in dogs, a (1-year) oral toxicity study in rats, and embryotoxicity/teratogenicity studies in rats and rabbits. In the studies with oral administration, gamma-CD was given at dietary concentrations of up to 20%. All these studies demonstrated that gamma-CD is well tolerated and elicits no toxicological effects. Metabolic studies in rats showed that gamma-CD is rapidly and essentially completely digested by salivary and pancreatic amylase. Therefore, the metabolism of gamma-CD closely resembles that of starch and linear dextrins. A human study with ingestion of single doses of 8 g gamma-CD or 8 g maltodextrin did not reveal a difference in gastrointestinal tolerance of these two products. An interaction of ingested gamma-CD with the absorption of fat-soluble vitamins or other lipophilic nutrients is not to be expected because the formation of inclusion complexes is a reversible process, gamma-CD is readily digested in the small intestine, and studies with beta-CD, a non-digestible cyclodextrin, have shown that the bioavailability of vitamins (A, D, and E) is not impaired. On basis of these studies it is concluded that gamma-CD is generally recognized as safe (GRAS) for its intended uses in food.

The FEMA GRAS assessment of pyrazine derivatives used as flavor ingredients. Flavor and Extract Manufacturers Association.

This is the fifth in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, pharmacokinetics and toxicology. Flavor ingredients are evaluated individually taking into account the available scientific information on the group of structurally related substances. Scientific data relevant to the safety evaluation of the use of pyrazine derivatives as flavoring ingredients is evaluated.

Treatment with soybean-derived Bowman Birk inhibitor increases serum prostate-specific antigen concentration while suppressing growth of human prostate cancer xenografts in nude mice.

BACKGROUND: Bowman Birk inhibitor (BBI) is an anticarcinogenic serine protease inhibitor that may inhibit the protease activity of prostate-specific antigen (PSA) and the growth of human prostate cancer xenografts in nude mice. METHODS: Human prostate cancer xenografts were established by implanting LNCaP cells into the prostate glands of NCRNU-M athymic nude mice. The animals with established tumors were maintained on a control diet or diets supplemented with 1% BBI or 1%, 2%, or 3% BBI concentrate (BBIC) for 6 weeks. The serum PSA concentrations were determined before and after the BBI or BBIC treatment period. The final tumor loads were determined at autopsy. RESULTS: Treatment with BBI or BBIC decreased the final tumor load and increased the tumor doubling time and PSA density in the nude mice bearing human prostate cancer xenografts. CONCLUSIONS: BBI and/or BBIC could be useful for prostate cancer treatment.

Esophageal carcinogenesis in the rat: zinc deficiency and alcohol effects on tumor induction.

Sprague-Dawley male rats were fed zinc-deficient or supplemented diets for 2 weeks, administered a carcinogenic dose of methylbenzylnitrosamine and observed over 20 or more weeks for effects of superimposing excess zinc or alcohol on development of esophageal tumors. In three separate experiments it was shown that (1) excess zinc offered no protection, (2) switching diets during or after carcinogen exposure pointed toward involvement of zinc in both initiation and promotion, (3) neither ethanol nor 3-methyl butanol alone affected tumorigenesis but the two combined and superimposed on a zinc deficiency resulted in a significant enhancement of neoplasia. In one group of rats fed the zinc-deficient diet only, with no carcinogen, 4 rats developed neoplasms, one of which was malignant. Cell proliferation, an integral component of zinc deficiency, appears to be an important contribution to tumor induction in this model.

Esophageal carcinogenesis in the rat: zinc deficiency, DNA methylation and alkyltransferase activity.

Rats fed zinc-deficient diets and given an esophageal carcinogen, methylbenzylnitrosamine, develop tumors in greater incidence and with increased frequency compared to zinc-supplemented rats. This greater susceptibility is associated with a unique esophageal lesion, parakeratosis, with markedly increased epithelial necrosis and cell proliferation. Recent studies have shown that the increased susceptibility to tumorigenesis was further associated with a number of metabolic and biochemical alterations including increased binding of the carcinogen to DNA, shifts in O6-methylguanine (O6MeG)/7-methylguanine ratios and suggestions that the promutagen O6MeG lesion is not repaired effectively in the zinc-deficient esophagus; the latter was not reflected in the amount of O6-methyltransferase activity, however. The weight of evidence supports a presumption that zinc deficiency interferes with normal DNA repair mechanisms, the nature of which is not clear. An interesting additional finding was that zinc deficiency alone was associated with esophageal tumor induction, without carcinogen, which indicates that genetic material in the zinc-deficient esophageal epithelium is damaged sufficiently, without further chemical injury, to result in loss of control of cell proliferation. Manipulation of the time of exposure to zinc deficiency and carcinogen exposure defined the initiation period as most affected by the deficiency. Furthermore, reduced carcinogen exposure (and less toxicity), along with zinc deficiency, permits development of more tumors of the endophytic type, the form more relevant to human esophageal tumors. The groundwork, as described in this paper, has now been prepared to directly address the latter issue, endophytic tumors, and the putative relation of zinc deficiency to esophageal cancer in human populations.

