Genotoxic effects in the Eastern mudminnow (Umbra pygmaea) after prolonged exposure to River Rhine water, as assessed by use of the in vivo SCE and Comet assays.

The production of drinking water from river water requires a certain minimal river water quality. The Association of River Rhine Water Works (RIWA), therefore, operates a monitoring network. In vitro mutagenicity studies have shown that the genotoxicity of the River Rhine water steadily decreased from 1981 until 2001. Compared to a study in 1978, a decrease in genotoxicity was also observed in an in vivo genotoxicity study in 2005, in which Eastern mudminnows (Umbra pygmaea) were exposed to River Rhine water, and gill cells were used for the Sister Chromatid Exchange (SCE) test and the Comet assay. In this 2005 study, the in vivo genotoxicity increased upon extending exposure of the fish from 3 to 11 days. Therefore, the objectives of this study were to investigate (i) whether new data corroborate that in vivo genotoxicity of River Rhine water is at present lower than in 1978, (ii) whether the Comet assay is a suitable alternative to the SCE assay, and (iii) whether further prolonged exposure results in a further increase in in vivo genotoxicity. The new data corroborate that in vivo genotoxicity of River Rhine water is at present lower than in 1978. The Comet assay is a useful addition but does not provide a substitute for the SCE endpoint in these in vivo genotoxicity studies. Prolonging the exposure time of Eastern mudminnows to River Rhine water from 11 to 42 days did not give a significant increase in SCEs and DNA damage (Comet assay) in gill cells.

Genotoxic effects in the Eastern mudminnow (Umbra pygmaea L.) after exposure to Rhine water, as assessed by use of the SCE and Comet assays: a comparison between 1978 and 2005.

Surface water used for drinking-water preparation requires continuous monitoring for the presence of toxic compounds. For monitoring of genotoxic compounds fish models have been developed, such as the Eastern mudminnow (Umbra pygmaea L.) because of its clearly visible 22 meta-centric chromosomes. It was demonstrated in the late seventies that Rhine water was able to induce chromosome aberrations and sister chromatid exchange in this fish species. Although in vitro mutagenicity studies of the RIWA (Rhine Water Works, The Netherlands) have shown that the genotoxicity of the river Rhine steadily decreased during the last decades, there is still concern about the presence of some residual mutagenicity. In addition, in most studies the water samples have been tested only in in vitro test systems such as the Salmonella-microsome test. For this reason, and in order to be able to make a comparison with the water quality 27 years ago, a study was performed with the same experimental design as before in order to measure the effect of Rhine water on the induction of SCE in the Eastern mudminnow. As a new test system the single cell gel electrophoresis assay (Comet assay) was performed. Fish were exposed to Rhine water or to groundwater for 3 and 11 days in flow-through aquaria. Fish exposed for 11 days to Rhine water had a significantly higher number of SCE and an increased comet tail-length compared with control fish exposed to groundwater. After exposure for three days to Rhine water there was no difference in SCE and a slightly increased comet tail-length compared with the control. It was concluded that genotoxins are still present in the river Rhine, but that the genotoxic potential has markedly decreased compared with 27 years ago. Furthermore, the Comet assay appears to be a sensitive assay to measure the genotoxic potential of surface waters in fish.

Effects of polyunsaturated fatty acids on prostaglandin synthesis and cyclooxygenase-mediated DNA adduct formation by heterocyclic aromatic amines in human adenocarcinoma colon cells.

Dietary heterocyclic aromatic amines (HCA) and polyunsaturated fatty acids (PUFA) are both believed to play a role in colon carcinogenesis, and are both substrate for the enzyme cyclooxygenase (COX). In HCA-7 cells, highly expressing isoform COX-2, we investigated the effects of PUFA on prostaglandin synthesis and DNA adduct formation by the HCA 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Furthermore, we studied the role of COX, COX-2 in particular, and cytochrome P4501A2 (CYP1A2) by using the enzyme inhibitors indomethacin (IM), NS-398, and phenethyl isothiocyanate (PEITC), respectively. COX-mediated formation of prostaglandin E2 (PGE2) from linoleic acid (LA) showed that HCA-7 cells can convert LA into arachidonic acid (AA). Alternatively, eicosapentaenoic acid (EPA) was found to compete with AA for COX. Strongly decreased PGE2 levels by addition of IM demonstrated involvement of COX in PUFA metabolism. Both IM and NS-398 inhibited adduct formation by HCA to nearly the same extent, indicating involvement of COX-2 rather than COX-1, while CYP1A2 activity in HCA-7 cells was demonstrated by addition of PEITC. Overall, inhibiting effects were stronger for PhIP than for IQ. HCA-DNA adduct formation was stimulated by addition of PUFA, although high PUFA concentrations partly reduced this stimulating effect. Finally, similar effects for n-3 and n-6 fatty acids suggested that adduct formation may not be the crucial mechanism behind the differential effects of PUFA on colon carcinogenesis that have been described. These results show that COX, and COX-2 in particular, can play a substantial role in HCA activation, especially in extrahepatic tissues like the colon. Furthermore, the obvious interactions between PUFA and HCA in COX-2 expressing cancer cells may be important in modulating colorectal cancer risk.

