Expression profiling of colon cancer cell lines and colon biopsies: towards a screening system for potential cancer-preventive compounds.

Interest in mechanisms of colon cancer prevention by food compounds is strong and research in this area is often performed with cultured colon cancer cells. In order to assess utility for screening of potential cancer-preventive (food) compounds, expression profiles of 14 human cell lines derived from colonic tissue were measured using cDNA microarrays with 4000 genes and compared with expression profiles in biopsies of human colon tumours and normal tissue. Differences and similarities in the gene expression profiles of the cell lines were analysed by clustering and principal component analysis (PCA). Cytoskeleton genes and immune response genes are two functional classes of genes that contributed to the differences between the cell lines. A subset of 72 colon cancer-specific genes was identified by comparing expression profiles in human colon biopsies of tumour tissue and normal tissue. A separation of the cell lines based on the tumour stage of the original adenocarcinoma was observed after PCA of expression data of the subset of colon cancer-specific genes in the cell lines. The results of this study may be useful in the ongoing research into mechanisms of cancer prevention by dietary components.

Safety evaluation of chemical mixtures and combinations of chemical and non-chemical stressors.

Recent developments in hazard identification and risk assessment of chemical mixtures are reviewed. Empirical, descriptive approaches to study and characterize the toxicity of mixtures have dominated during the past two decades, but an increasing number of mechanistic approaches have made their entry into mixture toxicology. A series of empirical studies with simple chemical mixtures in rats is described in some detail because of the important lessons from this work. The development of regulatory guidelines for the toxicological evaluation of chemical mixtures is discussed briefly. Current issues in mixture toxicology include the adverse health effects of ambient air pollution; the application of such modern, sophisticated methodologies as genomics, bioinformatics, and physiologically based pharmacokinetic modeling; and databases for mixture toxicity. Finally, the state of the art of our knowledge on the potential adverse health effects of combined exposures to chemicals and non-chemical stressors (noise, heat/cold, microorganisms, immobilization, restraint, or transportation), research initiatives in these fields, and the development of an indicator for the cumulative health impact of multiple environmental exposures are discussed.

Do aberrant crypt foci have predictive value for the occurrence of colorectal tumours? Potential of gene expression profiling in tumours.

The effects of different dietary compounds on the formation of aberrant crypt foci (ACF) and colorectal tumours and on the expression of a selection of genes were studied in rats. Azoxymethane-treated male F344 rats were fed either a control diet or a diet containing 10% wheat bran (WB), 0.2% curcumin (CUR), 4% rutin (RUT) or 0.04% benzyl isothiocyanate (BIT) for 8 months. ACF were counted after 7, 15 and 26 weeks. Tumours were scored after 26 weeks and 8 months. We found that the WB and CUR diets inhibited the development of colorectal tumours. In contrast, the RUT and BIT diets rather enhanced (although not statistically significantly) colorectal carcinogenesis. In addition, the various compounds caused different effects on the development of ACF. In most cases the number or size of ACF was not predictive for the ultimate tumour yield. The expression of some tumour-related genes was significantly different in tumours from the control group as compared to tumours from the treated groups. It was concluded that WB and CUR, as opposed to RUT and BIT, protects against colorectal cancer and that ACF are unsuitable as biomarker for colorectal cancer. Effects of the different dietary compounds on metalloproteinase 1 (TIMP-1) expression correlated well with the effects of the dietary compounds on the ultimate tumour yield.

Effects of dietary fat on virus-induced pancreatic carcinogenesis in guinea fowl.

