Liver Metallothionein Research Articles

Combining Native Mass Spectrometry and Proteomics to Differentiate and Map the Metalloform Landscape in Metallothioneins.

Within the intricate landscape of the proteome, approximately 30% of all proteins bind metal ions. This repertoire is even larger when considering all the different forms of a protein, known as proteoforms. Here, we propose the term "metalloforms" to refer to different structural or functional variations of a protein resulting from the binding of various hetero- or homogeneous metal ions. Using human Cu(I)/Zn(II)-metallothionein-3 as a representative model, we developed a chemical proteomics strategy to simultaneously differentiate and map Zn(II) and Cu(I) metal binding sites. In the first labeling step, N-ethylmaleimide reacts with Cysteine (Cys), resulting in the dissociation of all Zn(II) ions while Cu(I) remains bound to the protein. In the second labeling step, iodoacetamide is utilized to label Cu(I)-bound Cys residues. Native mass spectrometry (MS) was used to determine the metal/labeling protein stoichiometries, while bottom-up/top-down MS was used to map the Cys-labeled residues. Next, we used a developed methodology to interrogate an isolated rabbit liver metallothionein fraction containing three metallothionein-2 isoforms and multiple Cd(II)/Zn(II) metalloforms. The approach detailed in this study thus holds the potential to decode the metalloproteoform diversity within other proteins.

Regulation of Inorganic Zinc Supplementation on Intestinal Absorption, Metabolism, and Muscle Development in Broilers Fed Low-Protein Diets

The issue of nitrogen fertilizer contamination resulting from high-protein diets can be effectively solved by adopting low-protein diets. The objective of this study was to investigate the effects of inorganic zinc supplementation in low-protein diets on 42-day-old broilers across a multitude of parameters. To determine the optimal dose of inorganic zinc in broiler diets with a 1.5% reduction in crude protein, 1-day-old Arbor Acres broilers (n = 270) were randomly assigned to five groups, each containing 54 broilers. Our results revealed that inorganic zinc supplementation at levels of 130 mg/kg elevated growth performance and carcass traits (p < 0.05). It also significantly increased the ratio of intestinal villi heights to crypt depths (p < 0.001), changed intestinal morphology, and significantly increased albumin content in serum (p < 0.05). Furthermore, analysis of mRNA expression showed that 130 mg/kg and 150 mg/kg of inorganic zinc improved the myogenic differentiation involved in muscle development, as well as intestinal tight junction and liver metallothionein capacity (p < 0.001). Additionally, these groups exhibited lower zinc excretion compared with other treatments (p < 0.001). In summary, our findings suggest that inorganic zinc supplementation in low-protein diets holds the potential to support muscle and intestinal development in broilers, presenting a viable nutritional strategy.

Toxicological effects of copper on bioaccumulation and mRNA expression of antioxidant, immune, and apoptosis-related genes in Chinese striped-necked turtle (Mauremys sinensis).

Heavy metals are among the most ubiquitous environmental pollutants of recent decades. Copper is commonly used to control algal blooms or macrophyte and waste infestations, its ambient concentration has increased significantly, indicating possible environmental risk. To investigate the effects of copper exposure on bioaccumulation, antioxidant defense, immune response, and apoptosis in the Chinese Striped-necked Turtle Mauremys sinensis, three experimental groups, control (0.0mg/L), Cu2 (2mg/L) and Cu4 (4mg/L) were designed, and sampled at 14 and 28days. Results showed that copper accumulates in different organs depending on the concentration and exposure time, Liver > Kidney > Gut > Heart > Brain > Muscle and the time order was 28days > 14days. The liver enzymes AST, ALT, and ALP decreased when the turtles were exposed to copper stress, while the contents of bilirubin TBIL, DBIL, IBIL, and LDH showed a significant upward trend. Similarly, the mRNA expression level of acetylcholinesterase AChE in the brain was significantly downregulated upon copper exposure. An upward trend was noticed in the liver Metallothionein MT mRNA expression levels compared to the control group. The mRNA expression levels of antioxidant enzymes CAT, SOD, MnSOD, and GSH-PX1 in the liver increased initially and then significantly decreased. Furthermore, the relative mRNA expression levels of inflammatory cytokines IL-1β, IL-8, TNF-α, and IFN-γ involved in inflammatory response significantly upregulated. Copper significantly increased the hepatic mRNA transcription of heat shock proteins HSP70 and HSP90 at different exposure durations. In addition, the relative mRNA levels of caspase3, caspase8, and caspase9 related to the caspase-dependent apoptotic pathway significantly increased under copper stress. These results explain that copper toxicity causes bioaccumulation, promotes oxidative stress, obstructs immunity, and induces inflammation and apoptosis by altering their gene expression levels in M. sinensis.