Diet: the neglected variable in chemical safety evaluations.

The objectives of this review are to alert the community of toxicologic pathologists to the types and significance of various diets fed to experimental animals used in safety evaluations of drugs and other chemicals, to acquaint investigators with dietary nutrients and contaminants of significance to research animals, to review some of the consequences of improper or inadequate diets on results of safety evaluations, and, finally, to offer a brief introduction to a highly promising new digital specimen technology for evaluating histopathology of tissues from animals used in safety evaluations with the aid of computers. Results that form the basis for this review have accumulated for more than 4 decades during research conducted during appointments at the University of Missouri, Columbia; Auburn University; Massachusetts Institute of Technology; and Boston University School of Medicine. The methods used in these studies have generally been state-of-the-art at the time the studies were conducted and involved an integration of techniques and methods used in fundamental nutrition, toxicology, and experimental pathology. Some of these investigations have shown that contaminants, naturally occurring and man-made, intentional and nonintentional, do occur in commercial animal diets; that essential nutrients, including total calories, protein and fat, can be provided in less or more than adequate amounts for the species of animal used in safety evaluations; and that such variables can influence responses to chemicals through effects on xenobiotic metabolism, immunocompetence, secondary diseases, and other factors. Under such conditions, serious differences in final interpretation of human risk for a drug or other chemical can contribute to failure to gain approval for a potentially valuable therapeutic agent. This review discusses the significance of various dietary components with examples of their effects on response of the animal to chemicals and demonstrates why nutrients, new technologies for assessing their effects, and other components in the diet should be accorded greater consideration by investigators and regulators.

Suppression of carcinogenesis in the intestines of min mice by the soybean-derived Bowman-Birk inhibitor.

We have performed experiments to determine whether the soybean-derived protease inhibitor, Bowman-Birk inhibitor (BBI), has the ability to affect intestinal carcinogenesis in Min mice. Min mice have an autosomally dominantly inherited predisposition to multiple intestinal neoplasms and are known to have a very high spontaneous rate of tumor development in both the small intestine and colon. BBI was administered in the diet as BBI Concentrate (BBIC), the form of BBI which is currently being evaluated in human trials as a cancer chemopreventive agent. We observed that 0.5% dietary BBIC led to a 42-50% reduction in the number of tumors/mouse in the small intestine and colon and a 41% reduction of tumorigenesis in the colon when the data are analyzed in terms of the fraction of mice bearing tumors. Thus, tumor-development in both the small intestine and colon of Min mice can be significantly suppressed by BBIC, despite the fact that the animals carry a predisposing mutation that leads to a markedly increased intestinal tumor incidence and mortality rate.

Zinc: health effects and research priorities for the 1990s.

This review critically summarizes the literature on the spectrum of health effects of zinc status, ranging from symptoms of zinc deficiency to excess exposure. Studies on zinc intake are reviewed in relation to optimum requirements as a function of age and sex. Current knowledge on the biochemical properties of zinc which are critical to the essential role of this metal in biological systems is summarized. Dietary and physiological factors influencing the bioavailability and utilization of zinc are considered with special attention to interactions with iron and copper status. The effects of zinc deficiency and toxicity are reviewed with respect to specific organs, immunological and reproductive function, and genotoxicity and carcinogenicity. Finally, key questions are identified where research is needed, such as the risks to human health of altered environmental distribution of zinc, assessment of zinc status in humans, effects of zinc status in relation to other essential metals on immune function, reproduction, neurological function, and the cardiovascular system, and mechanistic studies to further elucidate the biological effects of zinc at the molecular level.

Preparation and production of a cancer chemopreventive agent, Bowman-Birk inhibitor concentrate.

We describe our studies to produce an extract of soybeans with anticarcinogenic activity that we believe will be useful as a human cancer chemopreventive agent for several different organs. The anticarcinogenic activity of the extract is thought to be due to chymotrypsin inhibitor activity, which is due to the Bowman-Birk protease inhibitor (BBI) present in the extract, termed BBI concentrate (BBIC). We describe the contents of BBIC, the ability of BBIC to inhibit malignant transformation in vitro in terms of its chymotrypsin inhibitor activity, and the results of long-term toxicity studies in which mice and rats were exposed to high levels of BBIC for long periods of time.

