Changes in plasma antioxidant status following consumption of diets high or low in fruit and vegetables or following dietary supplementation with an antioxidant mixture.

The aim of the present study was to examine the effect of consumption of a high-fruit and vegetable diet, or a spray-dried extract of selected fruits and vegetables of high antioxidant content, on indices of antioxidant status of individuals consuming a background diet with minimal antioxidant intake. Plasma antioxidant concentrations were determined in twenty-five men following a 2-week depletion period during which they consumed self-selected low-antioxidant diets (less than three servings of fruit and vegetables with no tea, coffee, red wine or fruit juice). Following this period the volunteers consumed either a self-selected diet containing five to seven servings of fruit and vegetables/d, or 30 g of a spray-dried supplement designed to provide the equivalent antioxidant activity of five to seven servings of fruit and vegetables for 2 weeks in a crossover trial. Following consumption of a high-antioxidant diet for 2 weeks, plasma concentrations of ascorbic acid, alpha- and beta-carotene and lutein+zeaxanthin were all significantly increased (P < 0.05) over the depletion period. However, concentrations of lycopene, retinol and tocopherol were not affected. Consumption of the supplement also raised the concentrations of these same antioxidants in plasma. Despite the increases in the concentrations of measured antioxidant nutrients, the 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid-equivalent antioxidant capacity of plasma, as estimated by inhibition of metmyoglobin activity, was not significantly affected by any of the dietary treatments.

Iron, alpha-tocopherol, oxidative damage and micronucleus formation in rat splenocytes.

The influence of low and high alpha-tocopherol diets in concert with a high polyunsaturated fat content and a modest increase in dietary iron has been studied. Iron supplementation at five times the recommended dietary level was not associated with any increased sensitivity of the splenocytes to any of the oxidative challenges. Despite the significantly higher alpha-tocopherol concentrations in the plasma and liver of animals supplemented with this vitamin, there was no apparent protection against oxidative genotoxicity, as judged by the formation of micronuclei in splenocytes subjected to oxidative stress ex vivo. These results add to the evidence that vitamin E supplementation has little effect against oxidative genomic damage, at least as demonstrated by an increase in micronucleus frequency.

Iron, alpha-tocopherol, oxidative damage and micronucleus formation in rat splenocytes.

The influence of low and high alpha-tocopherol diets in concert with a high polyunsaturated fat content and a modest increase in dietary iron has been studied. Iron supplementation at 5 times the recommended dietary level was not associated with any increased sensitivity of splenocytes to any of several oxidative challenges ex vivo. Despite the significantly higher alpha-tocopherol concentrations in plasma and liver in animals supplemented with this vitamin, there was no apparent protection against oxidative genotoxicity as judged by the formation of micronuclei in splenocytes subjected to oxidative stress ex vivo. These results add to the accumulating evidence that vitamin E supplementation has little effect against oxidative genomic damage, at least as demonstrated by an increase in micronucleus frequency.

Coarse brown rice increases fecal and large bowel short-chain fatty acids and starch but lowers calcium in the large bowel of pigs.

Young male pigs were fed a diet formulated from human foods including either boiled white rice plus rice bran or heat-stabilized brown rice at equivalent levels of fiber for 3 wk. Stool and starch excretion were low in pigs fed white rice during the first 2 wk of the experiment. In pigs fed brown rice, their excretion was high during wk 1 but declined in wk 2 while short-chain fatty acid (SCFA) excretion was higher at both times. Large bowel digesta mass, measured during wk 3, was higher in pigs fed brown rice but only in the proximal colon. Large bowel and fecal starch concentrations were higher in pigs fed brown rice but the difference was insufficient to explain the increase in large bowel digesta mass. In pigs with a cecal cannula, digesta starch concentrations were equally higher when white or brown rice was fed compared with the corresponding rice which had been finely milled, indicating that particle size was a determinant of ileal digestibility. Concentrations and pools of total and individual SCFA were higher in all regions of the colon but not the cecum of pigs fed brown rice. Large bowel Ca(2+) concentrations were lower in pigs fed brown rice, suggesting greater absorption. The data confirm earlier findings that brown rice raises large bowel digesta mass and SCFA through greater fermentation of starch but show that starch itself makes a relatively small contribution to digesta and stool mass. Apparently, the rate of passage of digesta is a determinant of the concentrations and pools of SCFA in the distal colon and in feces.