Chronic Corticosterone Exposure Suppresses Copper Transport through GR-Mediated Intestinal CTR1 Pathway in Mice.

Numerous studies have discovered that chronic stress induces metabolic disorders by affecting iron and zinc metabolism, but the relationship between chronic stress and copper metabolism remains unclear. Here, we explore the influence of chronic corticosterone (CORT) exposure on copper metabolism and its regulatory mechanism in mice. Mice were treated with 100 μg/mL CORT in drinking water for a 4-week trial. We found that CORT treatment resulted in a significant decrease in plasma copper level, plasma ceruloplasmin activity, plasma and liver Cu/Zn-SOD activity, hepatic copper content, and liver metallothionein content in mice. CORT treatment led to the reduction in duodenal expression of copper transporter 1 (CTR1), duodenal cytochrome b (DCYTB), and ATPase copper-transporting alpha (ATP7A) at the mRNA and protein level in mice. CORT treatment activated nuclear glucocorticoid receptor (GR) and down-regulated CRT1 expression in Caco-2 cells, whereas these phenotypes were reversible by an antagonist of GR, RU486. Chromatin immunoprecipitation analysis revealed that GR bound to the Ctr1 promoter in Caco-2 cells. Transient transfection assays in Caco-2 cells demonstrated that the Ctr1 promoter was responsive to the CORT-activated glucocorticoid receptor, whereas mutation/deletion of the glucocorticoid receptor element (GRE) markedly impaired activation of the Ctr1 promoter. In addition, CORT-induced downregulation of Ctr1 promoter activity was markedly attenuated in Caco-2 cells when RU486 was added. These findings present a novel molecular target for CORT that down-regulates intestinal CTR1 expression via GR-mediated trans-repression in mice.

Comparison of zinc bioavailability in zinc-glycine and zinc-methionine chelates for broilers fed with a corn-soybean meal diet.

The objective of this study was to compare the bioavailability of zinc (Zn) from zinc-glycine (Zn-Gly) and zinc-methionine (Zn-Met) as compared with zinc sulfate (ZnSO4) used as a standard in broilers. A total of 1,200 one-day-old male broilers (Cobb 500) were randomly allotted to one of 10 treatments with eight replicate cages of 15 birds each. The broilers were fed a corn-soybean meal basal diet (containing 26.46mg Zn/kg; control) or the basal diet added with 40, 80, and 120mg Zn/kg as Zn-Gly, Zn-Met, or ZnSO4 for 14days. The relative bioavailability value (RBV) was calculated based on multiple linear regression slope ratios of Zn concentrations in tibia and pancreas, pancreas metallothionein (MT) concentration, and pancreas MT mRNA abundance on added Zn intake. When comparing the control with all Zn-supplemented treatments, Zn addition did not significantly affect average feed intake and bodyweight gain during days 1-14 (p > 0.10). However, Zn concentrations in the tibia, pancreas, and liver and pancreas MT concentration and MT mRNA abundance increased in all Zn-supplemented treatments compared with the control (p < 0.05), and these indices increased linearly (p < 0.001) with increasing added Zn levels on days 7 and 14. The RBV of Zn as Zn-Met was similar to that as Zn-Gly or ZnSO4 (p > 0.40) on days 7 and 14, based on tibia and pancreas Zn. In contrast, on days 7 and 14, the RBVs of Zn were in the following order: Zn-Met > Zn-Gly > ZnSO4 (p < 0.05), based on pancreas MT concentration. The bioavailable Zn from Zn-Met was 1.20 or 1.25 times that from Zn-Gly on day 7 or 14, respectively, evaluated by pancreas MT content. The RBV of Zn as Zn-Met was similar to that as Zn-Gly or ZnSO4 on day 7, whereas it was higher than that as Zn-Gly or ZnSO4 on day 14, based on pancreas MT mRNA abundance. In conclusion, Zn-Met had higher bioavailable Zn than Zn-Gly for the starter broilers fed with the corn-soybean meal diet, using pancreas MT concentration as the response criterion.