Dietary lipids and immune function.

The influence of dietary lipids on immune function has come under serious study only within the past two decades. It is clear from whole-animal studies that obesity and consumption of diets high in fat, particularly unsaturated fat, depress immunocompetence and enhance risk for serious infectious disease and cancer. In vitro systems, cell cultures and the tools of molecular biology are moving nutrition and immunology closer together with promise of significant benefits.

Inhibition of N-nitrosomethylbenzylamine-induced esophageal neoplasms by the Bowman-Birk protease inhibitor.

Model systems in which carcinogenesis by given agents can be prevented or reduced offer a means of gaining insight into the mechanism(s) of action of carcinogens and the feasibility of chemoprevention in humans. In the current study, the ability of the soy-bean derived Bowman-Birk protease (BBI) to suppress esophageal carcinogenesis induced by N-nitrosomethylbenylamine (NMBzA) was examined. Esophageal lesions were produced in male Sprague-Dawley rats by i.p. injection of 2 mg/kg NMBzA twice weekly for 3 weeks. Groups receiving BBI were fed three tablets a week containing 180 mg BBI each in a mixture of Witepsol H15 and peanut butter for the duration of the experiment. The frequency of papillomas and carcinomas was reduced 45% in groups receiving BBI. Furthermore, the frequency of appearance of five separate characteristics of preneoplastic lesions was significantly reduced in the esophagi of BBI-treated animals. The most significant reduction was in the total number of lesions with simple hyperplasia. Groups receiving NMBzA and placebo tablets, containing only Witepsol H15 and peanut butter, did not display statistically significant differences in the frequency of esophageal lesions as compared to animals receiving NMBzA alone. These results demonstrate that BBI can effectively inhibit NMBzA-induced esophageal tumors when given in tablet form separate from the regular diet.

Protease inhibitor suppression of colon and anal gland carcinogenesis induced by dimethylhydrazine.

In the present study, we examined the ability of chymostatin, a highly specific inhibitor of chymotrypsin, to suppress dimethylhydrazine-induced colon carcinogenesis, and the dose-response relationship for an extract of soybeans containing the Bowman-Birk inhibitor (BBI) to suppress dimethylhydrazine-induced colon carcinogenesis, when added to the diet of mice. Our results showed that: (i) diets containing 0.1% BBI reduced the incidence of adenocarcinomas of the colon approximately 50%, but had no effect on the incidence of squamous cell carcinomas of the anal gland; (ii) the suppressive effect requires protease inhibitor activity, as the autoclaved BBI, in which all protease inhibitory activity has been destroyed, was ineffective at suppressing the incidence of adenocarcinomas; (iii) chymostatin suppressed the incidence of squamous cell carcinomas of the anal gland, but not adenocarcinomas of the colon; and (iv) the growth rates of the animals were the same in each of the experimental groups. Our results indicate that the levels of anticarcinogenic protease inhibitors present in the diets of these animals do not have any adverse effects on the growth or general health of the animals.

Renal mineralization--a ubiquitous lesion in chronic rat studies.

Renal mineralization is a commonly encountered lesion in old rats and its presence at times complicates the interpretation of data derived from chronic rat studies. The feeding of sucralose, a new and high-intensity sweetener under regulatory review, resulted in caecal enlargement and an increase in the incidences of renal mineralization and pelvic epithelial hyperplasia. These responses prompted a review of the literature focusing on the relationships, if any, between the caecal and renal changes. The literature supports the contention that caecal and renal changes occur frequently in response to feeding poorly absorbed osmotically active substances to rats. Some possible mechanisms that may be involved in the development of the renal lesion are discussed.

Roles of micronutrients in cancer prevention: recent evidence from the laboratory.

In addition to differences in needs for dietary quality and quantity, humans, as individuals and as subsets of the population, are exposed to variations in climate, stress, environmental contaminants and other confounding factors which likely impinge on susceptibility to cancer. Despite the complexity of lifestyles and dietary habits, it is impressive to review available data on the relation of nutrients to cancer. There is sufficient parallelism between controlled animal studies and human behavior that we are compelled to believe that a variety of essential nutrients can modify carcinogenesis in humans and in lower animals. The micronutrients which appear to meet criteria for classifying them as protective agents in animal models include vitamin A and some of the synthetic retinoids; beta carotene; folic acid; vitamin C; choline/methionine; zinc, and selenium. Some of the others have suggestive effects but in the view of this author, the data are often equivocal, inadequate, or conflicting. These observations clearly support the proposal that animal studies have made enormous contributions in the past 15-20 years to our understanding of carcinogenesis and that this will continue into the future. From the data now available we can state with confidence that animal studies have shown that nutrients can modify the carcinogenesis process at specific sites and through a variety of mechanisms. These include effects on the formation of carcinogens from precursors; effects on metabolism of the carcinogen; effects on one or more stages of initiation, promotion, and progression; host defense mechanisms; cellular differentiation and on growth and metastasis of the tumor. The tools of the molecular biology, just now emerging in the field of nutrition, should have an immense impact on determining more accurately where nutrients exert their effects, how this is accomplished, and to suggest appropriate prevention and intervention techniques. Using molecular biology, combined with traditional and newer methods of toxicology and pathology, we should be able within a few years to better understand carcinogenesis and with such knowledge in hand to make sound recommendations about dietary habits to the public.

