Effect of dietary supplementation on the frequency of spontaneous lacZ mutations in the developing colon

Epidemiological studies have demonstrated that dietary modifications can reduce the incidence of cancer. Specifically, diets high in vegetables and fruits are associated with lower rates of cancer at many sites. Somatic mutations have a critical role in carcinogenesis suggesting the use of in vivo mutation assays as an alternative approach to studying the relationship between diet and cancer. Since the rate of accumulation of spontaneous mutations is highest during growth and development early in life, we tested whether certain foods as dietary supplements could reduce the rate of mutation during this period using lacZ transgenic mice. Pregnant female mice were placed on a control diet or a diet supplemented to 20% final dry weight with broccoli, cabbage, carrots, flaxseed, green peas, green peppers, oranges or strawberries for the entire duration of their pregnancy and lactation. Mutation frequencies were subsequently measured at the lacZ transgene in colonic epithelial cells of the offspring at 3 weeks of age. A small number of measurements were also made on siblings at 8 weeks of age. While the control AIN-96G diet on its own resulted in lower mutant frequencies than had been observed in earlier experiments with lab chow, no significant reduction in mutant frequencies was detected for any of the foods tested as compared to the AIN-93G diet alone. Significantly more mutations were found at 3 weeks of age in mice fed diets supplemented with broccoli or oranges, but the result with oranges may be the result of jackpot mutations.     

Role of diet modification in cancer prevention

Carcinogenesis encompasses a prolonged accumulation of injuries at several different biological levels and include both genetic and biochemical changes in the cells. At each of these levels, there are several possibilities of intervention in order to prevent, slow down or even halt the gradual march of healthy cells towards malignancy. Diet modification is one such possibility. A number of natural foodstuffs, especially fruits and vegetables contain substantial quantities of molecules that have chemopreventive potential against cancer development. Such compounds include vitamins, trace elements and a variety of other molecules with antioxidant properties. Carotenoids, flavanoid polyphenols, isoflavones, catechins, and several other components that found in cruciferous vegetables are molecules that are known to protect against the deleterious effect of reactive oxygen species. A number of epidemiological and experimental studies have shown that vitamin C and E, Beta-carotene and the essential trace element selenium can reduce the risk of cancer. Consistent observations during the last few decades that cancer risk is reduced by a diet rich in vegetables, fruits, legumes, grains and green tea have encouraged research to identify several plant components especially phytochemicals that protect against DNA damage. Many of these substances block specific carcinogen pathways. Dietary supplements are part of an overall health program, along with a high intake of fruits and vegetables that help to combat damage to cells, which in turn may initiate cancer development. This paper will review current knowledge concerning diet modification and cancer prevention with special reference to minerals and trace elements.     

Dietary modulation of DNA damage in human

Manipulation of human diet can modulate urinary biomarkers of oxidative DNA base damage (UBODBD), reflecting changes in levels of DNA damage. When dietary composition is maintained but caloric intake is decreased (caloric restriction), UBODBD excretion is suppressed. At isocaloric dietary intake the level of damage depends on diet composition. For diets consisting of foods containing carbohydrates, proteins, and fats but lacking fruits and vegetables, the level of damage is higher than for diets including fruits and vegetables, which are rich in natural antioxidants. Assay of urinary biomarkers is suggested as a potential test for quantitative assessment of the carcinogenic or anticarcinogenic properties of foods, food components, and diets and for individual responses to nutritional regimens.     

Ascorbic acid reduces the frequency of iron induced micronuclei in bone marrow cells of mice

Iron is a potent oxidant that can lead to the formation of genotoxic lipid peroxides. Ascorbic acid, which enhances dietary iron absorption, has been suggested to enhance the oxidant effects of iron and to directly lead to the formation of lipid peroxides. The combined effects of dietary iron and ascorbic acid on genotoxicity were investigated by measuring the frequency of micronuclei in the bone marrow cells of C3H/He mice. In addition, liver iron concentration was measured in all treated groups. Three weeks old mice were fed diets for 3 weeks containing iron at 100 or 300 mg/kg diet in the form of FeSO₄ that were supplemented either with or without ascorbic acid (15 g/kg diet). The results of the bone marrow micronucleus test revealed that the high iron diet resulted in an increased frequency of micronucleated polychromatic erythrocytes (MnPCEs) as compared to low iron. Ascorbic acid supplementation in the low iron diet did not show any effect on incidence of MnPCEs and protected against the increased frequency of MnPCEs induced by the high iron diet. However, liver iron concentration was significantly increased only in the high iron treated and ascorbic acid supplemented group as compared to all other groups. These results demonstrate that ascorbic acid protects against the clastogenic effects of iron.     

