Prediction and Effect of Maternal Marginal Zinc Deficiency on Development, Redox Status, and Gene Expression Related to Oxidation and Apoptosis of Embryos

BackgroundMaternal severe zinc (Zn) deficiency induced oxidative damage and apoptosis in embryos, resulting in growth retardation. Therefore, it is crucial to assess maternal marginal Zn nutritional status for poultry breeders to prevent embryos subjected to severe Zn deficiency. MethodsIn Exp. 1, twelve egg embryos were sampled at incubation day 17 (E17), E20, E23, E26, E29, and E32 (day of hatch) respectively, with 6 replicates of 2 embryo each. The developmental changes of Zn mobilization and Zn transport gene mRNA expression were determined. In Exp. 2, 324 laying duck breeders were randomly allotted into 3 dietary Zn levels (0, 60, and 120 mg Zn/kg diet) with 6 replicates of 18 ducks per replicate. Plasma Zn concentration and erythrocytic Zn metalloenzyme activities in breeders as well as the development, redox status, and gene expression related to oxidation and apoptosis in embryos were measured. Blood samples were collected at the 2th, 4th and 6th weeks of the experiment. ResultsIn Exp. 1, the overall Zn mobilization rates were increased in yolk sac and embryonic liver in response to the increased incubation period, associated with the decreased ZIP10, 13, and 14 mRNA expressions in embryonic liver (P < 0.05). In Exp. 2, with the prolonged dietary Zn depletion, maternal Zn deficiency decreased plasma Zn concentration and erythrocytic alkaline phosphatase activity at the 6th week and inhibited erythrocytic 5'-nucleotidase (5’-NT) activity at 2th week (P < 0.05). On E29 of the maximal rate of Zn mobilization, maternal marginal Zn deficiency increased middle and late embryonic mortality and contents of superoxide anion radical, MDA and PPC, as well as decreasedMT content, CuZnSOD activity, and MT1 mRNA expression in embryonic livers (P < 0.05). Additionally, maternal marginal Zn deficiency increased BCL2-associated X protein and Caspase-9 mRNA expression and decreased B-cell lymphoma-2 mRNA expression in embryonic liver (P < 0.05). ConclusionErythrocytic 5’-NT activity was more rapid and reliable to assess marginal Zn-deficient status in duck breeders. Marginal Zn deficiency impaired hatchability and antioxidant defense system and then induced the oxidative damage and apoptosis in embryonic liver, contributing to the greater loss of embryonic death.

Secondary Zinc Deficiency via Exposure to the Environmental Toxicant Di-2-ethylhexyl phthalate Affects Neurogenesis at Embryonic Day 19

Objectives Zinc deficiency can affect early brain development. We previously found that developmental marginal zinc deficiency affected neurogenesis leading to a lower number of neurons and altered neural specification in the adult rat brain. Zinc deficiency can occur as low dietary zinc intake and secondary to diseases, infections, and exposure to environmental toxicants such as phthalates. This work investigated if gestational exposure to toxicant Di-2-ethylhexyl phthalate (DEHP) could decrease zinc availability to the fetus and altered neurogenesis. Methods Rats were fed an adequate (25 µg zinc/g diet) (C) or a marginal zinc deficient (MZD) (10 µg zinc/g diet), without or with DEHP (300 mg/kg BW) (C + DEHP, MZD + DEHP) from gestational day zero until embryonic day 19 (E19). Zinc concentration was analyzed by atomic absorption spectrometry (AAS). Neurogenesis was evaluated in the offspring at E19 measuring parameters of neural progenitor cells (NPC) proliferation and differentiation by Western blot and/or immunofluorescence. Results Fetal brain zinc concentration was significantly decreased in MZD, C + DEHP and MZD + DEHP than in C. Protein Markers of neurogenesis (NeuN, PAX6, SOX2, TBR2) were lower in MZD and C + DEHP than C, and lowest in MZD + DEHP. The excitatory neuron marker vesicular glutamate transporter 1 (VGLUT1) was lower in C + DEHP, MZD and MZD + DEHP than in C, while the marker of inhibitory neurons glutamic acid decarboxylase (GAD65) level were similar among groups. The ERK1/2 pathway, crucial to neurogenesis, was affected by MZD and DEHP. ERK1/2 activation was lower, and at a similar extent in C + DEHP and MZD groups than in C, while it was markedly lower in the MZD + DEHP group compared to all other groups. Lower ERK1/2 activation could be due to activation of the ERK1/2 phosphatase 2A (PP2A). We found that PP2A activation was higher, in MZD and DEHP than in C, being highest in the MZD + DEHP group. Conclusions Gestational exposure to DEHP in rats causes a secondary zinc deficiency in the fetal brain and altered neurogenesis. This can be due to the inhibition of the ERK1/2 signaling pathway. DEHP exposure can adversely affect the offspring's brain development and result in irreversible consequences to adult brain structure and function. Funding Sources Supported by grants from NIFA CA-D-XXX-7244-H, Packer-Wentz foundation, NIEHS T 32 training grant (T32 ES 0,07059).

