Liver mitochondria from manganese-deficient and pallid mice: function and ultrastructure.

Oxidative phosphorylation was studied in isolated liver mitochondria from manganese-deficient mice and in those from a mutant strain, pallid. In mitochondria from manganese-deficient mice, ratios of adenosine triphosphate formed to oxygen consumed were normal, but oxygen uptake was reduced. Electron microscopy of these mitochondria revealed ultrastructural abnormalities including elongation and reorientation of cristae. No biochemical or structural abnormalities were found in mitochondria from pallid mice.

Zinc intake, neoplastic DNA synthesis, and chemical carcinogenesis in rats and mice.

DNA synthesis in a transplanted hepatoma induced by 3'-methyl-4-dimethylaminoazobenzene was significantly reduced (P less than 0.01) in rats maintained on diets low (0.4 mug/g) or high (greater than or equal to 500 mug/g) in zinc when compared with control animals given 60 mug zinc/g. 3-Methylcholanthrene-induced carcinogenesis was considerably lowered in mice receiving the same low or high zinc diets during the induction periods.

Radioisotopic studies concerning the efficacy of standard washing procedures for the cleansing of hair before zinc analysis.

Various standard procedures were investigated in relation to the removal of exogenously applied 65Zn from human hair and endogenously incorporated 65Zn from rat hair. Human hair was found to adsorb zinc and a variety of other metal ions from aqueous solutions in a manner which suggested some ion-exchange capacity. Uptake of zinc varied considerably between human hair samples, but in most cases accumulation of zinc occurred rapidly and often resulted in hair zinc levels several-fold higher than found in control samples. Extraction of zinc and other metal ions was greatest after treatment with disodium EDTA and sodium lauryl sulfate than after washing with water or aqueous Triton X-100. However, no procedure effectively removed all exogenous zinc, while all treatments extracted varying proportions of the endogenous zinc component. Because of the inability of standard washing procedures to remove exogenous zinc without reducing endogenous or indicator zinc levels, use of hair zinc analyses to indicate nutritional zinc status are inadvisable if hair zinc contamination is likely to have occurred.

Cancer biomarkers in the field of tea.

Suitable intermediate end-point biomarkers are needed in order to reduce the time and expense associated with cancer chemoprevention studies. Because of the multi-step nature of cancer intermediate biomarkers reflect a continuum of events associated with different stages of disease development, and range from genetic damage linked to cancer initiation, to tumor growth and expansion during disease progression. With tea, experimental evidence points to potential protection at several stages of carcinogenesis including endogenous carcinogen formation, carcinogen activation and detoxification, DNA damage and destabilisation, cell proliferation, neoplastic growth and metastatic spread. If used effectively in tea research, biomarkers should contribute to a better understanding of the possible extent of cancer protection afforded by tea, as well as the putative mechanisms involved and the principal components of tea with prophylactic potential.

Nutrition, cancer, and aging.

The parallel increase in cancer risk with advancing age is well recognized, and several pathophysiological mechanisms common to both conditions have been proposed to explain this interrelationship. The importance of nutrition, both in delaying the aging process and in protecting against cancer is also well recognized, and it is therefore of interest to compare the relative impact several of the more widely studied dietary manipulations may have on each of these conditions. For example, caloric restriction, which putatively reduce oxidative stress and effectively increases life span in animals also seems to reduce the incidence of many cancers, possibly due to diminished mitogenesis. Likewise, oxidative damage to DNA appears to be common to both processes but may be more important in the mitochondria with respect to aging and in the nucleus in relation to cancer. Inadequate dietary folate and impaired DNA methylation status are closely associated with increased cancer risk, and recently defective somatic cell methylation and accumulated genetic instability have been proposed as key mechanisms contributing to senescence. Several other well-established anticancer dietary strategies, which include increased fiber intake and the consumption of more fruits and vegetables, have not been studied extensively in relation to aging, although many of the phytochemicals considered important as chemopreventive agents for cancer may well contribute to delaying the aging process. Although not directly related to nutrition, but nevertheless highly relevant, is the question of physical activity, which has been strongly linked to a reduction in risk of some cancers. Although less is known with respect to exercise and biological markers of aging, physical activity does appear to retard the age-related decline in the muscle strength and in the bone density.

Regional effects of hypothyroidism on 5'-nucleotidase and cyclic nucleotide phosphohydrolase activities in developing rat brain.

