The effect of butylated hydroxytoluene on selected immune surveillance parameters in rats bearing enzyme-altered hepatic preneoplastic lesions.

Selected immune function parameters were examined in male Fischer 344 rats following (a) induction of enzyme-altered preneoplastic liver foci (EAF), and (b) growth modulation of EAF by 30-day feeding with the food antioxidant butylated hydroxytoluene (BHT). Glutathione S-transferase-P (GSTP)-positive EAF were observed in livers of rats receiving diethylnitrosamine (DEN), 2-acetylaminofluorene (2-AAF) and partial hepatectomy (PH) (Solt-Farber procedure), with or without BHT treatment. The induction of EAF and/or 0.5% BHT treatment resulted in a significant reduction in the natural killer (NK) cell activity of splenocytes. PH did not affect NK activity significantly compared with control (no PH) rats. The concanavalin A-induced lymphoproliferative activity of splenocytes was increased in rats with PH compared with those without. A lag in time needed to attain maximum calcium release was observed only in the rats with PH compared with those without PH. None of the treatments affected the phagocytic activity of resident peritoneal macrophages. Only EAF-bearing rats without BHT treatment had increased granulocyte and monocyte levels, while the leucocyte and lymphocyte levels were reduced by the initiator DEN. but not by BHT treatment. Further investigations are necessary to determine whether the observed suppression of NK cell activity during EAF induction and growth modulation by BHT is a contributing factor in enhancement of rodent liver neoplasia by this non-genotoxic food antioxidant.

Carcinogen risk assessment: a necessary dilemma?

Carcinogen risk assessment is the process by which an attempt is made to estimate human risk due to carcinogens, from the results of animal studies. It is based upon a number of prudent default assumptions, that is, assumptions that cannot be proved scientifically because either the basic concept is philosophical in nature or because the amount of scientific evidence required is too costly to obtain even on a world-wide basis. Recently, scientific effort has shown that more and more examples have been described suggesting these examples do not behave in the way indicated by the default assumptions. Since carcinogen risk assessment processes were initiated, it has been demonstrated that cancer may arise by four or more different mechanisms. It is the purpose of this paper to enquire whether consideration of these basically different mechanisms may facilitate carcinogen risk assessment.

Cancer risk assessment at the crossroads: the need to turn to a biological approach.

Mathematically based carcinogen risk assessment is based on a number of prudent default assumptions which are becoming progressively less tenable as new scientific evidence is adduced. For example, the assumptions that all rodent carcinogens will be carcinogenic in humans and that there is no safe dose of any carcinogen may, in specific examples, be shown to be untrue. The mechanisms by which carcinogens exert their effects, especially the induction of DNA lesions, DNA repair of these lesions, and cell proliferation, are considered; it is suggested that with recently developed experimental techniques they might be employed to develop a more biologically based approach to risk assessment and might avoid at least, some of the pitfalls associated with the present mathematically based carcinogen risk assessment models. They might lead to an improved appreciation of the shape of the carcinogen dose-response curve, at least at medium to high exposure levels.

The effect of different levels of dietary alpha-linolenic and other fatty acids on mammary gland ductular cell proliferation in female Swiss Webster mice.

In previous work we have shown that changing the fatty acid composition of a constant amount of fat in a modified AIN-76A diet affected the level of ductular cell proliferation in the mammary glands of young virgin female Swiss Webster mice. In particular, linoleic acid concentrations of 5-10% of the total fat in the diet led to variable but appreciably higher levels of proliferation than did higher levels of linoleic acid. In this report it is shown that feeding low levels of the total fat as alpha-linolenic acid (0-5%) resulted in a similar effect. In addition the effects of other fats including menhaden oil, were further investigated.

The effect of butylated hydroxytoluene on the growth of enzyme-altered foci in male Fischer 344 rat liver tissue.