The present study was performed to assess the effects of diets supplemented with low (5%) and high (20%) corn oil on a Pts 56 retrovirus-induced model of pancreatic carcinogenesis in guinea fowl. The early microscopic lesions appear after 3 mo after virus treatment and progress over time. Eight to 10 mo after initiation, up to 100% of virus-inoculated birds develop multiple hyperplastic and neoplastic pancreatic lesions of duct/ductular phenotype. Short-term (1-4 mo) feeding of low- or high-fat diets, beginning at Month 3, had no significant effects on body and pancreatic weight. However, the incidence, multiplicity, and areas of the pancreatic tissue occupied by intra- and interlobular aggregates of hyperplastic ducts with mucinous metaplasia of the lining cells were significantly increased compared with the birds fed the common diet. At the same time, development of ductular neoplasms, particularly carcinomas, was retarded compared with the common diet-fed controls. Long-term (5-7 mo) fat intake resulted in an increase in body weight gain, while absolute pancreatic weights remained relatively constant. Furthermore, the high- and low-fat diets caused a significant increase in areas of retrovirus-induced pancreatic lesions, as well as an increase in multiplicity of ductular neoplasms compared with short-term fat feeding. It is concluded that short-term feeding of diets supplemented with 5% or 20% corn oil delayed the development of the common virus-induced ductular neoplasms, particularly carcinomas, and had an enhancing effect on development of hyperplastic inter- and intralobular aggregates of ducts. This finding was not observed, however, during the long-term feeding period of the study.

An analysis of human response to the irritancy of acetone vapors.

Studies on the irritative effects of acetone vapor in humans and experimental animals have revealed large differences in the lowest acetone concentration found to be irritative to the respiratory tract and eyes. This has brought on much confusion in the process of setting occupational exposure limits for acetone. A literature survey was carried out focusing on the differences in results between studies using subjective (neuro)behavioral methods (questionnaires) and studies using objective measurements to detect odor and irritation thresholds. A critical review of published studies revealed that the odor detection threshold of acetone ranges from about 20 to about 400 ppm. Loss of sensitivity due to adaptation and/or habituation to acetone odor may occur, as was shown in studies comparing workers previously exposed to acetone with previously unexposed subjects. It further appeared that the sensory irritation threshold of acetone lies between 10,000 and 40,000 ppm. Thus, the threshold for sensory irritation is much higher than the odor detection limit, a conclusion that is supported by observations in anosmics, showing a ten times higher irritation threshold level than the odor threshold found in normosmics. The two-times higher sensory irritation threshold observed in acetone-exposed workers compared with previously nonexposed controls can apart from adaptation be ascribed to habituation. An evaluation of studies on subjectively reported irritation at acetone concentrations < 1000 ppm shows that perception of odor intensity, information bias, and exposure history (i.e., habituation) are confounding factors in the reporting of irritation thresholds and health symptoms. In conclusion, subjective measures alone are inappropriate for establishing sensory irritation effects and sensory irritation threshold levels of odorants such as acetone. Clearly, the sensory irritation threshold of acetone should be based on objective measurements.

Health risks associated with inhaled nasal toxicants.

Health risks of inhaled nasal toxicants were reviewed with emphasis on chemically induced nasal lesions in humans, sensory irritation, olfactory and trigeminal nerve toxicity, nasal immunopathology and carcinogenesis, nasal responses to chemical mixtures, in vitro models, and nasal dosimetry- and metabolism-based extrapolation of nasal data in animals to humans. Conspicuous findings in humans are the effects of outdoor air pollution on the nasal mucosa, and tobacco smoking as a risk factor for sinonasal squamous cell carcinoma. Objective methods in humans to discriminate between sensory irritation and olfactory stimulation and between adaptation and habituation have been introduced successfully, providing more relevant information than sensory irritation studies in animals. Against the background of chemoperception as a dominant window of the brain on the outside world, nasal neurotoxicology is rapidly developing, focusing on olfactory and trigeminal nerve toxicity. Better insight in the processes underlying neurogenic inflammation may increase our knowledge of the causes of the various chemical sensitivity syndromes. Nasal immunotoxicology is extremely complex, which is mainly due to the pivotal role of nasal lymphoid tissue in the defense of the middle ear, eye, and oral cavity against antigenic substances, and the important function of the nasal passages in brain drainage in rats. The crucial role of tissue damage and reactive epithelial hyperproliferation in nasal carcinogenesis has become overwhelmingly clear as demonstrated by the recently developed biologically based model for predicting formaldehyde nasal cancer risk in humans. The evidence of carcinogenicity of inhaled complex mixtures in experimental animals is very limited, while there is ample evidence that occupational exposure to mixtures such as wood, leather, or textile dust or chromium- and nickel-containing materials is associated with increased risk of nasal cancer. It is remarkable that these mixtures are aerosols, suggesting that their "particulate nature" may be a major factor in their potential to induce nasal cancer. Studies in rats have been conducted with defined mixtures of nasal irritants such as aldehydes, using a model for competitive agonism to predict the outcome of such mixed exposures. When exposure levels in a mixture of nasal cytotoxicants were equal to or below the "No-Observed-Adverse-Effect-Levels" (NOAELs) of the individual chemicals, neither additivity nor potentiation was found, indicating that the NOAEL of the "most risky chemical" in the mixture would also be the NOAEL of the mixture. In vitro models are increasingly being used to study mechanisms of nasal toxicity. However, considering the complexity of the nasal cavity and the many factors that contribute to nasal toxicity, it is unlikely that in vitro experiments ever will be substitutes for in vivo inhalation studies. It is widely recognized that a strategic approach should be available for the interpretation of nasal effects in experimental animals with regard to potential human health risk. Mapping of nasal lesions combined with airflow-driven dosimetry and knowledge about local metabolism is a solid basis for extrapolation of animal data to humans. However, more research is needed to better understand factors that determine the susceptibility of human and animal tissues to nasal toxicants, in particular nasal carcinogens.