An integrated biomarker approach: Non-monotonic responses to cadmium exposure in the suckermouth catfish Hypostomus plecostomus

Environmental risk assessment in aquatic ecosystems typically uses biomarkers to detect interactions between potential hazards and biological systems. Next to knowing environmental contaminant levels in tissues and the environment, it is important to link to potentially deleterious effects at higher levels of biological organization such as biochemistry, physiology, and overall health status. In this laboratory study we assessed the toxicity of waterborne cadmium (Cd) over an exposure range of 0 - 100 µg l-1 for nine days to the loricariid suckermouth catfish Hypostomus plecostomus. We evaluated the integrated response of the fish at the biochemical to physiological level by means of a suite of tissue biomarkers of exposure and effects, including Cd concentrations in gills, liver metallothioneins (MT) and cholinesterase activity (ChE) in brain, before and after the inhibition of the alkaloid eserine, as well as whole-fish resting oxygen consumption rates and ingestion rate. Tissue biomarkers (MT and ChE) showed a non-monotonic relationship, with maximum/minimum responses at intermediate doses. i.e. 10 and 50 µg l-1, whereas biomarker responses of fish exposed at 100 µg l-1 more closely resembled biomarker responses seen at lower concentrations (< 10 µg l-1). Conversely, the oxygen consumption rate peaked at 100 µg l-1, suggesting a higher metabolic cost for higher metal exposure, with no significant correlation with fish body condition and food intake. Integrated Biomarker Response (IBR) values peaked at the intermediate exposure concentration of 50 µg l-1 Cd. The non-monotonic dose-response of the biochemical biomarkers of exposure, together with the higher metabolic rates of fish exposed to 50 - 100 µg l-1 of Cd and the non-significant effects on the more relevant physiological and histological variables suggests that H. plecostomus is capable of biochemically and physiologically regulating moderately high Cd concentrations, thus representing a suitable indicator organism to monitor metal pollution by Cd.

Broiler responses to copper levels and sources: growth, tissue mineral content, antioxidant status and mRNA expression of genes involved in lipid and protein metabolism

BackgroundFive hundred 8-d old male broilers Cobb500 were randomly allotted into 10 treatments in factorial arrangement with 5 Cu levels (0, 4, 8, 12, and 16 mg/kg), and 2 sources (Cu proteinate, CuPro and Cu sulphate, CuSO4.5H2O) for a 10-d-experiment.ResultsFeed conversion ratio (FCR) was better (P < 0.05) in CuPro fed chicks compared with CuSO4.5H2O group. Average daily feed intake (ADFI) decreased linearly (P < 0.05) as dietary Cu increased. A quadratic response (P < 0.05) to Cu levels was found for FCR, being optimized at 9.87 and 8.84 mg Cu/kg in CuPro and CuSO4.5H2O diets, respectively. Copper supplementation linearly increased liver Cu content (P < 0.05) and tended to linearly increase (P = 0.07) phosphorus (P) and copper in tibia. Manganese and zinc were higher (P < 0.05) in tibia of CuPro fed birds. Broilers fed CuPro exhibited lower liver iron (P < 0.05) content, lower activities of Cu, Zn superoxide dismutase (CuZnSOD) in breast muscle and liver, and glutathione peroxidase in liver. Glutathione peroxidase reduced linearly (P < 0.05) with CuPro levels and increased linearly (P < 0.05) with CuSO4.5H2O levels and were lower (P < 0.05) in all CuPro levels in breast muscle. Breast muscle malondialdehyde concentration tended to be higher (P = 0.08) in broilers fed CuSO4.5H2O. Copper levels linearly increased (P < 0.05) metallothionein (MT) and malate dehydrogenase (MDH) expression in liver, and six-transmembrane epithelial antigen of the prostate-1 (STEAP-1) in the intestine. Copper elicited a quadratic response (P < 0.050) in AKT-1 and mammalian target of rapamycin (mTOR) in breast muscle, CuZnSOD in liver and antioxidant 1 copper chaperone (ATOX 1) in intestine. Broilers fed CuPro exhibited higher mRNA expression of mTOR in muscle breast and lower CuZnSOD in liver and ATOX 1 in intestine. Interaction (P < 0.05) between levels and sources was found in mRNA expression for GSK-3β, MT, and CuZnSOD in breast muscle, FAS and LPL in liver and MT and CTR1 in intestine.ConclusionsCuPro showed beneficial effects on feed conversion and bone mineralization. Organic and inorganic Cu requirements are 9.87 and 8.84 mg Cu/kg, respectively.