[Nutrition and health--toxic substances in food]. / Voeding en gezondheid--toxische stoffen in de voeding.

With respect to food, the most important factors causing adverse health effects are: an unbalanced diet, resulting in obesity or vitamin deficiencies, overconsumption of alcohol or fat, the presence of microbial contamination and the presence of natural toxins. Two additional factors, the presence of environmental contaminants and products formed on heating food, may also be of importance. It is generally assumed that, when combined, food-related factors contribute to around 35% of overall cancer incidence. The most important groups of health-threatening compounds to be found in the food chain include natural toxins, such as those produced by plants (phytotoxins), fungi (mycotoxins), marine algae (phycotoxins) and by bacteria, and toxins present in animals for human consumption, especially fish. A second important group of toxic compounds in food consists of environmental contaminants, including heavy metals and persistent organic pollutants, such as dioxins and polychlorinated biphenyls, all of which may unintentionally end up in the food chain. A third group of toxins present in food are those substances produced when food is heated, and include polycyclic aromatic hydrocarbons, heterocyclic amines and acrylamide.

Biochemical and morphological changes in lung tissue and isolated lung cells of rats induced by short-term nitrogen dioxide exposure.

To investigate the effects of repeated exposure to nitrogen dioxide (NO2) on antioxidant enzymes in lung tissue and isolated lung cells, rats were continuously exposed to 20 mg/m3 NO2 (10.6 ppm) for 4 days. The activities of glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase (GR), and glutathione peroxidase (GSHPx) were measured in the cytosolic fraction of lung tissue of both control and NO2-exposed rats as well as in isolated alveolar macrophages (AMs) and type II cells. Qualitative and quantitative changes in AM and type II cells were studied by electron microscopy and by morphometric analyses using enzyme and immunohistochemistry. NO2 exposure resulted in significantly increased pulmonary activities of G6PDH, GR, and GSHPx, both expressed per lung and per gram of lung weight. Morphometric data show that NO2 exposure significantly increased the number of type II cells, predominantly in the centriacinar region, indicating proliferation of epithelium following cellular injury. Type II cells in lungs of NO2-exposed rats had a squamous, less cuboidal appearance with more lamellar bodies compared to type II cells in lungs of control rats. Compared to control lungs, a higher number of macrophages could be isolated from NO2-exposed lungs, while numbers of type II cells isolated from lungs of control and NO2-exposed rats were the same. Isolated type II cells from control and NO2-exposed rats were polymorphic, with a small number of lamellar bodies and without polarity. Isolated macrophages were rounded and contained many filopodia. NO2 exposure caused increases in the activities of G6PDH and GSHPx in isolated type II cells and of GSHPx in isolated macrophages, when expressed per number of cells. Macrophages and type II cells isolated from control and NO2-exposed rats and re-exposed in vitro to NO2, showed no differences in phagocytosis and viability features. Our results indicate that NO2-induced increases in pulmonary antioxidant enzymes are also reflected in isolated AM and type II cells. Since these lung cells do not display a decreased sensitivities toward an in vitro NO2 exposure, overall increase in antioxidant enzyme activities do not seem to play the most pivotal role in controlling cellular NO2 sensitivity and oxidant defence. Combined data from biochemical, morphological, and morphometric analyses of lungs and lung cells suggest that lung cell and tissue oxidant sensitivity and defence largely depends on the cell and tissue organisation, i.e., cell numbers and morphology as well as the ratio of surface area to cytoplasmic volume.

Mutagenicity of heated sugar-casein systems: effect of the Maillard reaction.