Butylated hydroxytoluene (BHT) is a synthetic, food-use, phenolic antioxidant. It has previously been demonstrated to be operationally non-genotoxic and, in addition, failed to induce biologically significant increases in cellular proliferation in the liver, urinary bladder and thyroid gland on feeding to young adult Wistar rats. Nevertheless, it has been reported to enhance the yield of liver tumors when fed to rats or mice that developed an appreciable background incidence of these tumors without treatment. In order to resolve this situation, cell proliferation in response to BHT treatment was studied in enzyme-altered foci (EAF) induced in male Fischer 344 rats using the Solt-Farber procedure. It was demonstrated that feeding 0.5% dietary BHT for 30 days after the induction of EAF led to a 20- to 30-fold increase in the gamma-glutamyltranspeptidase-positive areas in both DEN- and saline-initiated rat livers, but to no major effects in glutathione S-transferase placental form (GSTP)-positive foci. Cell proliferation rates within EAF and surrounding normal liver were measured using different histological techniques. Nuclear labeling with [3H]thymidine and proliferating cell nuclear antigen (PCNA) over the total hepatocyte population indicated that BHT approximately doubled nuclear labeling in rats initiated with DEN. PCNA labeling in GSTP-positive foci was not affected by BHT. In GSTP-positive foci, evaluation of nucleolar organizer regions (AgNOR), which reflect cell proliferative in addition to transcriptional activity of ribosomal RNA, was achieved using a novel double staining technique. BHT diet did not affect the number of AgNOR per nucleus or the percentage AgNOR area/nucleus. Nevertheless, both PCNA labeling and the AgNOR area per nucleus were significantly greater in GSTP-positive foci compared with non-focal regions in rats fed either BHT or control diets. These results are discussed in the light of further experimental work required to determine the relevance of these data to possible human risk assessment for BHT.

Mechanistic and other considerations in cancer prevention.

Possibilities for the prevention of cancer, particularly in relation to the food supply, are considered. It is suggested that the growing realization that cancer may be induced by more than one mechanism combined with a present lack of knowledge of the nature and level of naturally-occurring carcinogens in food crops, makes successful prevention in humans exceedingly difficult.

Effect of addition of dried healthy or diseased parsnip root tissue to a modified AIN-76A diet on cell proliferation and histopathology in the liver, oesophagus and forestomach of male Swiss Webster mice.

Umbelliferous crop plants, including the parsnip (Pastinaca sativa L.), elaborate enhanced levels of furocoumarins, including psoralens, when subjected to biotic or abiotic stress. These furocoumarins are recognized to lead to phototoxicity. In this study, the effect of these agents, which are present in diseased parsnip root tissue, on the liver and two tissues on the route of entry to the body (the oesophagus and forestomach) were investigated. Young male Swiss Webster mice were fed for approximately 30 days with modified AIN-76A diets containing 32.5% dried healthy, 32.5% apparently healthy or 32.5% fungicide-treated parsnip root tissue, and 8, 16 or 32.5% dried diseased (Phoma complanata-infected) parsnip root tissue. As controls, three modified AIN-76A diets differing in their edible starch-to-sucrose ratios (C1-C3) were administered for an equal time. Dried healthy parsnip root tissue, compared with controls, did not significantly affect any of the indices of cellular proliferation or histopathological parameters that were assessed. Histopathological examination of the oesophagus and forestomach demonstrated no significant changes as a result of feeding any of the diets containing parsnip tissue. In the liver, the highest level (but neither of the two lower levels) of dried diseased parsnip root tissue led to swelling of the cytoplasm in cells surrounding the central vein of hepatic lobules, with consequent compression of the peripheral cells. Using [3H]thymidine radioautography, a dose-related increase in cell labelling with the level of diseased parsnip root tissue was demonstrated in the liver. Compared with control diet C2 only, the extent of [3H]thymidine labelling in the liver was increased in mice receiving apparently healthy parsnip tissue; a slight, not statistically significant, increase was also noted with fungicide-treated parsnip tissue. Increased [3H]thymidine labelling with the feeding of diseased parsnip tissue was also found in the greater curvature of the forestomach and the region of the oesophageal-forestomach junction, but not at the glandular junction of the forestomach nor in the mid-oesophagus.

International Commission for Protection Against Environmental Mutagens and Carcinogens. Oxidative DNA damage--the effects of certain genotoxic and operationally non-genotoxic carcinogens.