Effects of ethyl acetate extract of Moringa oleifera leaf and vitamin c co-exposures on acute cadmium chloride-induced toxicopathological changes in Clarias gariepinus

Cadmium (Cd2+) is reportedly one of the most toxic metals, which is increasingly polluting our aquatic environments. The need to modify Cd2+ toxicity led to the investigation of the possible effects of ethyl acetate extract of Moringa oleifera (M. oleifera) leaf (EAEMOL) and vitamin C on cadmium chloride (CdCl2) co-exposures of adult Clarias gariepinus over a 96-hour period. The exposure involved 105 fish in five groups of seven fish each in triplicates. Group I contained the control unexposed fish, Group II contained Tween 80, and Groups III-V contained CdCl2, EAEMOL co-exposure, and the vitamin C co-exposure, respectively. A total of 52.38%, 28.57%, and 47.62% mortalities occurred in Group III, IV, and V exposed fish, respectively, with marked deterioration in fish water quality over the exposure period. Although CdCl2 exposure caused considerable lipid peroxidation, the EAEMOL and vitamin C co-exposures insignificantly (p>0.05) aggravated it rather than ameliorate it. Similarly, the co-exposures slightly elevated the recorded drop in liver metallothionein (MT) level induced by the CdCl2 exposure. Although CdCl2 exposure caused severe gill damage, only moderate gill damages occurred in both co-exposures based on the degree of tissue change protocol. Therefore, the EAEMOL and vitamin C co-exposures, as administered, ameliorated the CdCl2 toxicity in the exposed fish based on the recorded mortality and gills cellular damage as the induced biochemical changes were insignificant (p >0.05). However, the rationale behind the recorded slightly elevated serum lipid peroxidation and liver MT levels by the co-exposures need further elucidation.

Gestational Cd Exposure in the CD-1 Mouse Sex-Specifically Disrupts Essential Metal Ion Homeostasis.

In CD-1 mice, gestational-only exposure to cadmium (Cd) causes female-specific hepatic insulin resistance, metabolic disruption, and obesity. To evaluate whether sex differences in uptake and changes in essential metal concentrations contribute to metabolic outcomes, placental and liver Cd and essential metal concentrations were quantified in male and female offspring perinatally exposed to 500 ppb CdCl2. Exposure resulted in increased maternal liver Cd+2 concentrations (364 µg/kg) similar to concentrations found in non-occupationally exposed human liver. At gestational day (GD) 18, placental Cd and manganese concentrations were significantly increased in exposed males and females, and zinc was significantly decreased in females. Placental efficiency was significantly decreased in GD18-exposed males. Increases in hepatic Cd concentrations and a transient prenatal increase in zinc were observed in exposed female liver. Fetal and adult liver iron concentrations were decreased in both sexes, and decreases in hepatic zinc, iron, and manganese were observed in exposed females. Analysis of GD18 placental and liver metallothionein mRNA expression revealed significant Cd-induced upregulation of placental metallothionein in both sexes, and a significant decrease in fetal hepatic metallothionein in exposed females. In placenta, expression of metal ion transporters responsible for metal ion uptake was increased in exposed females. In liver of exposed adult female offspring, expression of the divalent cation importer (Slc39a14/Zip14) decreased, whereas expression of the primary exporter (Slc30a10/ZnT10) increased. These findings demonstrate that Cd can preferentially cross the female placenta, accumulate in the liver, and cause lifelong dysregulation of metal ion concentrations associated with metabolic disruption.