The formation of mutagens after the heating of sugar-casein model systems at 120 degrees C was examined by the Ames test, using Salmonella typhimurium strain TA100. Several sugars (glucose, fructose, galactose, tagatose, lactose, and lactulose) were compared in their mutagenicities. Mutagenicity could be fully ascribed to Maillard reaction products and strongly varied with the kind of sugar. The differences in mutagenicity among the sugar-casein systems were caused by a difference in reaction rate and a difference in reaction mechanism. Sugars with a comparable reaction mechanism (glucose and galactose) showed a higher mutagenic activity corresponding with a higher Maillard reactivity. Disaccharides showed no mutagenic activity (lactose) or a lower mutagenic activity (lactulose) than their corresponding monosaccharides. Ketose sugars (fructose and tagatose) showed a remarkably higher mutagenicity compared with their aldose isomers (glucose and galactose), which was due to a difference in reaction mechanism.

Effect of airborne particles from selected indoor and outdoor environments on gap-junctional intercellular communication.

The effect of airborne particles from diesel exhaust, rubber and metal industry, urban air and biological sources (poultry, pig farming, compost industry) on gap-junctional intercellular communication (GJIC) were compared, using HEPA1c1c7 cells. Particles as such were compared with aqueous and organic extracts. Significant inhibition of GJIC by particle suspensions was only observed for the diesel and rubber samples, and for one biological sample (compost). Up to 83% of the inhibition of the whole suspension could be attributed to the particles as such. Washing the particles with organic solvents (aceton, methanol, hexane) did not result in a significant loss of activity from the particles, although the organic fractions showed a significant activity towards GJIC. More active organics was eluted from the rubber industry particles than from the diesel particles by the organic solvent. It is suggested that cancer promoting potential as measured by inhibition of GJIC may vary widely depending on the particle source, and that this effect may be exerted by the particles as such and/or by means of tightly bound bio-active material to the surface.

Effects of dietary fat and a vegetable-fruit mixture on the development of intestinal neoplasia in the ApcMin mouse.

The variation in colorectal cancer (CRC) incidence worldwide strongly suggests a role for dietary influences. Based on epidemiological data, protective effects of vegetables and fruit intake on CRC are widely claimed, while other data indicate a possible increased CRC risk from (higher) dietary fat intake. Therefore, we have investigated single and interactive effects of dietary fat and a vegetable-fruit mixture (VFM) in the ApcMin mouse, a mouse model for multiple intestinal neoplasia. In this study, four different diets (A-D) were compared, which were either low in fat (20% energy diets A/B) or high in fat (40% energy diets C/D). In addition, 19.5% (wt/wt) of the carbohydrates in diets B and D were replaced by a freeze-dried VFM. The diets were balanced so that they only differed among each other in fat/carbohydrate content and the presence of specific plant-constituents. Because the initiation of intestinal tumors in ApcMin mice occurs relatively early in life, exposure to the diets was started in utero. Without the addition of VFM, mice maintained at a high-fat diet did not develop significantly higher numbers of small or large intestinal adenomas than mice maintained at a low-fat diet. VFM added to a low-fat diet significantly lowered multiplicity of small intestinal polyps (from 16.2 to 10.2/mouse, 15 animals/group), but not of colon tumors in male ApcMin mice only. Strikingly, addition of VFM to female mice maintained on a low-fat diet and to both sexes maintained on a high-fat diet significantly enhanced intestinal polyp multiplicity (from 16.5 to 26.7 polyps/mouse). In conclusion, our results indicate that neither a lower fat intake nor consumption of VFM included in a high-fat diet decreases the development of polyps in mice genetically predisposed to intestinal tumor development.

Effects of a vegetables-fruit mixture on liver and colonic 1,2-dimethylhydrazine-metabolizing enzyme activities in rats fed low- or high-fat diets.

The effects of a vegetables-fruit mixture (19.5% w/w) were studied on hepatic (h) and colonic (c) 1,2-dimethylhydrazine (DMH)-metabolizing enzyme activities (ethoxy- and pentoxyresorufine-O-deethylation, NDMA-demethylase, glutathione-S-transferase, UDP-glucuronyltransferase) in rats fed low- or high-fat diets (20 or 40 energy%). A vegetables-fruit mixture added to the diets resulted in altered enzyme activities in animals either treated or not treated with DMH. Remarkably, the vegetables-fruit mixture given to DMH-treated rats decreased glutathione-S-transferase (h) and increased NDMA-demethylase activities (c), whereas the mixture given to controls increased glutathione-S-transferase (h) and decreased NDMA-demethylase activities (c). The high-fat diets only modulated enzyme activities in animals not treated with DMH.

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.

Protection of UV-induced suppression of skin contact hypersensitivity: a common feature of flavonoids after oral administration?