A wide variety of oxidative DNA lesions are commonly present in untreated human and animal DNA. One of these lesions, 8-hydroxydeoxyguanosine, has been shown to lead to base mispairing (mutation) on DNA replication. Other lesions remain to be investigated in this respect. Oxidative DNA lesions on cell replication may, in appropriate circumstances, lead to proto-oncogene activation. Oxidative DNA damage, on fixation, may also lead to cytotoxicity followed by regenerative proliferation. The probable or possible importance of oxidative DNA damage is reviewed for various classes of carcinogens and natural processes, including metal ions, high-energy radiation, miscellaneous chemicals, tumor-promoting agents, polyhydroxyphenols/quinones, lipid metabolism, peroxisome proliferators and thyroid function. It is concluded that although the evidence needs considerable strengthening in many of these examples, the available information indicates the potential importance of oxidative DNA damage in the induction of tumors by these agents. It is also possible that non-cancerous degenerative diseases associated with aging are the result of the accumulation of lesions resulting from unrepaired oxidative DNA damage.

Food safety: are human activities really worse than nature's?

Those chemicals that are added to food during modern processing are most stringently examined for toxic effects and, if they demonstrate toxicity, are strictly controlled. Despite this, a considerable proportion of the North American human cancer burden has been associated with diet and nutrition. The possible contributions of excess calories, excess and wrongly balanced fats, natural contaminants, and naturally occurring carcinogens within the food supply to this horrendous burden of cancer is considered. The theoretically possible use of bioengineering techniques to modify the composition of food crops and thus to minimize the levels of carcinogens in the food supply is discussed. This is considered important since failure to monitor the effect of bioengineering may lead to an increase in the level of such noxious agents, especially if the goal of such bioengineering is to develop food crops with increased intrinsic resistance to pests and other spoilage organisms.

Effect of varying the type of fat in a semi-purified AIN-76A diet on cellular proliferation in the mammary gland and intestinal crypts in female Swiss Webster mice.

Young virgin female Swiss Webster mice were fed AIN-76A semi-purified diets containing equal weights of different fats for approximately 30 days. Using [3H]thymidine radioautography, it was established that mice fed 100% lard or high levels of fish oils (menhaden oil or cod liver oil) developed elevated cellular proliferation in the duct cells of the mammary gland and an increased number of labeled cells/crypt in the crypts of the colo-rectum accompanied by an increase in the size of the proliferative compartment. A possible inverse correlation between the level of [3H]thymidine labeling in the mammary gland, but not in the colo-rectum, and the linoleic acid content of individual diets may help to explain the significance of these observations. The effect of adding an antioxidant mixture to these diets was to reduce the excess proliferation induced in the intestinal crypts by lard or fish oil to the level induced by soybean oil, but only partially so in the duct cells of the mammary gland.

Calories, fat, fibers, and cellular proliferation in Swiss Webster mice.

Increased cellular proliferation has been associated with the enhanced expression of several key stages in carcinogenesis. A standard protocol was used to investigate the effect of specific dietary regimens on cellular proliferation. Young adult Swiss Webster mice were fed for 30 days with modified AIN-76A semi-purified diets designed to illustrate the effects of the levels of dietary or calorie restriction, different fibers and bulking agents, and different fats on cellular proliferation. Female mice were used for the restriction and fat studies, males for the fiber and bulking agent studies. Vaginal smears were taken from females from treatment day 15, and the mice killed 2 days following the first estrus following 30 days feeding; males were killed on the 30th day. One hour before death, mice were injected ip with 0.25 micro Ci/g 3[H]-thymidine. Slides were prepared for radioautography and histopathology. Both dietary and calorie restriction led to reduced 3[H]-thymidine labeling indices in each of the seven tissues studied, the mammary gland being the most severely affected. Different fibers and bulking agents, in specific cases, reduced labeling in the duodenum but not to a consistent statistically significant extent in the colon or colo-rectal region. In the duodenum, oat bran and oat gum were the most effective while wood cellulose (alphacel) had no effect. Investigations on the effects of different fats is continuing. High levels of lard, menhaden oil, or cod liver oil as the fat component of the AIN-76A diet, led to much higher levels of labeled cells in the mammary gland or colo-rectal region than did fat components rich in vegetable oils. The labeling indices appeared to be inversely correlated with the level of linoleic acid in the diet, a presumption that has been confirmed by investigating a series of diets containing different levels of this acid. Anti-oxidants were not used in any of these fat-modified diets. The overall results obtained in these studies clearly indicate the utility of cellular proliferation studies in investigating the effects of dietary modifications.