The benchmark approach applied to a 28-day toxicity study with Rhodorsil Silane in rats. the impact of increasing the number of dose groups.

The OECD study design, aimed at obtaining a no-observed-adverse-effect level (NOAEL), may be suboptimal for deriving a benchmark dose. Therefore the present subacute (28-day) study was carried out to evaluate a multiple dose study design and to compare the results with the common OECD design. Seven groups of 10 female rats each were intragastrically administered corn oil without (controls) or with 50, 150, 300, 450, 600 or 750 mg Rhodorsil Silane/kg body weight/day, once daily (7 days/week) for 4 weeks. From the complete dataset, two subsets were selected, one representing a study design with seven dose groups of five animals (7 x 5 design), the other representing a study design with four dose groups of 10 animals (4 x 10 design). Under the conditions of the present study, the NOAEL for Rhodorsil Silane 198 was assessed at 50 mg/kg body weight/day, based on the data of the 4 x 10 design. The benchmark approach resulted in a benchmark dose of 19 mg/kg body weight/day, based on the data of the 7 x 5 design. Comparison of the results demonstrated that the multiple dose (7 x 5) design led to a more reliable result than the OECD (4 x 10) design, despite the smaller total number of animals. The dose-response analysis showed that at "the NOAEL" the effect on relative spleen weight was larger than 10%, illustrating that at the NOAEL, adverse effects may occur.

Disposition, accumulation and toxicity of iron fed as iron (II) sulfate or as sodium iron EDTA in rats.

A study was performed to provide data on the disposition, accumulation and toxicity of sodium iron EDTA in comparison with iron (II) sulfate in rats on administration via the diet for 31 and 61 days. Clinical signs, body weights, food consumption, food conversion efficiency, hematology, clinical chemistry and pathology of selected organs were used as criteria for disclosing possible harmful effects. Determination of iron and total iron binding capacity in blood plasma and non-heme iron analysis in liver, spleen and kidneys were used to assess the disposition and accumulation of iron originating from sodium iron EDTA or iron (II) sulfate. It was concluded that, under the conditions of the present study, iron is accumulated from the diet in liver, spleen and kidneys in a dose-dependent manner, and iron derived from FeEDTA is taken up and/or accumulated less efficiently in liver and spleen than iron from FeSO(4). Moreover, feeding iron up to 11.5 and 11.2 mg/kg body weight/day, derived from FeSO(4) and FeEDTA, respectively, did not result in tissue iron excess nor in any other toxicologically significant effects.

Effect of dietary galacto-oligosaccharides on azoxymethane-induced aberrant crypt foci and colorectal cancer in Fischer 344 rats.