Effects of Moringa oleifera leaves extract, vitamin C, and taurine co-exposures on calcium and metallothionein levels, oxidative stress, and gill histopathological changes in Clarias gariepinus exposed to sub-lethal cadmium

Cadmium aquatic environmental pollution poses great threats to fish and their would-be consumers. The present work investigated the effects of ethyl acetate extract of Moringa oleifera leaves (EAEMOL), vitamin C, and taurine co-exposures on calcium and metallothionein levels, oxidative stress, and gill histopathological changes in Clarias gariepinus exposed to sub-lethal cadmium (CdCl2) for 28 days. Fish were exposed to CdCl2 only (1.048 mg/L) as well as co-exposed with EAEMOL (20 mg/L), vitamin C (5 mg/L), and taurine (5 mg/L) separately. There was significant (p < 0.05) deterioration in fish water quality with increasing exposure period but no significant (p > 0.05) changes occurred between the exposed groups. However, the co-exposure of EAEMOL, vitamin C, and taurine did not significantly (p > 0.05) improve the CdCl2-induced fish water quality deterioration. Sub-lethal exposure to CdCl2 only caused significant (p < 0.05) increase in the serum malondialdehyde (MDA) and liver metallothionein (MT) levels with significant (p < 0.05) decrease in serum catalase activity only. However, EAEMOL, vitamin C, and taurine co-exposures did not significantly (p > 0.05) improve the MDA, superoxide dismutase, catalase, and glutathione activities, as well as MT and calcium (Ca2+) levels, condition factor (CF), hepatosomatic index (HSI), and gill histopathological changes induced by the CdCl2 exposure. Similarly, none of the present exposures, CdCl2 only or its co-exposures with EAEMOL, vitamin C, and taurine significantly (p > 0.05) altered the normal functioning of the gills despite the observed histopathological changes based on the degree of tissue change protocol. Therefore, EAEMOL, vitamin C, and taurine co-exposures, as administered in the present case, did not considerably alter the physicochemical parameters of the experimental fish water. However, outside significantly (p < 0.05) increasing MDA level, EAEMOL, vitamin C, and taurine co-exposures did not significantly (p > 0.05) improve the CdCl2-induced Ca2+, MT, CF, HSI, and gill histopathological alterations by sub-lethal CdCl2 exposure of C. gariepinus.

Co-administration of zinc for treating and preventing arsenism in common carp Cyprinus carpio: An alternative to avoid physiological and cellular damages

Arsenic (As) contamination in water bodies has adverse effects on aquatic organisms. Zinc (Zn), as an important enzyme cofactor and efficient reactive oxygen species scavenger, plays an indispensable role in cellular homeostasis and biological functions. However, there has been less investigation on AsZn interaction in common carp (Cyprinus carpio). We hypothesize that Zn is a candidate for the treatment and prevention of arsenism. In general, feed efficiency, growth performance and organ indices did not significantly differ between the As or/and Zn group and the control groups (p > 0.05). However, arsenite significantly altered the intestinal morphological features and suppressed intestinal tight junction proteins (claudins, occluding, and zonula occludens) as indicated by mRNA and protein levels. Moreover, oxidative stress (malondialdehyde and organic 8-hydroxy-2-deoxyguanosine), inflammatory (nuclear factor kappa-B and pro-inflammatory cytokines) and detoxification parameters (metallothionein) in intestine and liver increased following arsenite exposure (p < 0.05), concomitant with innate immunity suppression (C4 and IgM; p < 0.01). These anomalies led to abnormal lipid metabolism (SREBP-1c pathway; p < 0.01) and endoplasmic reticulum stress (PERK-eIF2α, IRE1α and ATF6; p < 0.01). Overall, As impaired general physiological and cellular functions, and compromised the immune system of carp. Zn2+ restored these alterations, as evidenced by the reduction in inflammation and endoplasmic reticulum stress via the activation of the antioxidant system. Thus, Zn supplementation can be used to treat and prevent arsenism; it is a useful alternative to prevent physiological and cellular damage in the inland culture of carp.