Ascorbic acid reduces the frequency of iron induced micronuclei in bone marrow cells of mice

Iron is a potent oxidant that can lead to the formation of genotoxic lipid peroxides. Ascorbic acid, which enhances dietary iron absorption, has been suggested to enhance the oxidant effects of iron and to directly lead to the formation of lipid peroxides. The combined effects of dietary iron and ascorbic acid on genotoxicity were investigated by measuring the frequency of micronuclei in the bone marrow cells of C3H/He mice. In addition, liver iron concentration was measured in all treated groups. Three weeks old mice were fed diets for 3 weeks containing iron at 100 or 300 mg/kg diet in the form of FeSO(4) that were supplemented either with or without ascorbic acid (15 g/kg diet). The results of the bone marrow micronucleus test revealed that the high iron diet resulted in an increased frequency of micronucleated polychromatic erythrocytes (MnPCEs) as compared to low iron. Ascorbic acid supplementation in the low iron diet did not show any effect on incidence of MnPCEs and protected against the increased frequency of MnPCEs induced by the high iron diet. However, liver iron concentration was significantly increased only in the high iron treated and ascorbic acid supplemented group as compared to all other groups. These results demonstrate that ascorbic acid protects against the clastogenic effects of iron.     

Comparison of the effect of raw and blanched‐frozen broccoli on DNA damage in colonocytes

Consumption of cruciferous vegetables may protect against colorectal cancer. Cruciferous vegetables are rich in a number of bioactive constituents including polyphenols, vitamins and glucosinolates. Before consumption, cruciferous vegetables often undergo some form of processing that reduces their content of bioactive constituents and may determine whether they exert protective effects. The aim of this study was to compare the ability of raw and blanched‐frozen broccoli to protect colonocytes against DNA damage, improve antioxidant status and induce xenobiotic metabolizing enzymes (XME). Fifteen Landrace × Large White male pigs were divided into five age‐matched and weight‐matched sets (79 days, SD 3, and 34·7 kg, SD 3·9, respectively). Each set consisted of siblings to minimize genetic variation. Within each set, pigs received a cereal‐based diet, unsupplemented (control) or supplemented with 600 g day^?1 of raw or blanched‐frozen broccoli for 12 days. The consumption of raw broccoli caused a significant 27% increase in DNA damage in colonocytes (p = 0·03) relative to the control diet, whereas blanched‐frozen broccoli had no significant effect. Both broccoli diets had no significant effect on plasma antioxidant status or hepatic and colonic XME. This study is the first to report that the consumption of raw broccoli can damage DNA in porcine colonocytes. Copyright © 2015 John Wiley & Sons, Ltd.     

Insulin resistance, low-fat diets, and low-carbohydrate diets: time to test new menus

PURPOSE OF REVIEW: Insulin resistance increases the risk of cardiovascular disease and diabetes, and the risk of cardiovascular disease increases further once diabetes has developed. As insulin resistance is a precursor to diabetes, it is critically important to identify cost-effective means, such as dietary changes, by which to reduce insulin resistance. The purpose of this review is to evaluate recent findings concerning dietary composition and insulin resistance, with particular focus on low-fat diets compared with the currently popular low-carbohydrate diets. RECENT FINDINGS: Recent findings indicate little support for the value of low-carbohydrate diets as therapies for insulin resistance. In contrast, the limited data available suggest that the higher fat content of typical low-carbohydrate diets may exacerbate insulin resistance in the long term. Preliminary data indicate that proteins from different sources may have differing effects on insulin resistance. Preliminary data also suggest the potential value of whole grains, fruits and vegetables in therapeutic diets to reduce insulin resistance. SUMMARY: Current evidence supports the inclusion of whole grains, fruits and vegetables, and lean sources of animal proteins including low-fat dairy products in dietary therapies for insulin resistance. Those who wish to follow a low-carbohydrate diet should be encouraged to follow a new menu low in fat, and with most of the protein derived from plant sources.     