The aim of the present study was to investigate the effects of galacto-oligosaccharides (GOS, Elix'or) on the development of aberrant crypt foci (ACF) and colorectal tumours in rats treated with azoxymethane (AOM). Two groups of 102 male Fischer 344 rats were injected twice with AOM to induce colorectal tumours, and fed diets containing either a low [5% (w/w); LGOS] or a high [20% (w/w); HGOS] concentration of GOS. Four weeks after the last AOM injection, 18 animals from each group were killed and their colon was removed for scoring ACF. Half of the animals in the LGOS group were switched to an HGOS diet (L/HGOS) and half of those in the HGOS group to an LGOS diet (H/LGOS). Six weeks after the change in diet, nine animals per group were killed for scoring ACF. Ten months after the start of the study the remaining animals were killed for scoring colorectal tumours. The aberrant crypt multiplicity scored after 13 weeks and the colorectal tumour incidence in rats fed an HGOS diet were significantly lower than those in rats fed an LGOS diet. However, the induction of ACF by AOM, the proliferation rate and apoptotic index of the adenomas, and the size and multiplicity of colorectal tumours were not influenced by the amount of GOS in the diet. The aberrant crypt multiplicity, scored after 13 weeks, was predictive for the tumour outcome at the end of the study. It was concluded that an HGOS diet has a protective effect against the development of colorectal tumours in rats and that this protective effect is exerted during the promotion phase rather than the initiation phase of carcinogenesis.

Modulation of pancreatic carcinogenesis by antioxidants.

Previously performed short-term (4-month) studies demonstrated that vitamins C and E, beta-carotene and selenium modulate growth of early putative preneoplastic acinar lesions induced in rat pancreas by azaserine. The present paper summarizes the results of long-term studies performed with azaserine-treated rats maintained on diets high in either beta-carotene, vitamins C and E or selenium. It appeared that rats given a diet high in beta-carotene, vitamin C or selenium, but not vitamin E, developed fewer pancreatic tumours than controls. The chemopreventive effects of these micronutrients were most pronounced when beta-carotene and/or selenium were given during the promotion phase of the carcinogenic process. Surprisingly, cell proliferation in azaserine-induced preneoplastic acinar lesions was higher in rats given beta-carotene and/or selenium via the diet in comparison to controls. It is considered unlikely that any antioxidant alone can be associated with protection against cancer. It is concluded that dietary supplementation of combinations of antioxidants may have practical application in chemoprevention of cancer.

Dietary fat and carcinogenesis.

Epidemiologic investigations have suggested a relationship between dietary fat intake and various types of cancer incidences. Furthermore, epidemiologic studies as well as studies with animal models have demonstrated that not only the amount but also the type of fat consumed is important. At present, the mechanism by which dietary fat modulates carcinogenesis has not been elucidated. The effects of dietary fat on the development of tumours have been summarized in the present review with emphasis on colorectal, pancreas, breast and prostate cancer. It is concluded that influence on synthesis of prostaglandins and leukotrienes may be the universal mechanism by which dietary fats modulate carcinogenesis.

A comparison of the effects of dietary cellulose and fermentable galacto-oligosaccharide, in a rat model of colorectal carcinogenesis: fermentable fibre confers greater protection than non-fermentable fibre in both high and low fat backgrounds.

The objective of this experiment was to compare the effects of diets with either a non-fermentable fibre source (cellulose) or a fermentable fibre source [galacto-oligosaccharide (GOS)], combined with different levels of dietary fat, on the development of colorectal cancer. Male Wistar rats were fed AIN76-based diets with either a low or high level of cellulose, or a low or high level of GOS, for 9 months. The fat content of the diets was low, medium or high. All rats were treated with 1,2-dimethylhydrazine to induce colorectal tumours. Generally, the tumour incidence increased with increasing fat content in the diet. Despite marked faeces bulking, dietary cellulose either had no effect or an enhancing effect on the formation of colorectal tumours in general, although the development of carcinomas was decreased. GOS appeared to be highly protective against the development of colorectal tumours, as was demonstrated by an inhibitory effect on tumour incidence, multiplicity and size, regardless of the fat content of the diet. Neither fibre source influenced the bromodeoxyuridine labelling index determined in colon crypts or tumours. In animals fed high-GOS diets, the caecal content was significantly increased in weight and significantly decreased in pH. It was concluded that tumorigenesis was enhanced by increased fat content of the diet, and that the diets containing fermentable GOS conferred a greater protection against colorectal cancer than did the diets containing non-fermentable cellulose.