Comparison of Thymulin Activity with Other Measures of Marginal Zinc Deficiency.

Activity of the immunoregulatory peptide thymulin reflects differences in zinc status. This study compared thymulin activity with four other zinc status measures in rats fed zinc at either 5 or 25ppm. Rats fed the lower zinc showed the following results compared with rats with adequate zinc intake: serum thymulin activity 61% lower, serum zinc 31% lower, serum extracellular superoxide dismutase 18% lower, serum 5'-nucleotidase activity 26% lower, and liver metallothionein 28% lower. Thus, thymulin activities showed more sensitivity to restricted zinc intake than did four other parameters.

Type I Interferons Ameliorate Zinc Intoxication of Candida glabrata by Macrophages and Promote Fungal Immune Evasion.

SummaryHost and fungal pathogens compete for metal ion acquisition during infectious processes, but molecular mechanisms remain largely unknown. Here, we show that type I interferons (IFNs-I) dysregulate zinc homeostasis in macrophages, which employ metallothionein-mediated zinc intoxication of pathogens as fungicidal response. However, Candida glabrata can escape immune surveillance by sequestering zinc into vacuoles. Interestingly, zinc-loading is inhibited by IFNs-I, because a Janus kinase 1 (JAK1)-dependent suppression of zinc homeostasis affects zinc distribution in macrophages as well as generation of reactive oxygen species (ROS). In addition, systemic fungal infections elicit IFN-I responses that suppress splenic zinc homeostasis, thereby altering macrophage zinc pools that otherwise exert fungicidal actions. Thus, IFN-I signaling inadvertently increases fungal fitness both in vitro and in vivo during fungal infections. Our data reveal an as yet unrecognized role for zinc intoxication in antifungal immunity and suggest that interfering with host zinc homeostasis may offer therapeutic options to treat invasive fungal infections.

Effects of corn supplementation on the antioxidant activity, selected minerals, and gene expression of selenoprotein and metallothionein in serum, liver, and kidney of sheep-fed palm kernel cake: urea-treated rice straw diets.

This study aimed to determine influence of corn inclusion on glutathion peroxidase (GPx) activity, selected minerals concentration, and gene expression in sheep-fed palm kernel cake (PKC) and urea-treated rice straw. Twenty-seven of Dorper sheep were divided into three groups and fed a basal diet of (20% rice straw and 80% concentrate) with addition of ground corn at either 0% (T1), 5% (T2), or 10% (T3), respectively. After 120days feeding trial, all animals were slaughtered and tissue samples of kidney, liver, and muscles were taken for enzyme and mineral analyses. The results showed that Cu concentration in the liver was lower treatment T3 compared to the control and T2. The serum activity of GPx was higher in T2 than in T3 at day 120 of experiment. Serum malondialdehyde (MDA) concentrations decreased at day 80 in sheep on T3, whereas MDA of liver increased linearly with increasing corn supplementation. The qRT-PCR analyses revealed significant up-regulation of ATP7A and MIa genes in T3, while hepatic Cu/Zn SOD, GPx1, and GPx4 mRNA showed a higher expression in lamb hepatocytes in T3 compared to those on T1. Present study results suggest that feeding PKC as basal diet can increase antioxidant activity, but cause liver dysfunction in sheep. Inclusion corn was found to regulate transcriptional levels of the GPx family and metallothionein genes. These genes may play a role in the antioxidant protection response and reduce incidence of toxicity associated with Cu.