Damage to lymphocytes by X-ray and bleomycin measured with the cytokinesis-block micronucleus technique.

Chromosome damage induced by X-irradiation or bleomycin was measured using the cytokinesis-block micronucleus assay in the peripheral blood lymphocytes of 6 newborn, 8 young and 10 elderly individuals. An increase in the frequency of spontaneous micronuclei with age was observed. There was no difference in the X-irradiation-induced micronucleus frequency between the 3 groups. There was a significant increase with age in the number of micronuclei induced by bleomycin. Kinetochore-labelling studies revealed that the percentage of kinetochore-positive induced micronuclei was higher for bleomycin (36.2-43.3%) than for X-irradiation (17.1-19.7%). The age-related increase in frequency of spontaneous or bleomycin-induced micronuclei was due to increases in both kinetochore-positive and kinetochore-negative micronuclei. The frequency of kinetochore-positive or -negative micronuclei induced by X-irradiation was not different between the 3 age groups. These results suggest that bleomycin is more potent in inducing whole-chromosome loss than X-rays, and that lymphocytes from aged individuals are more sensitive to bleomycin in terms of both chromosome breakage and whole chromosome loss.     

Biomarkers of DNA damage in marine mammals.

Certain environmental contaminants found in marine mammals have been shown to cause DNA damage and cancer. The micronuclei (MN), sister chromatid exchange (SCE) and/or chromosome aberration (CA) assays were used to assess baseline (spontaneous) levels of DNA damage in blood lymphocytes of individuals of the relatively healthy and lightly contaminated Arctic beluga whale (Delphinapterus leucas), Sarasota Bay, FL, bottlenose dolphin (Tursiops truncatus) and Northwestern Atlantic grey (Halichoerus grypus) and harp (Phoca groenlandicus) seal populations. MN cell (MNC) frequencies ranged between 2 and 14/1000 binucleated (BN) cells and were statistically similar between species. In bottlenose dolphins, MNC frequency was correlated with age and was significantly higher in females than in males. No intraspecific variation in MNC frequency was found in beluga whales. Intraspecific variation was not tested in seals due to the small sample size. Frequencies of SCEs and total CAs, excluding gaps, ranged, respectively, between 1 and 15 SCE(s)/per cell and 4-6 CAs/100 cells in beluga whales. SCE and CA frequencies did not vary with age or sex in beluga whales. The MN, SCE and CA assays were found to be practical tools for the detection of DNA damage in marine mammals and could be used in the future to compare DNA damage between relatively lightly and highly contaminated populations.     

Anticancer effects of a plant lignan 7-hydroxymatairesinol on a prostate cancer model in vivo

Clinical intervention studies and experimental studies with lignan-rich diets suggest that lignans may have inhibitory effects on prostate cancer, but no clinical or experimental studies with purified lignans have been published. The purpose of this study was to investigate the effect of a plant lignan 7-hydroxymatairesinol (HMR) on LNCaP human prostate cancer xenografts in athymic mice. Athymic nude male mice were injected subcutaneously with LNCaP cells. Starting 3 days after tumor cell injections, a control diet or a control diet supplemented with 0.15% or 0.30% of HMR was administered to mice and the tumor take rate and growth was observed for 9 weeks. HMR diet inhibited the growth of LNCaP tumors. Mice treated with HMR had smaller tumor volume, lower tumor take rate, increased proportion of nongrowing tumors, and higher tumor cell apoptotic index compared with controls. Furthermore, the cell proliferation index was reduced in mice receiving the 0.30% HMR diet compared with mice receiving the control diet. Our results suggest that dietary HMR started at the early phase of the tumor development inhibits the growth of the LNCaP human prostate cancer xenografts in athymic male mice.     