The activities of 5'-nucleotidase (EC 3.1.3.5) and of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) (EC 3.1.4.37) were measured in crude membrane preparations from 6 brain regions of hypothyroid rats 31 days and 42 days after conception (8-9 days and 19-20 days after birth respectively) and from normal rats 31, 36 and 42 days after conception. At 42 days the activities of both enzymes were markedly and significantly lower than in controls in all hypothyroid brain regions. The pattern of increase in CNP activity in the brain regions of normal animals reflected the caudo-rostral progress of myelination. At 42 days postconception in the more caudal regions of hypothyroid brain, myelination, as assessed by CNP activity, was delayed by 5-7 days. In the more rostral regions where normal activity had not yet developed, the delay due to hypothyroidism was not clearly defined. The lowered activity of 5'-nucleotidase observed using crude membrane preparations from hypothyroid animals was shown not to be due to a redistribution of enzyme between cytosol and membranes or to losses into the supernatant fraction during preparation of the particulate fraction used for assays. In addition the lowered activity of 5'-nucleotidase could not be accounted for in terms of the delayed myelination and consequent absence of myelin-associated enzyme. In contrast to the results with CNP, the effects of hypothyroidism on 5'-nucleotidase in the 6 regions showed no caudo-rostral pattern. Although it was clear that the lowered activity of 5'-nucleotidase in hypothyroid brain at 20 postnatal days was not directly attributable to the state of delayed myelination, no identification of the structures or cell types affected could be made.

Zinc deficiency in the 11 day rat embryo: a scanning and transmission electron microscope study.

Zinc deficient rat embryos were obtained on the 11th day of pregnancy and examined by scanning and transmission electron microscopy. Scanning electron microscopy revealed an increase in the number of deformed embryos, as well as embryonic growth retardation. In addition, the epithelium of zinc deficient embryos displayed a marked increase in surface microvilli, as well as the presence of blebbing. Transmission electron microscopy indicated extensive cell death in the neural epithelium which was apparently more severely damaged by zinc deficiency than were mesenchymal cells. Mitochondrial cristae were affected to a greater degree than any other membrane of the cell and cristael disintigration appeared to represent the principal cellular lesion preceding necrosis of neural cells and neural tube teratology.

Interactions of cadmium and zinc in cultured rat embryos.

Rat embryos were cultured in serum taken from animals dosed with cadmium, or serum with cadmium added in vitro in the presence or absence of additional zinc. Embryos explanted at day ten and grown in serum taken from animals sooner than 4 h after dosing had a reduced DNA content after 24 h culture. In one-hour serum, the yolk sac had become thick and brittle. Zinc ameliorated the effects but had no stimulatory effect on post eight-hour serum when serum zinc levels were at their lowest. The hypothesis that cadmium induces a maternal zinc deficiency sufficient to cause teratogenic changes could not be sustained. Embryos explanted at nine days were much more susceptible to cadmium added in vitro than ten-day embryos. The principal anomaly, apart from a reduced DNA content, was a thickening of the yolk sac similar to that seen in embryos grown in serum taken from animals one hour after cadmium dosing. Addition of zinc to the medium prevented both of these effects. The suggestion is made that the cadmium-induced dysgenesis of the yolk sac precludes appropriate embryonic nutrition.

Recommended dietary intakes of iron, zinc, and other inorganic nutrients and their chemical form and bioavailability.

The bioavailability of a nutrient can, in some cases, be significantly influenced by the chemical form in which it appears in the diet and by the presence of other factors in food that may enhance or depress mineral absorption and utilization. With inorganic nutrients in particular, it is important to understand the physicochemical factors that underlie the differences in bioavailability of some trace elements from diets of differing composition, so that meaningful recommended dietary intakes (RDIs) may be set for these nutrients in relation to the dietary profiles of the countries in which they are to be used. Iron and zinc are especially noteworthy in this regard because their absorption can be greatly influenced by chemical form, ionic competition for uptake mechanisms, and the presence of favorable or unfavorable chelating and binding agents in the diet. Recognizing this variability, several national and international authorities recommending RDIs for these nutrients list a range of desirable intakes that relate to the estimated bioavailability of the mineral from various dietary regimens.

Zinc and the gene.

A significant portion of cellular zinc is found in the nucleus where it appears to be critically involved in maintaining genetic stability and in the process of gene expression. With regard to gene expression zinc functions mechanistically at several levels but recent interest has focussed especially on the involvement of zinc in DNA transcription through the activity of transcription factors which contain specific zinc-finger regions which bind to DNA and, in conjunction with other families of transcription factors, control cell proliferation, differentiation and cell death. Because of the central importance of zinc in cell division and growth, considerable attention is paid to zinc as an essential trace element and much has been written concerning dietary sources of zinc and recommended dietary intakes of the metal.

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.

Induction of micronuclei in cultured murine splenocytes exposed to elevated levels of ferrous ions, hydrogen peroxide and ultraviolet irradiation.