Protection by black tea and green tea against UVB and UVA + B induced skin cancer in hairless mice.

The effects of green and black tea consumption on the early indices of UVB and UVA + B skin damage in hairless mice have been studied in the absence of any chemical tumour initiators or promoters. Black tea consumption was associated with a reduction in the number of sunburn cells in the epidermis of mice 24 h after UVA + B irradiation, although there was no effect of green tea. Other indices of early damage such as necrotic cells or mitotic figures were not affected. Neutrophil infiltration as a measure of skin redness was slightly lowered by tea consumption in the UVB group. Consumption of either green or black tea resulted in significantly fewer skin papillomas and tumours induced by UVA + B light, however black tea provided better protection against UVB-induced tumours than green tea. This study confirms earlier reports that tea consumption can reduce the incidence of skin cancer in hairless mice, and indicates that black tea may afford more protection against simulated solar irradiation than green tea.

Protection by tea against UV-A + B-induced skin cancers in hairless mice.

Consumption of tea, especially green tea, has been shown to reduce the incidence of ultraviolet (UV)-related skin tumors in hairless mice. Because milk is added to much of the tea consumed in Western cultures, we have studied the effects of including milk in the tea consumed by hairless mice receiving simulated solar radiation. Under these conditions, mice consuming tea with 10% whole milk had 30% fewer papillomas, 50% fewer tumors, and 55% smaller lesions than mice consuming water. Mice consuming tea alone had fewer papillomas and tumors than mice consuming tea with milk; however, the difference in area affected was not statistically significant. In separate experiments, there was a significant dose response to black tea as a preventive against UV-related skin lesions, and also consumption of black tea was associated with a small but significant reduction in the incidence of papillomas in mice previously exposed to UV radiation. The results of these studies demonstrate that, in hairless mice, black tea can inhibit the formation of UV-induced skin tumors in a dose-dependent manner and, even with the addition of milk, can still inhibit the growth of UV-related skin tumors.

Genistein inhibits growth of B16 melanoma cells in vivo and in vitro and promotes differentiation in vitro.

Consumption of soy products has been linked to a reduced mortality and morbidity from a number of cancers. Genistein, one of the principal soy isoflavones, has been shown to inhibit the growth of a number of tumour cell lines in vitro; however, a role of genistein in retarding tumour growth in vivo is less well documented. In this study, in addition to examining the effects of genistein on the growth of murine B16 melanoma cells in vitro, we have examined the effects of feeding a genistein-rich diet on s.c. growth of these tumour cells in mice. In vitro, the melanoma cells showed an increase in sensitivity to genistein with increasing time of exposure, culminating in a 50% growth inhibition (IC50) at 12.5 microM after 7 days. Genistein at 25 microM induced micronucleus formation after 24 hr and at concentrations as low as 2.5 microM induced morphological changes indicative of differentiation. Growth of solid tumours implanted into female C57BL/6J mice was inhibited by 50% when mice were fed genistein for 1 week before and for 1 week after inoculation with B16 melanoma cells. Plasma genistein concentrations at the time of tumour removal were 1.1 microM, which is similar to levels reported in humans consuming diets high in soybeans or soybean products, while control animals had no detectable genistein in plasma. Our results provide additional in vivo evidence suggesting that genistein retards the growth of implanted tumours, adding further to studies suggesting that this isoflavonoid is a biologically active component of soy foods.

Protection by zinc against UVA- and UVB-induced cellular and genomic damage in vivo and in vitro.