Effects of long term feeding of raw soya bean flour on virus-induced pancreatic carcinogenesis in guinea fowl.

The effects of a diet enriched with 25% raw soya bean flour (RSF) on the pancreas and on the avian retrovirus Pts 56-induced pancreatic carcinogenesis in guinea fowl were studied. It has been shown that prolonged RSF feeding of new-hatched virus-infected and uninfected guinea fowl-poults induced enlargement of the pancreas, which was less pronounced when administration of the RSF supplemented diet started at the age of 75 days. Time-dependent multifocal inter- and intralobular hyperplasia of pleomorphic ducts lined by mucin-producing epithelium in the exocrine pancreas of virus-infected guinea fowls fed a RSF supplemented diet was regularly observed. Enlargement of virus-induced ductular neoplasms has been shown only after simultaneous RSF and virus administration.

Safety evaluation of synthetic beta-carotene.

The safety of beta-carotene was reassessed by evaluating the relevant literature on the beneficial and adverse effects of beta-carotene on cancer and, in particular, by evaluating the results of toxicity studies. Beta-carotene appeared neither genotoxic nor reprotoxic or teratogenic, and no signs of organ toxicity have been found in subacute, subchronic, or chronic oral toxicity studies in experimental animals receiving doses of up to 1000 mg/day beta-carotene per kg body weight via the diet. Synthetic beta-carotene did not exert any carcinogenic effect in Sprague-Dawley rats or in CD1 mice. An enhanced risk of lung cancer was found in two human intervention studies. Although dose and (timing of) exposure, smoking status, and imbalance of antioxidant defense have been recognized as potential factors accounting for the outcome of these studies, a conclusive explanation has not yet been found. It is concluded that exposure to beta-carotene resulting in mean plasma concentrations of no more than 2.2 micromol/l (1.2 microg/ml) is safe to the general population. By contrast, in heavy smokers higher plasma concentrations may be associated with a higher lung cancer risk.

Toxicologic profile of acrylonitrile.

Acrylonitrile is a monomer used extensively as a raw material in the manufacturing of acrylic fibers, plastics, synthetic rubbers, and acrylamide. It has been classified as a probable human carcinogen according to the results of numerous chronic rat bioassays. The present report summarizes the toxicity data on acrylonitrile and reviews available data concerning the mechanism (genetic versus epigenetic) by which acrylonitrile is carcinogenic in rats. From the evaluation of the relevant toxicity data, it can be concluded that acrylonitrile is indeed carcinogenic to rats after either oral or inhalational exposure. However, information on other mammalian species is lacking, and, moreover, the exact mechanism of the carcinogenic process is unclear. Therefore, it is recommended to conduct an additional long-term inhalation carcinogenicity study with acrylonitrile in mice, as well as studies into the mechanism by which acrylonitrile induces (brain) tumors in rats (genetic versus epigenetic).

Absence of an inhibitory effect of a vegetables-fruit mixture on the initiation and promotion phases of azoxymethane-induced colorectal carcinogenesis in rats fed low- or high-fat diets.