Classification of carcinogens: polemics, pedantics, or progress?

Rodent carcinogens may, for physiological or other reasons, induce cancer by a variety of mechanisms which vary in their ability to affect humans. While the current approach of some regulatory agencies to carcinogen risk assessment and regulation may possibly be justified with most genotoxic carcinogens, this is not true with all nongenotoxic carcinogens. Mechanisms attributable to high dose toxicity occasioned by misuse of the maximum tolerated dose concept, imbalancing of homeostasis, unphysiological conditions, and induced cellular proliferation are reviewed. The greatest present need for meaningful regulation of carcinogens is to obtain public acceptance of the fact that some carcinogens are species specific and probably will not exert their effects in humans.

Early indicators of potential neoplasia produced in the rat forestomach by non-genotoxic agents: the importance of induced cellular proliferation.

Forestomach neoplasia induced by the apparently non-genotoxic carcinogens, butylated hydroxyanisole and propionic acid, appears to arise by way of sustained high levels of cellular proliferation. Several other inducers of enhanced cellular proliferation, or the consequential incidence of hyperplastic lesions, have been identified in the rodent forestomach but the requisite carcinogenicity bioassays remain undone. In other tissues, such as the male rat kidney, the rodent thyroid follicular cell and the bladder epithelium, there is also evidence supporting the concept that sustained enhanced cellular proliferation may be an important early marker for non-genotoxic carcinogens. This reaction is, however, not likely to be the only marker necessary for the identification of non-genotoxic carcinogens.

An appreciation of the maximum tolerated dose: an inadequately precise decision point in designing a carcinogenesis bioassay?

Cancers arise in specific tissues. One difficulty with the present definitions of the Maximum Tolerated Dose (MTD), as they pertain to the rodent cancer bioassay, is that they base MTD on relatively crude parameters associated with the well-being of the entire animal rather than with the lack of specific tissue toxicity. Additional factors that could be included in the MTD definition, or could be separately determined, are addressed. Many of these factors refer to toxic behavior in one or a few tissues and, if used in setting the MTD, may mask more relevant events occurring at higher dose levels in other tissues. Reducing the MTD to a level that fails to take into account pesticide or drug-related toxicity may lead to the loss of relevant information in the bioassay. It is concluded, therefore, that there are two possible approaches to a more appropriate use of the MTD. The highest dose of the test agent (MTD) may be chosen (i) to lie below the thresholds of carcinogenicity-related non-genotoxic toxicity or (ii) the present high level MTD may continue to be used and tumors that arise may be classified as being irrelvant to humans at some or all exposure levels. The latter approach is to be preferred. It has the potential to avoid missing high level effects of the test agent that may be relevant to the human population.

International Commission for Protection Against Environmental Mutagens and Carcinogens. ICPEMC publication No. 19. The classification of carcinogens identified in the rodent bioassay as potential risks to humans: what type of substance should be tested next?

The relevance of rodent cancer bioassay data to humans is discussed in relation to the needs of regulatory agencies. The usefulness of in vivo and in vitro genotoxicity testing in this connection is also discussed. In the case of rodent carcinogens that do not elicit genotoxicity, it is suggested that homeostatic imbalance, cell proliferation, and other processes may play a major role in tumor development and its importance to the possible ability of the test agent to induce human cancer. These possibilities need to be evaluated on a case by case basis. The methods by which chemicals are selected for the rodent cancer bioassay are also discussed and it is pointed out that naturally-occurring constituents of human foods should in future receive greater priority as a consequence of anticipated changes resulting from biotechnology.