The frequency of micronuclei in cultured mouse splenocytes increased positively and in a dose-related manner to exposure to ferrous ions and ultraviolet irradiation, but not to hydrogen peroxide. Combined treatments, especially when ferrous ions were present with hydrogen peroxide or with ultraviolet irradiation, led to a synergistic enhancement in micronucleus frequency. The results indicate that a significant level of chromosome damage is associated with exposure to ultraviolet light and to general cellular pro-oxidative stress, and indicate that under these conditions the micronucleus assay can provide an effective in vitro model for the study of genotoxicity in relation to oxygen-derived free radicals.

The teratogenic effects of nicotine in vitro in rats: a light and electron microscope study.

Cigarette smoking in pregnant women has been shown to lead to intrauterine growth retardation and fetal death, and possibly also to neural dysmorphology and long-term learning deficits in the offspring. Because the teratogenic agent in cigarette smoke remains controversial, the present study on rat embryos in culture examined specifically whether nicotine can cause neural dysmorphology and, hence, act as a nervous system teratogen. This in vitro study confirmed previous reports in utero that nicotine leads to growth retardation, and, in addition, demonstrated that development of the nervous system, particularly the forebrain, as well as the branchial arches was impaired, possibly leading to microcephaly and cleft palate respectively in term fetuses. Cellular disruption and necrosis occurred in the neuroepithelium and underlying mesenchyme, with the effect being dose dependent. Ultrastructurally, there was severe disruption of cell and organelle membranes, with many healthy cells containing engulfed, whole condensed or remnants of dead cells. This study demonstrates that nicotine acts as a nervous system teratogen leading to gross and cellular dysmorphology. Suggested mechanisms for nicotine action include the possibility that the highly lipid soluble teratogen may exert its effects directly on the membranes or indirectly through oxidative membrane damage.

A prospective study of serial maternal serum zinc levels and pregnancy outcome.

A prospective study of 878 pregnant women in Adelaide, South Australia, examined the relationship of maternal serum zinc concentration to pregnancy outcome. Blood samples were obtained at weeks 18 and 32 of pregnancy, and from the umbilical cord. Additional detailed data were obtained, via standardised antenatal interview and review of clinical records, on antenatal care, personal, behavioural, and sociodemographic characteristics, and, subsequently, on delivery and neonatal assessment. Maternal mid-pregnancy zinc status was negatively correlated, although very weakly, with duration of gestation and with birthweight (including, in particular, the 18 recorded cases of intrauterine growth retardation). This finding accords with recent suggestions that, in non-experimental human studies in populations in which frank zinc deficiency is absent, maternal serum zinc level may be an outcome, rather than a determinant, of fetal growth later in pregnancy. Low mid-pregnancy zinc levels were associated with increased risk of intrapartum haemorrhage. In general, the greatest risk of having some complication or abnormality of delivery or neonatal functional status occurred in pregnant women who had both an initially below-average, and subsequently decreasing, serum zinc concentration. This corroborates other recent research. While it might be inferred that above-average fetal growth, by depressing maternal zinc status, could itself impair delivery and neonatal functioning, our data indicate that these effects operated independently.

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.

Zinc, ethanol, and lipid peroxidation in adult and fetal rats.

Studies were performed on adult and fetal rats receiving either a zinc-deficient (<0.5 ppm) diet and/or ethanol (20%) throughout pregnancy. Liver zinc levels were depressed in fetuses exposed toin utero zinc deficiency, but brain zinc levels were unchanged. Ethanol had no effect on the concentration of zinc in the several fetal and adult tissues studies. Lipid peroxidation, as measured by endogenous levels of malondialdehyde (MDA) increased following food restriction, zinc improverishment, and alcoholism in adult and fetal livers, but not in fetal brains. Generally, levels of MDA were highest when both zinc deficiency and the ingestion of alcohol occurred concurrently. Glutathione (GSH) was depressed by zinc restriction in several adult and fetal tissues, but not in the fetal brain. Ethanol alone had no effect on GSH levels. The activity of the enzyme glutathione peroxidase (GSH-Px) was not changed in either organism by alcohol or zinc deficiency.Overall, the data point to increased lipid peroxidation in maternal and fetal rat tissues following zinc depletion and/or treatment with alcohol and draw attention to the apparent vulnerability of the fetal liver toin utero alcoholism. By contrast, the fetal brain seems to be especially resistant to alcohol and zinc-related lipoperoxidation. An association is suggested between the increased lipoperoxidation accompanying zinc deficiency and reduced levels of GSH, but this does not appear to relate to changes in the activity of GSH-Px. A similar relationship is not evident with respect to the increased levels of MDA in fetal and adult livers following chronic alcohol intoxication. A possible basis for the zinc-GSH interaction is discussed.

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.

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.

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.

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.