For many years, zinc salts have been used both topically and orally to treat minor burns and abrasions as well as to enhance wound repair in man and animals. In this study we describe the protective effects of zinc against UV-induced genotoxicity in vitro and against sunburn cell formation in mouse skin in vivo. Cultured skin cells from neonatal mice showed a dramatic increase in the number of micronuclei as a result of UVA and UVB irradiation. Inclusion of zinc at 5 micrograms/mL in the medium significantly reduced the frequency of micronuclei and of micronucleated cells. In hairless mice, topical application of zinc chloride for 5 consecutive days or a single application 2 h prior to UV exposure reduced the number of sunburn cells in the epidermis as did application of zinc 1 h after exposure. Application 2 h after irradiation also tended to have a protective effect, although there was a large variation between animals. It is proposed that an influx of zinc can protect epidermal cells against some of the more delayed effects of UV-induced damage.

Black tea, green tea, and tea polyphenols. Effects on trace element status in weanling rats.

Previous studies have shown that tea consumption can impair trace element metabolism, particularly iron status, and increase the risk of anemia in humans and animals. More recently, however, evidence has been accumulating to show that, in animals, consumption of green tea or its polyphenols is associated with a reduction of the incidence and severity of a variety of experimentally induced cancers. In this study we have monitored the growth, trace element status, including hematological parameters of weanling rats given either (1) water, (2) 1% black tea, (3) 1% green tea, or (4) 0.2% crude green tea extract as their sole drinking fluid while consuming diets containing either adequate or low amounts of iron. With the exception of manganese, none of the trace elements studied (iron, copper, zinc, and manganese) or the hematological indices measured were affected by the type of beverage supplied, even though the polyphenol extract was shown to chelate metals in vitro and all the animals fed the low iron diet were shown to be anemic. There appeared to be an effect of black and green teas on manganese balance in both the first and last weeks of the study. A lower level of brain manganese was associated with green tea consumption, and a higher level of this element in the kidneys of animals fed black tea. The results demonstrate that both black and green teas and a green tea polyphenol extract do not represent a risk to animals consuming the beverages as their sole fluid intake with respect to iron availability, although the interactions with manganese deserve further study.

Induction of micronucleus formation in mouse splenocytes by the soy isoflavone genistein in vitro but not in vivo.

The effects of genistein (one of the major soybean isoflavones), genistein (the glucosylated form of genistein) and etoposide (a topoisomerase 11 inhibitor) have been studied in mouse splenocytes in culture. Genistein (25 microM), genistein (25 microM) and etoposide (0.1 microM) all induced the production of large numbers of micronuclei; however, genistein at 12.5 or 2.5 microM had no clastogenic effect. In a second study, mice were gavaged with 20 mg genistein/kg body weight/day for 5 days (approximately equivalent to a 70 kg human consuming 2.8 kg soybeans/day) and the micronucleus frequency was determined. There was no observable increase in the micronucleus frequency even though the plasma genistein levels in the treated animals were found to be 9.2 +/- 2.0 microM compared with 0.1 +/- 0.0004 microM in the control animals. The results show that even though genistein is capable of inducing micronucleus formation, an event associated with genetic damage, plasma levels are unlikely to be sufficiently elevated to produce such an effect.

A comparative study of micronucleus frequency in peripheral blood lymphocytes of human subjects given dietary cis, trans and saturated fat.

The micronucleus test using peripheral blood lymphocytes has been used to study the clastogenic effects of saturated, cis or trans fatty acids in human volunteers. Consumption of diets rich in either saturated fats (palmitic acid), monounsaturated cis fats (oleic acid) or monounsaturated trans fats (elaidic acid) for 3 wk had no effect on either the number of micronuclei or the frequency of micronucleated cells in lymphocytes in culture. This study adds to the accumulating body of evidence to suggest that dietary intake of trans fatty acids is not especially linked to genetic damage.

The influence of topical and systemic vitamin E on ultraviolet light-induced skin damage in hairless mice.