Single-strand break repair and genetic disease

Hereditary defects in the repair of DNA damage are implicated in a variety of diseases, many of which are typified by neurological dysfunction and/or increased genetic instability and cancer. Of the different types of DNA damage that arise in cells, single-strand breaks (SSBs) are the most common, arising at a frequency of tens of thousands per cell per day from direct attack by intracellular metabolites and from spontaneous DNA decay. Here, the molecular mechanisms and organization of the DNA-repair pathways that remove SSBs are reviewed and the connection between defects in these pathways and hereditary neurodegenerative disease are discussed.     

Micronucleus induction in bone-marrow cells following consumption of cooked beef in mice. Preliminary investigations.

The bone-marrow micronucleus assay was used to investigate whether the consumption of cooked meat could induce chromosome damage. There was no difference in the micronucleus frequency of mice on normal diet (cereal-based, non-purified diet) and mice on normal diet supplemented with microwaved meat. However, supplementing the normal diet with well-done pan-fried meat or rare charcoal-barbecued meat or well-done charcoal-barbecued meat produced significant increments in the micronucleus frequency of polychromatic erythrocytes--the increments were of the order of 73% (p = 0.045), 90% (p = 0.047) and 136% (p = 0.001) respectively and they were observed after a 21-day feeding trial. These results suggest that ingestion of well-done pan-fried or barbecued meat may increase genetic damage, however, the accompanying decreased intake of vegetable constituents may have also contributed to the observed changes.     

Evolutionary aspects of omega-3 fatty acids in the food supply

Information from archaeological findings and studies from modern day hunter-gatherers suggest that the Paleolithic diet is the diet we evolved on and for which our genetic profile was programmed. The Paleolithic diet is characterized by lower fat and lower saturated fat intake than Western diets; a balanced intake of omega-6 and omega-3 essential fatty acids; small amounts of trans fatty acids, contributing less than 2% of dietary energy; more green leafy vegetables and fruits providing higher levels of vitamin E and vitamin C and other antioxidants than today's diet and higher amounts of calcium and potassium but lower sodium intake. Studies on the traditional Greek diet (diet of Crete) indicate an omega-6/omega-3 ratio of about 1/1. The importance of a balanced ratio of omega-6:omega-3, a lower saturated fatty acid and lower total fat intake (30-33%), along with higher intakes of fruits and vegetables leading to increases in vitamin E and C, was tested in the Lyon Heart study. The Lyon study, based on a modified diet of Crete, confirmed the importance of omega-3 fatty acids from marine and terrestrial sources, and vitamin E and vitamin C, in the secondary prevention of coronary heart disease, and cancer mortality.     

Mutagens in human urine: effects of cigarette smoking and diet

Human urine from smokers and nonsmokers on strictly controlled diets was assayed for mutagenic activity. Two distinct diets were employed in this study. Diet study A consisted of a high-meat, high-fat diet, observed for 5 days, followed by a vegan diet, adhered to for the next 5 days. The vegan diet contained no meat, fish, eggs, or dairy products. It was comprised of soy products, prepackaged vegan dinners, seeds, nuts, fruits, vegetables, beans and herbal teas. Diet study B consisted of 3 days on a typical western diet followed by a macrobiotic diet of grains and fresh vegetables for 5 days. Portions of 24-h urine samples were assayed in Salmonella typhimurium TA1538. The levels of urinary creatinine and cotinine were measured. Mutagenic activity was observed in the urine of most smokers. However, the levels of mutagens in the urine of light smokers were similar to those of nonsmokers. For both nonsmokers and smokers there was a significant increase in urine mutagenicity when volunteers were on the vegan diet. Several nonsmokers on the vegan diet in diet study A had pronounced mutagenic activity in their urine samples, in some instances at higher levels than that in the urine of smokers on a meat diet. In diet study B no clear differences were observed between the meat diet and the macrobiotic diet. In diet studies A and B the mutagenic potency of smokers' urine could not be correlated with cotinine levels alone or with urinary pH. These data suggest that dietary factors can play a dominant role in the mutagenicity of urine concentrates.     