In this study we investigated the effect of the dietary ingredients fruit and vegetable, green tea phenol extract (GTP) and the specific flavonoid components quercetin and chrysin on the UV-induced suppression of the contact hypersensitivity (CHS) response to picryl chloride (PCl). The SKH-1 mice were fed with test diet from 2 or 4 weeks before and during the UV irradiation (daily, 95 mJ/cm2) and tested for the CHS ear-swelling response 10 weeks after the onset of the irradiation. For the CHS, mice were immunized with PCl by epicutaneous application on nonirradiated sites. Four days after sensitization all mice were challenged on both sides of each ear by topical application of one drop PCl. In addition, from mice fed with the fruit and vegetable mixture the number of Langerhans cells (LC) were scored in the skin and from mice fed with quercetin, quercetin levels in plasma were measured at week 11 after the start of UV irradiation. It was found that fruit and vegetable (19% in the diet), GTP (0.1% and 0.01% in the drinking water), quercetin (1% in the diet) and chrysin (1% and 0.1% in the diet), prevented statistically significantly the UV-induced suppression of CHS to PCl. In the skin of mice fed with fruit and vegetables combined with UV irradiation the number of LC were comparable to the control mice, whereas the number of LC were significantly diminished in mice treated with UV only. This protective effect on the presence of LC in the epidermis after UV irradiation, which was also observed in a previous study with quercetin, may play a role in the prevention of UV-induced immunosuppression by the flavonoids tested. In conclusion, we found protection of flavonoids against UV-induced effects on CHS, which may be a common feature of most flavonoids.

Food plant toxicants and safety Risk assessment and regulation of inherent toxicants in plant foods.

The ADI as a tool for risk management and regulation of food additives and pesticide residues is not readily applicable to inherent food plant toxicants: The margin between actual intake and potentially toxic levels is often small; application of the default uncertainty factors used to derive ADI values, particularly when extrapolating from animal data, would prohibit the utilisation of the food, which may have an overall beneficial health effect. Levels of inherent toxicants are difficult to control; their complete removal is not always wanted, due to their function for the plant or for human health. The health impact of the inherent toxicant is often modified by factors in the food, e.g. the bioavailability from the matrix and interaction with other inherent constituents. Risk-benefit analysis should be made for different consumption scenarios, without the use of uncertainty factors. Crucial in this approach is analysis of the toxicity of the whole foodstuff. The relationship between the whole foodstuff and the pure toxicant is expressed in the `product correction factor' (PCF). Investigations in humans are essential so that biomarkers of exposure and for effect can be used to analyse the difference between animals and humans and between the food and the pure toxicant. A grid of the variables characterising toxicity is proposed, showing their inter-relationships. A flow diagram for risk estimate is provided, using both toxicological and epidemiological studies.

Effects of vegetables-fruit extracts and indole-3-carbinol on stearic acid-modulated intercellular communication and cytochrome P450-IA activity.

Modulatory effects were investigated of extracts of a vegetables-fruit mixture and indole-3-carbinol (I3C) on stearic acid-modulated gap junctional intercellular communication (GJIC) and cytochrome P450-IA activity (EROD). In V79 cells, pure water and hexane extracts of a vegetables-fruit mixture and 25 µg/ml I3C significantly protected against decreased GJIC caused by 10 µM stearic acid. Furthermore, pure, 10× and 100× diluted vegetables-fruit extracts significantly maintained their capacity to induce EROD activity in Caco-2 cells, but only when these extracts were added to the cells in media already containing 500 µM stearic acid for 48 h. Stearic acid itself did not induce EROD activity. I3C (10, 25, and 50 µg/ml) clearly induced EROD activity in Caco-2 cells, irrespective of the order at which I3C and stearic acid were added to the cells. In conclusion, the present in vitro study showed that vegetables-fruit extracts and I3C modulate effects of stearic acid on intercellular communication and cytochrome P450-IA activity.

Aflatoxin and liver cancer in Sudan.

This study investigated whether aflatoxin contamination of peanut products may contribute to the incidence of hepatocellular carcinoma (HCC) in Sudan. Thirty-seven peanut butter and peanut samples were collected from local markets. Aflatoxin concentrations were significantly higher in West Sudan [87.4 +/- 197.3 (SD) micrograms/kg], a high-risk area, than in Central Sudan (8.5 +/- 6.8 micrograms/kg), a low-risk area. In West Sudan, humid local storage conditions of peanut products were related to high aflatoxin concentrations. In a small case-control study of HCC patients (n = 24) and controls (n = 34), an odds ratio of 7.5 (95% confidence interval = 1.4-40.2) was observed for humid vs. dry local storage conditions. Development of an index of individual HCC exposure was less successful, probably because of year-to-year variability in aflatoxins in food. These preliminary findings justify further research into the role of aflatoxins and hepatitis in HCC incidence in Sudan.