Zinc Status Elicits Age-Dependent Effects in the Gut Microbiome

Objectives Zinc is an essential micronutrient critical for a variety of cellular processes, including immune function. In US, 12% of the population do not consume the EAR for zinc. In older populations the prevalence of inadequate zinc intake increases to ∼40%. Moreover, zinc levels are also often depressed in aged individuals, even when consuming a zinc-adequate diet. Thus, older populations can be highly susceptible to zinc deficiency. Both zinc deficiency and aging are associated with progressive immune dysfunction and chronic inflammation that could be correlated with the promotion of many age-related diseases. Increasing evidence indicates that the interaction among gut microbiota, the immune system, and diet contributes to age-related inflammation. Objectives: The goal of this study is to determine the importance of zinc status and age with respect to composition of the gut microbiome. We hypothesize that age and zinc status are correlated with specific taxa in the gut microbiome. Further, we expect to see additional correlations, both positive and negative, between these significant taxa and markers of host inflammation. Methods We studied the effects of dietary zinc supplementation and marginal zinc deficiency on changes in microbial communities in young and old mice. Young (2 mo) and old (24 mo) C57Bl/6 mice were fed a zinc adequate (30ppm Zn), zinc supplemented (300ppm Zn), or marginal zinc deficient (6 ppm Zn) diet for six weeks. 16S rRNA amplicon sequencing was performed on fecal samples at study start and end; cecal and colon samples at study end. Results Age correlated with overall microbial composition in the gut, according to a PERMANOVA test and a permutation test, regardless of zinc status. Gut microbiome content of young mice had significant overall correlation with zinc status, while the gut microbiome of old mice was not significantly affected by zinc status. Conclusions Age effects on the microbiome are significant and must be considered when studying effects of diet on the host gut microbiome. Zinc status, especially deficiency, elicits a varied effect on the microbiome that is dependent upon host age. Sampling site (i.e., colon, cecum, feces) had a small but significant effect on specific microbial taxa. Funding Sources NIFA, USDA.

Effects of Zinc Status and Aging on Age-Related Immune Dysfunction and Chronic Inflammation

Objectives Aging is associated with progressive immune dysfunction, including impaired adaptive response, increased susceptibility to infection, and reduced vaccination efficacy. Aging is also associated with chronic inflammation that correlated with the promotion of many age-related diseases. Zinc is an essential micronutrient critical for immune function. In US, 12% of the population do not consume the estimated average requirement for zinc. The prevalence of inadequate zinc intake is even higher among older populations, and are at increased risk for marginal zinc deficiency. Effects of zinc deficiency share similarities to age-related immune dysfunction, including impaired adaptive immunity and increased in proinflammatory response. The goal of this study is to understand the effects of zinc status and aging on age-related immune dysfunction and chronic inflammation. We hypothesize that age-related decline in zinc status contributes to immune dysregulation and chronic inflammation in the elderly. Methods We studied the effects of dietary zinc supplementation and marginal zinc deficiency on changes in mucosal immunity and inflammatory response in young and old mice. Young (2 mo) and old (24 mo) C57Bl/6 mice were fed a zinc adequate (ZA, 30 ppm Zn), zinc supplemented (ZS, 300 ppm Zn), or marginal zinc deficient (MZD, 6 ppm Zn) diets for 6 wks. Serum zinc status, cytokines, and naïve/memory T-cell phenotypes, were determined at the end of the study. Results Old mice had reduced zinc and increased proinflammatory cytokines MCP1 and IL6 in the serum, increased Th1/Th17/inflammatory cytokines (IFNγ, IL17, TNFα, respectively) and decreased naïve CD4 T-cells in the mesenteric lymph nodes (MLN). ZS significantly increased serum zinc levels, decreased TNFα, IFNγ, IL17 in MLN, and increased naïve T-cell populations in aged mice. MZD further reduced serum zinc and increased serum IL6 levels in aged mice. Conclusions ZS improved the immune function of aged mice and reduced inflammatory response, and MZD further increased age-related inflammation. Our data suggest that zinc status is an important contributing factor in age-related immune dysfunction and chronic inflammation. Funding Sources NIFA, USDA.