90-day dermal toxicity study and neurotoxicity evaluation of nitromusks in the albino rat.

Musk ketone, musk xylene, musk tibetene and moskene, synthetic musks used in fragrances, were applied dermally to rats in daily doses of 240 (musk ketone and musk xylene only), 75, 24 or 7.5 mg/kg body weight for 90 days. The chemically related musk ambrette, a known neurotoxin in rats, was used as a positive control. While musk ambrette was clearly neurotoxic and caused testicular atrophy, as had been previously reported, the other compounds tested caused neither effect. The only effects of application of these materials were some organ weight changes at the higher doses, but these were not associated with histopathological changes in any of the tissues. The no-effect levels were: musk ketone, 75 mg/kg for males and females; musk xylene, 75 mg/kg for males and 24 mg/kg for females; moskene, 24 mg/kg for males and 75 mg/kg (highest dose administered) for females; and musk tibetene, 75 mg/kg (highest dose) for males and females.

The influence of dietary levels of vitamin A and fat on colon cancer.

Rats fed diets high (24%) or low (5%) in fat were given dietary levels of vitamin A (retinyl acetate) ranging from 0.3 to 30 micrograms/g food. The lowest tumor incidence was in the group fed diets high in vitamin A and low in fat. When the diet was high in fat and low in vitamin A, tumor incidence and frequency were significantly increased over that in rats fed the high-fat diet with normal levels of vitamin A (10 micrograms/g feed). However, even with a high level of fat in the diet, raising the level of vitamin A above 10 micrograms/g feed had no further beneficial effect. Thus, although there was a significant interaction between vitamin A and fat, it is the latter that appears to require the most attention, once the vitamin A intake is adequate. These data support the view that we should set as a goal an adequate, diversified diet that is low in fat but that an excessive intake of vitamins such as vitamin A that are toxic should be avoided.

The influence of pre- and postnatal caloric intake on colon carcinogenesis.

Mother rats were allowed to litter under conventional conditions. They were fed a complete, semipurified diet during gestation, and at time of littering the numbers of pups were reduced to either eight per litter or four per litter in two additional groups. At weaning, all rats were continued on the same diet that their mothers had consumed. One group of those reduced to four per litter at birth was allowed to continue to eat ad libitum while the other group, reduced to four per litter, was pair fed to the ad libitum eight per litter group. The group reduced to four per litter at birth and allowed to eat ad libitum during postnatal life gained the most weight and were heaviest at the termination of the study. This group also had the greater incidence and frequency of colon tumors when exposed to dimethylhydrazine (DMH). The group pair fed to rats fed conventional diets, eight rats per litter, had an incidence and frequency of tumors between the other two groups. These data demonstrate that early exposure to excess calories increased risk for cancer and that early and late excess caloric intake further increased risk. Thus, pre- and perinatal caloric intake may have a significant influence on susceptibility to cancer later in life. Mechanisms are only speculative but may include differences in metabolism and modulation of hormonal balance.

Anguidine-induced testicular injury in Lewis rats.

Anguidine (diacetoxyscirpenol, DAS) and other trichothecene mycotoxins are potent inhibitors of protein synthesis and injure organs with rapidly dividing cell populations, including the testis. Testicular structure and function were studied in male Lewis rats 1, 3, 7, 30, 60, and 90 days after exposure at age 12 weeks to anguidine at 1.7 mg/kg body weight given by ip injection. The dose was equivalent to 75% of the ip LD50. Anguidine caused a gradual decline in testicular weight beginning 30 days after treatment. Sperm production was also reduced by 30 days, and the frequency of hypocellular seminiferous tubules increased by day 60. There was no evidence of recovery by 90 days. These changes are consistent with injury to proliferating cells early in the maturation sequence. Epididymal sperm reserves were reduced by 3 days after anguidine administration, prior to the reduction in sperm production, suggesting premature release of spermatozoa from the epididymis.