Hairless mice were fed diets containing different levels of vitamin E or received topical applications of the vitamin for three weeks before a single exposure equivalent to one minimal erythematous dose of ultraviolet light provided by an artificial sunlight source. Lipid peroxidation and suppression of incorporation of thymidine into DNA were used to estimate the degree of damage caused by the radiation. Restriction of dietary vitamin E had little effect on degree of epidermal lipid peroxidation or on thymidine incorporation into DNA. High dietary levels of the vitamin did not alter the degree of lipid peroxidation; however, the incorporation of thymidine was restored to levels comparable to those of unirradiated animals. Topical administration of a 1% solution of the vitamin in ethanol 1 or 24 hours before irradiation also restored thymidine incorporation and reduced the degree of lipid peroxidation. The results suggest that both dietary and topical vitamin E are effective in protecting the epidermis against some of the early damage induced by ultraviolet radiation.

Zinc, iron, vitamin E, and erythrocyte stability in the rat.

Previous studies have shown that deficiencies of zinc and vitamin E, as well as iron excess, contribute to peroxidative damage in several tissues in vivo. The present study reports on the sensitivity of red blood cells from young rats exposed to individual or concurrent imbalances of these three nutrients. For 21 d, rats were fed diets that were either deficient or replete in zinc and with or without excess iron or replete or deficient in vitamin E. When red blood cells from these rats were incubated in vitro, erythrocyte hemolysis, lipid peroxidation (assessed by MDA production), and hemoglobin degradation (assessed by alanine release), did not significantly increase unless vitamin E had been omitted from the diet. These results imply that either adequate tightly-bound zinc exists within the zinc-deficient cell to protect it from oxidative damage, or that other antioxidant defense mechanisms (including vitamin E) present within the plasma membrane and cytosol are sufficient to protect the cell from the otherwise damaging effects of zinc deficiency and/or iron excess.

Maternal metabolism and teratogenesis in zinc-deficient rats.

Embryos removed at 11.5 days gestation from pregnant rats allowed a zinc-deficient diet from the time of mating showed a high frequency of malformations of all organ systems. There were, however, large differences between litters of individual dams. Comparison of the daily food intake of zinc-deficient dams with the appearance of the embryos suggested that fluctuations in the maternal serum zinc levels induced by feeding or fasting influenced the availability of zinc to the embryos. By cyclically feeding zinc-deficient dams to a predetermined schedule, low maternal serum zinc levels were induced at selected stages of development. This was accompanied by specific malformations of the organs developing at that time.

Cellular necrosis in zinc-deficient rat embryos.

Abnormal cellular necrosis was studied in 9.5-11.5-day embryos obtained from zinc-deficient rats. At periods of low maternal zinc status induced by a high intake of a zinc-deficient diet, cell death was observed in those regions of the embryo that were most sensitive to teratogenic insult at that time. As the maternal serum zinc level increased during the fasting phase of the feeding cycle, the degree of necrosis decreased, leaving the tissue histologically more normal even though the embryos were grossly malformed. The mitotic index of cells in the neural epithelium and limb buds of zinc-deficient, non-necrotic embryos was found to be elevated, but there was no evidence of blockage at any particular stage of mitosis. It can be hypothesised that during the early stages of organogenesis, periods of low maternal zinc status initiate unscheduled cell death by some as yet undefined mechanism that in turn, gives rise to the morphological anomalies observed later.

Dietary zinc intake of pre-menopausal women.

Assessment of the diets of 73 pre-menopausal women completing dietary frequency questionnaires suggests that 11 per cent consume a daily average of less than 7.5 mg of zinc. A further 26 per cent consume less than 10 mg of zinc daily, an amount considered to approximate the average daily requirement. Dietary records from a second group of 18 working women (aged less than 35 y), covering periods from 6 to 76 days are also analysed. Of these, the zinc intake of 8 women averages less than 7.5 mg/day. The apparent zinc intake of all women shows large day-to-day fluctuations. The presence of such wide variability within individual eating patterns suggests that short-term estimates of dietary zinc intake cannot provide a reliable estimate of overall zinc nutriture.