Lessons Learned from Experimental Human Model of Zinc Deficiency

Zinc is an essential element for humans, and its deficiency was documented in 1963. Nutritional zinc deficiency is now known to affect over two billion subjects in the developing world. Conditioned deficiency of zinc in many diseases has also been observed. In zinc-deficient dwarfs from the Middle East, we reported growth retardation, delayed sexual development, susceptibility to infections, poor appetite, and mental lethargy. We never found a zinc-deficient dwarf who survived beyond the age of 25 y. In an experimental model of human mild zinc deficiency, we reported decreased thymulin (a thymopoietic hormone) activity in Th1 cells, decreased mRNAs of IL-2 and IFN-gamma genes, and decreased activity of natural killer cells (NK) and T cytotoxic T cells. The effect of zinc deficiency on thymulin activity and IL-2 mRNA was seen within eight to twelve weeks of the institution of zinc-deficient diet in human volunteers, whereas lymphocyte zinc decreased in 20 weeks and plasma zinc decreased in 24 weeks after instituting zinc-deficient diet. We hypothesized that decreased thymulin activity, which is known to proliferate Th1 cells, decreased the proliferation differentiation of Th1 cells. This resulted in decreased generation of IL-2 and IFN-gamma. We observed no effect in Th2 cell function; thus, zinc deficiency resulted in an imbalance of Th1 to Th2 function resulting in decreased cell-mediated immunity. Zinc therapy may be very useful in many chronic diseases. Zinc supplementation improves cell-mediated immunity, decreases oxidative stress, and decreases generation of chronic inflammatory cytokines in humans. Development of sensitive immunological biomarkers may be more sensitive than an assay of zinc in plasma and peripheral blood cells for diagnosis of marginal zinc deficiency in human.

Zinc Nutritional Status in Patients with Cystic Fibrosis

Background: Zinc is an essential nutrient for all forms of life and its deficiency affects the normal growth and development of human beings. Objective: The main aim was to investigate zinc nutritional status by serum zinc concentration (SZC) and dietary zinc intake and their association in cystic fibrosis (CF) patients. Methods: A cross-sectional study was conducted in CF patients. Anthropometric measurements and respiratory and pancreatic tests were conducted. Hypozincemia was determined by SZC while using atomic absorption spectrophotometry and dietary zinc deficiency by prospective 72-h dietary surveys. Results: Mean SZC (87.2 ± 16.7 μg/dL) and dietary zinc intake (97 ± 26.9% Dietary Reference Intake) were normal. Three of 17 patients with CF (17.6%) had hypozincemia and four (23.5%) had a dietary zinc deficiency. No patient with dietary zinc deficiency had hypozincemia. A positive and significant association was observed between SZC and Z-score of BMI-for-age (p = 0.048) and weight-for-height (p = 0.012) and between dietary zinc intake and energy intake (EI, p = 0.036) and Z-score of weight-for-high (p = 0.029). Conclusion: SZC was associated with the nutritional status, expressed as BMI (Body Mass Index) and weight-for-height Z score, and dietary zinc intake with EI and weight-for-height Z-score. No patient with hypozincemia had dietary zinc deficiency. This situation should alert us to a marginal zinc deficiency and it may explain why there were no overlapping cases between the two groups. We suggest that probably 41% of the cases in this study would be at elevated risk of zinc deficiency and a zinc supplementation may be considered.

Marginal Zinc Deficiency and Environmentally Relevant Concentrations of Arsenic Elicit Combined Effects on the Gut Microbiome

ABSTRACT Extensive research shows that dietary variation and toxicant exposure impact the gut microbiome, yielding effects on host physiology. However, prior work has mostly considered such exposure-microbiome interactions through the lens of single-factor exposures. In practice, humans exposed to toxicants vary in their dietary nutritional status, and this variation may impact subsequent exposure of the gut microbiome. For example, chronic arsenic exposure affects 200 million people globally and is often comorbid with zinc deficiency. Zinc deficiency can enhance arsenic toxicity, but it remains unknown how zinc status impacts the gut microbiome’s response to arsenic exposure and whether this response links to host toxicity. Using 16S amplicon sequencing, we examined the combinatorial effects of exposure to environmentally relevant concentrations of arsenic on the composition of the microbiome in C57BL/6 mice fed diets varying in zinc concentration. Arsenic exposure and marginal zinc deficiency independently altered microbiome diversity. When combined, their effects on microbiome community structure were amplified. Generalized linear models identified microbial taxa whose relative abundance in the gut was perturbed by zinc deficiency, arsenic, or their interaction. Further, we correlated taxonomic abundances with host DNA damage, adiponectin expression, and plasma zinc concentration to identify taxa that may mediate host physiological responses to arsenic exposure or zinc deficiency. Arsenic exposure and zinc restriction also result in increased DNA damage and decreased plasma zinc. These physiological changes are associated with the relative abundance of several gut taxa. These data indicate that marginal zinc deficiency sensitizes the microbiome to arsenic exposure and that the microbiome associates with some toxicological effects of arsenic. IMPORTANCE Xenobiotic compounds, such as arsenic, have the potential to alter the composition and functioning of the gut microbiome. The gut microbiome may also interact with these compounds to mediate their impact on the host. However, little is known about how dietary variation may reshape how the microbiome responds to xenobiotic exposures or how these modified responses may in turn impact host physiology. Here, we investigated the combinatorial effects of marginal zinc deficiency and physiologically relevant concentrations of arsenic on the microbiome. Both zinc deficiency and arsenic exposure were individually associated with altered microbial diversity and when combined elicited synergistic effects. Microbial abundance also covaried with host physiological changes, indicating that the microbiome may contribute to or be influenced by these pathologies. Collectively, this work demonstrates that dietary zinc intake influences the sensitivity of the microbiome to subsequent arsenic exposure.