Relative effectiveness of HZE iron-56 particles for the induction of cytogenetic damage in vivo

One of the risks of prolonged manned space flight is the exposure of astronauts to radiation from galactic cosmic rays, which contain heavy ions such as (56)Fe. To study the effects of such exposures, experiments were conducted at the Brookhaven National Laboratory by exposing Wistar rats to high-mass, high-Z, high-energy (HZE) particles using the Alternating Gradient Synchrotron (AGS). The biological effectiveness of (56)Fe ions (1000 MeV/nucleon) relative to low-LET gamma rays and high-LET alpha particles for the induction of chromosome damage and micronuclei was determined. The mitotic index and the frequency of chromosome aberrations were evaluated in bone marrow cells, and the frequency of micronuclei was measured in cells isolated from the trachea and the deep lung. A marked delay in the entry of cells into mitosis was induced in the bone marrow cells that decreased as a function of time after the exposure. The frequencies of chromatid aberrations and micronuclei increased as linear functions of dose. The frequency of chromosome aberrations induced by HZE particles was about 3.2 times higher than that observed after exposure to (60)Co gamma rays. The frequency of micronuclei in rat lung fibroblasts, lung epithelial cells, and tracheal epithelial cells increased linearly, with slopes of 7 x 10(-4), 12 x 10(-4), and 11 x 10(-4) micronuclei/binucleated cell cGy(-1), respectively. When genetic damage induced by radiation from (56)Fe ions was compared to that from exposure to (60)Co gamma rays, (56)Fe-ion radiation was between 0.9 and 3.3 times more effective than (60)Co gamma rays. However, the HZE-particle exposures were only 10-20% as effective as radon in producing micronuclei in either deep lung or tracheal epithelial cells. Using microdosimetric techniques, we estimated that 32 cells were hit by delta rays for each cell that was traversed by the primary HZE (56)Fe particle. These calculations and the observed low relative effectiveness of the exposure to HZE particles suggest that at least part of the cytogenetic damage measured was caused by the delta rays. Much of the energy deposited by the primary HZE particles may result in cell killing and may therefore be "wasted" as far as production of detectable micronuclei is concerned. The role of wasted energy in studies of cancer induction may be important in risk estimates for exposure to HZE particles.     

Developmental aluminum toxicity in mice can be modulated by low concentrations of minerals (Fe,Zn, P,Ca, Mg) in the diet

Female Swiss Webster mice were fed diets containing 7 (control) or 1000 μg Al/g diet from conception to weaning. Pregnancy weight gain, brith weight, litter size, postnatal mortality, and weaning weight were measured. In different groups, diets low in Fe, Zn, P, or Ca and Mg (CaMg) were used as basal diets, to which Al was added. Relative to controls, who received NRC recommended levels of these nutrients, all diets with marginal essential trace elements impacted development, as demonstrated by effects on birth weight (CaMg, Fe) or weaning weight (Fe, Zn, P). Compared to diets low in Al, the 1000-mg Al/g diet led to reduced weaning weight regardless of the essential element content of the diet. Other end points were influenced by Al only within the basaldiet group; pregnancy weight gain with the low-P diet, litter size with the low-Fe diet, pregnancy completion with the low-Zn diet, and postnatal mortality with the low-CaMg or low-Zn diet. Thus, diets marginal in selected minerals can differentially alter the toxicological profile of developmental Al exposures. A basal diet was also used in which the NRC diet was supplemented with ascorbic acid, which promotes Al absorption. No modification of Al toxicity was seen with ascorbic acid supplementation.     

Effect of diet on cancer development: is oxidative DNA damage a biomarker?

Free radicals and other reactive species are generated in vivo and many of them can cause oxidative damage to DNA. Although there are methodological uncertainties about accurate quantitation of oxidative DNA damage, the levels of such damage that escape immediate repair and persist in DNA appear to be in the range that could contribute significantly to mutation rates in vivo. The observation that diets rich in fruits and vegetables can decrease both oxidative DNA damage and cancer incidence is consistent with this. By contrast, agents increasing oxidative DNA damage usually increase risk of cancer development. Such agents include cigarette smoke, several other carcinogens, and chronic inflammation. Rheumatoid arthritis and diabetes are accompanied by increased oxidative DNA damage but the pattern of increased cancer risk seems unusual. Other uncertainties are the location of oxidative DNA damage within the genome and the variation in rate and level of oxidative damage between different body tissues. In well-nourished human volunteers, fruits and vegetables have been shown to decrease oxidative DNA damage in several studies, but data from short-term human intervention studies suggest that the protective agents are not vitamin C, vitamin E, beta-carotene, or flavonoids.     