In vitro development of zinc-deficient and replete rat embryos.

The development of the zinc-deficient rat embryo has been studied in vitro using embryo culture techniques. Normal 9.5 day embryos cultured for 48 h in serum obtained from zinc-deficient rats grew and developed to the same extent as those cultured in zinc-replete serum. Embryos from dams which had been fed a zinc-deficient diet since mating were also removed for culture. Such zinc-deficient embryos fell into two broad morphological categories. One group appeared identical to the normal embryos, while the others had apparently normal visceral yolk sacs but small embryonic poles and retarded or abnormal embryonic development. Culture of the first group in either zinc-deficient or replete serum produced morphologically normal embryos; however, those which appeared abnormal at day 9.5 were grossly malformed after 48 h incubation in either sera. When embryos were cultured in the presence of 65Zinc, the most severely affected zinc-deficient embryos accumulated as much zinc as the zinc-replete and apparently unaffected zinc-deficient embryos, indicating that the malformations do not arise from an inability of the embryo or yolk sac to accumulate zinc from the surrounding fluid. The results from these studies suggest that the teratogenic effects of zinc deficiency cannot be induced by direct culture of zinc-replete embryos in zinc-deficient serum. Furthermore, it would appear that maternal zinc deficiency can exert its teratological influence prior to day 9.5 of gestation and that these effects are not readily reversible.

Zinc deficiency and the developing embryo.

The effect ofin utero zinc deficiency on fetal development in rats is reviewed. Attention is paid to the primary biochemical lesion associated with zinc-related teratogenesis and special consideration is given to the central nervous system. Evidence is presented that the thymidine kinase salvage pathway, used for the synthesis of thymidine monophosphate in DNA synthesis, is depressed more in fetal brain tissue than in the liver. In addition, greater reliance appears to be placed on this pathway than onde novo synthesis in the fetal brain than in other tissues. Some consideration is given to the use of in vitro embryo culture in studies relating to neurogenesis, but evidence is presented of a greater capacity of explanted rat embryos to obtain zinc from maternal serum than occurs in vivo.The rapid onset of a teratogenic zinc deficiency following dietary zinc restriction is again highlighted and further studies are described which demonstrate the critical impact of a single feeding cycle, of 4 d duration, on maternal plasma zinc levels and on the extent and nature of the observed fetal abnormalities. Evidence is presented that by shifting the timing of the high dietary intake/low plasma zinc peak to coincide with a particular 48 h period between days 6 and 10 of pregnancy, the pattern of malformations thus obtained reflected the coincidence of the high dietary intake of zinc-deficient diet and the critical time of morphogenesis of several organ systems.Whereas diminished plasma zinc levels at term in zinc-deficient animals are generally well correlated with reduced growth and dysmorphogenesis of the offspring, the same is not always found in human studies. In some cases, elevated plasma zinc levels at parturition are found in mothers with growth-retarded children, or vice versa. Experimental studies with rats are reported that suggest that maternal zinc status at term may be higher in dams bearing pups stunted by exposure to a transient zinc deficiency early in pregnancy, which in turn may have reduced the demand for maternal zinc in the later stages of gestation.The protective effect of zinc on cadmium-induced teratogenesis is discussed, particularly in relation to findings concerning an interaction of these metals in the embryonic yolk sac and thus on preplacental embryonic nutrition. Possible interactions between alcohol and zinc deficiency are also considered and data are presented pointing to increased fetotoxicity and teratogenesis in the presence of both treatments and to a more specific interaction with respect to reduced cell numbers in the developing rat hippocampus. Malondialdehyde levels, which reflect the extent of lipid peroxidation in tissue, are reported to be substantially higher in microsomes from fetal rat livers whenin utero deficiency and gestational alcoholism are combined. The suggestion is made that alcohol and zinc deficiency act independently in the body, but overlap to some extent at the common biochemical locus of membrane lipid peroxidation.