Antimutagenic activity of casein against MNNG in the E. coli DNA repair host-mediated assay.

The effect of caseinate and soy protein in the diet on the mutagenicity induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was assessed in-vivo and ex-vivo in the DNA-repair host-mediated assay and liquid suspension assay, respectively. Of the two proteins only casein showed a strong antimutagenic activity over the whole digestive tract, except in the stomach. It is suggested that the molecular structure of a protein determines its protective effect against mutagens: casein lacks secondary and tertiary structure so that amino acids are more readily available for interaction with the mutagen than with the amino acids in soy protein which is a globular protein.

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.

Interaction of dietary fat with a vegetables-fruit mixture on 1,2-dimethylhydrazine-induced colorectal cancer in rats.

The aim of the present investigation was to study the interaction of dietary fat in combination with a vegetables-fruit mixture on 1,2-dimethylhydrazine (DMH)-induced colorectal carcinogenesis in rats. For this purpose, 120 weanling male. Wistar rats received a semisynthetic diet without (Groups A and C) or with a vegetables-fruit mixture (Groups B and D; vegetables and fruit content 19.5% wt/wt) for 35 weeks. Diets of Groups A and B contained 20 energy percent (20e%) fat, whereas diets of Groups C and D contained 40e% fat. The vegetables and fruit used the amount of fat, and its fatty acid composition were chosen according to the mean consumption values of The Netherlands. After the animals were maintained for four weeks on the respective diets, they were given 10 weekly injections of DMH at 50 mg/kg body wt sc. After sacrifice, their colons were removed and examined macroscopically and microscopically for the presence of tumors. Rats fed high-fat diets developed significantly more tumors than rats fed low-fat diets. Furthermore, although not statistically significant, a lower number of colorectal tumors was observed in rats fed a low- or a high-fat diet containing the vegetables-fruit mixture than in rats fed diets without the vegetables-fruit mixture. No differences were observed in intestinal tumor incidences among all groups. The results suggest that the vegetables-fruit mixture used in this experiment, present in an amount comparable with the mean consumption in The Netherlands, has no significant inhibitory effect on the development of colorectal tumors induced by DMH in rats maintained on diets low or high in fat.

Modulation of dietary fat-enhanced colorectal carcinogenesis in N-methyl-N'-nitro-N-nitrosoguanidine-treated rats by a vegetables-fruit mixture.

The modulation of a vegetables-fruit mixture on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced colorectal carcinogenesis was studied in rats maintained on a low- or a high-fat diet. For this purpose, 120 rats received a semisynthetic diet without (Groups A and C) or with a vegetables-fruit mixture (19.5% wt/wt, Groups B and D) for 35 weeks. Diets of Group A and B contained 20 (low) energy percent (20e%) fat, whereas diets of Groups C and D contained 40e% (high) fat. Between Weeks 4 and 9 the animals were given weekly intrarectal instillations of 6 mg MNNG/kg body wt. The colorectal adenocarcinoma incidences showed a significant decrease in animals fed high-fat diets with a vegetables-fruit mixture compared with animals fed a high-fat diet alone. Furthermore, without a vegetables-fruit mixture, diets high in fat caused a significant increase in adenocarcinoma incidence compared with diets low in fat. Although not significant, the adenoma incidences tended to be lower in animals fed a vegetables-fruit mixture than in animals maintained on a diet without this mixture. The results demonstrate that a vegetables-fruit mixture has a significant inhibitory potency on the development of colorectal tumors induced by MNNG in rats fed diets high in fat.

Carcinogenicity study of outdoor airborne particulate matter in newborn male NMRI mice.

An organic extract of airborne particulate matter (APM) was tested for carcinogenicity at two dose levels in the newborn mouse bioassay. The samples used were taken under specific polluted conditions. The doses tested corresponded with 0.75 and 1.5 times the amount of air man inhales during lifetime. Benzo(a)pyrene, which was used as a positive control, significantly increased the lung tumor incidence. No evidence was found for a carcinogenic activity of the organic extract of APM. Considering the high dose of APM applied in this animal model and the much lower actual cumulative dose to which man is exposed to in many areas, the conclusion can be drawn that exposure to APM alone probably does not represent an important cancer risk for man.