Diet and colorectal cancer prevention

The majority of cancers are sporadic and epidemiological estimates suggest that up to 80% of colorectal cancer is attributable to diet. Epidemiologically, cross-sectional comparisons, case-control studies and trends in food intake show high rates of colorectal cancer in populations consuming diets high in meat and fat, and low in starch, NSP (non-starch polysaccharides, fibre) and vegetables. In general, prospective studies tend to support these findings although estimates of relative risk are not high. Existing prospective studies have however used crude indices of diet subject to substantial measurement error, and interactions with genetic polymorphisms in, for example, phase-I and -II enzymes have been studied only rarely. The association between meat consumption and colorectal cancer is usually attributed to the formation of heterocyclic amines in meat when it is cooked. In addition, in humans high-meat diets increase the level of nitrosatable material entering the colon so that faecal N-nitroso compounds (NOCs) increase in a dose-responsive manner following endogenous synthesis in the colon. Some of the mutations and guanine adducts accumulated during colorectal cancer progression are characteristic of alkylative damage, which would be compatible with NOC exposure. To date, NSP, resistant starch and vegetables have not reduced faecal NOC levels.     

Methylation patterns in sentinel genes in peripheral blood cells of heavy smokers: Influence of cruciferous vegetables in an intervention study

Changes in DNA methylation patterns are a hallmark of tobacco-induced carcinogenesis. We have conducted a randomized 4-week intervention trial to investigate the effects of three dietary regimens to modify DNA methylation patterns in peripheral white blood cells of heavy smokers. A group of 88 smokers were randomly assigned to and distributed among three diets, including (1) normal isocaloric diet (balanced in fruits and vegetables), according to international guidelines; (2) a diet enriched in flavonoids and isothiocyanates (particularly cruciferous vegetables); (3) a regimen consisting of diet 1 supplemented with flavonoids (green tea and soy products). Methylation patterns were analyzed by pyrosequencing in LINE1 (Long Interspersed DNA Elements), RASSF1A, ARF and CDKN2a (tumor suppressor genes), MLH1 (mismatch DNA repair) and MTHFR (folate metabolism). Three distinct patterns of methylation were observed. In LINE1, methylation showed a small but reproducible increase with all three regimens. MTHFR was constitutively methylated with no significant modulation by diets. The four other loci showed low basal levels of methylation with no substantial change after intervention. These data suggest that the isocaloric diet may stabilize global epigenetic (LINE1 DNA methylation) patterns in peripheral white blood cells but does not provide evidence for methylation changes in specific genes associated with this short-term dietary intervention.     

Micronucleus frequency in peripheral blood lymphocytes and exfoliated buccal cells of untreated cancer patients

Some genetic diseases may increase the cellular instability. Since most human tumors have some genetic base, this study was undertaken for the genetic instability in cancer patients by micronucleus analysis, a mutation-screening test, which is more practical and economic technique than metaphase analysis carried out for chromosomal aberrations. Genetic changes were assessed in untreated cancer patients (lung, stomach and colon cancer) by different genotoxical screening methods: the cytokinesis-block micronucleus test and the buccal mucosa cell micronucleus test. The evaluation of micronuclei number in peripheral blood lymphocytes and buccal cells showed genomic instability in somatic cells. There was a significant increase in the number of micronuclei in cancer patients prior to the initiation of chemotherapy, and/or radiotherapy compared with healthy human subjects. Furthermore, there was no significant difference between smokers and non-smoking groups or male and female groups. These results suggest that cancer in humans is characterized by an increase of chromosomal damage and thus, the micronucleus assay carried out here may be useful in routine cytogenetic studies of cancer. The text was submitted by the authors in English.