The potential inhibitory effects of a vegetables-fruit mixture on the initiation and promotion phases of azoxymethane-induced colorectal carcinogenesis were examined in rats fed low- or high-fat diets. Rats were fed low-fat diets (20 energy percent, Diets A and B) or high-fat diets (40 energy percent, Diets C and D), supplemented with a vegetables-fruit mixture (19.5% wt/wt, Diets B and D) or unsupplemented (Diets A and C) for 36 weeks. After the animals were maintained on the respective diets for four weeks, they were given three weekly injections of azoxymethane at 15 mg/kg body wt sc. Eight weeks after the start of the study, animals maintained on Diet A were switched to Diet B or C or maintained on the same diet. Animals maintained on Diet B or D were switched to Diet A or C, respectively. Furthermore, animals maintained on Diet C were switched to Diet A or D or maintained on the same diet. Multiplicity of colorectal tumors did not differ between groups fed a vegetables-fruit mixture during the initiation or the promotion phase (Group B-->A vs. Group A-->B; Group D-->C vs. Group C-->D). However, multiplicity was significantly lower in animals fed low-fat diets than in animals fed high-fat diets in combination with a vegetables-fruit mixture (Group A-->B/B--A vs. Group C-->D/D-->C). Furthermore, multiplicity was significantly increased in groups fed a high-fat diet during the promotion phase only in comparison with animals fed a low-fat diet during the whole experiment (Group A-->C vs. Group A-->A). No other differences in multiplicity or tumor incidences were observed among the eight experimental groups.

Carcinogenic response of the nasal cavity to inhaled chemical mixtures.

Nasal cancers occur in experimental animals following chronic exposure to a wide range of inhaled chemicals. Although exposure to several of these chemicals is common in industrial as well as domestic environments, epidemiological studies have not provided convincing evidence that exposure to these chemicals individually is associated with nasal cancer in humans. The reverse seems to be true for inhalation of chemical mixtures. The evidence for nasal carcinogenicity of inhaled chemical mixtures in experimental animals is very limited, while there is ample evidence in humans that occupational exposure to certain chemical mixtures is associated with increased risk of nasal cancer. Examples of such (complex) chemical mixtures are wood dust, textile dust, chromium- and nickel-containing materials and leather dust. It is remarkable that these mixtures are aerosols, suggesting that a 'dusty working environment' may increase nasal cancer risk.

Pancreatic cancer in rats and hamsters does not induce IAPP-related hyperglycaemia.

Many patients with exocrine pancreatic cancer develop diabetes mellitus due to insulin resistance. This may relate to concurrent over-production of islet amyloid polypeptide (IAPP) by the pancreatic beta cells. We investigated the effects of pancreatic cancer on circulating IAPP and glucose homeostasis in azaserine-treated rats (developing acinar pancreatic tumours) and BOP-treated hamsters (developing ductular pancreatic tumours). Glucose, insulin and IAPP levels in plasma were neither affected in azaserine-only treated rats nor in animals with enhanced carcinogenesis after chronic caerulein treatment. Azaserine-treated rats on a high-fat diet had decreased insulin levels and enhanced IAPP/insulin ratios in plasma, without hyperglycaemia. All BOP-treated hamsters showed pancreatic carcinogenesis at 6 months post-treatment. Supranormal plasma glucose levels in animals on a low-fat diet were the only change observed. After a second 6-month period, subnormal plasma glucose levels, at least 4-fold decreased plasma insulin and up to 2-fold decreased plasma IAPP levels were present in all hamsters. Remarkably, both in azaserine-treated rats on high-fat and in BOP-treated hamsters, decreased insulin levels and elevated IAPP/insulin ratios are not associated with hyperglycaemia. In contrast to humans with pancreatic cancer, IAPP over-production and hyperglycaemia do not develop in rats and hamsters with (pre-)neoplastic pancreatic lesions.

Carcinogenicity testing of complete human diets in rats.

So far, in most animal experimental studies isolated food components have been tested. However, as components may interact with each other at different mechanistic levels, testing complex food mixtures more representative for human consumption patterns may better predict the ultimate carcinogenic risk. Studies were performed in Wistar rats using human and rat control diets to assess the effect of relevant food factors such as heat processing and the presence of non-nutrients in vegetables and fruit. The complete human diets, containing meat, bread and eggs, with or without vegetables and fruit, were composed according to mean consumption figures, balanced for macro- and micronutrients. Experiments were performed with spontaneous as well as with chemical-induced tumor models. Heat processing had no effect on tumor induction, while vegetables and fruit only exerted a protective effect on chemically induced tumors in rats fed low-fat animal diets. Data suggest interaction between major food factors in the human diet on colon carcinogenesis.