A multibiomarker approach for evaluating environmental contamination: Common carp (Cyprinus carpio) transplanted along a gradient of metal pollution

Environmental monitoring and risk assessment approaches which include a more holistic view on the effects of pollutants on biota are increasingly sought by regulators and policy makers. Therefore, caged carp juveniles (Cyprinus carpio) were transplanted for 7 weeks along a known Cd and Zn pollution gradient. Metal (Cu, Cd and Zn) accumulation in gill and liver tissue and effect biomarkers (growth, condition factor (CF), hepatosomatic index (HSI), oxygen consumption, swimming capacity, Na+/K+-ATPase activity (NKA) and metallothionein (MT) levels) were compared.Up to 10-fold higher cadmium concentrations were measured in the gills of the fish at the most polluted locations compared to the laboratory control fish. Similarly, cadmium concentrations in liver tissues of field-exposed fish were significantly higher than those measured in laboratory control fish. Cu and Zn concentrations in the gills were not significantly different between field-exposed and control organisms, whereas higher levels in liver tissues were measured in carps deployed in some locations. Effects on liver MT levels were up to 10 times greater for organisms exposed to the field, whereas no clear effect of the metal exposure on NKA in the gill tissue was observed. A decrease in muscle glycogen stores was observed for all organisms deployed in the field, while liver glycogen levels decreased only in fish exposed to two of the 5 sites compared to the laboratory control fish. Additionally, significant drops in liver protein- and lipid stores were observed. No effect on oxygen consumption rates and swimming capacity was observed. The CF and HSI of caged fish reflected the pollution gradient in the river and considerable loss of weight was observed for fish transplanted in the most polluted site. Overall, this active biomonitoring study successfully revealed differences in metal accumulation, physiological and organismal endpoints as a direct consequence of field exposure.

Effects of organic zinc on tibia quality, mineral deposit, and metallothionein expression level of aged hens

ABSTRACTThe study aimed to determine the effects of methionine hydroxy analog chelate zinc on the tibia quality, mineral deposit, apparent retention of nutrients, and liver metallothionein (MT) expression level of aged laying hens. A total of 960 layers (Hy-Line Grey, 57 wk old) were randomly assigned into 4 groups, and each group had 8 replicates of 30 hens. During the first 2 wk, groups were fed a basal diet without extra zinc (Zn: 35.08 mg/kg). During the ensuing 14 wk, 4 levels of Zn (inorganic Zn: 80 mg/kg; organic Zn: 20, 40, 80 mg/kg) were added to the diet. The results indicated that both the Zn source and level did influence tibia strength and calcium (Ca) and Zn concentrations of tibia (P < 0.05), whereas there were no differences in the copper (Cu) and phosphorus (P) concentrations of the tibia and the tibia length (P > 0.05). Moreover, dietary supplementation with 40 or 80 mg/kg of organic Zn showed higher Zn and Ca concentrations in the tibia and higher tibia strength. The Cu concentration in the liver showed no difference among the 4 treatments, whereas the Zn concentration in the liver increased with the increasing Zn level. The apparent retention of P, iron (Fe), and manganese (Mn) was not affected by the Zn level or source (P > 0.05). However, the organic Zn group increased the apparent retention of Cu, Zn, Ca, crude protein (CP), and energy, and the group supplemented with 40 or 80 mg/kg of organic Zn obtained significant effects (P < 0.05). Moreover, dietary supplementation with 40 or 80 mg/kg organic Zn increased the MT mRNA expression of the liver at week 72, whereas 20 mg/kg of organic Zn decreased it (P < 0.05). In conclusion, this study suggested that an optimum dietary (40 mg/kg) organic Zn level plays a key role in promoting the apparent retention of minerals and nutrients, trace element deposit, and MT mRNA expression.

Effect of supplementation of nicotinamide and sodium butyrate on the growth performance, liver mitochondrial function and gut microbiota of broilers at high stocking density.

This study was conducted to investigate the effects of stocking density and dietary nicotinamide (NAM) and butyrate sodium (BA) supplementation on the growth performance, liver mitochondrial function and gut microbiota of broilers at high stocking density. A total of 342, 26-d-old Cobb500 broilers were divided into 5 groups with 6 replicates. Treatments were as follows: (a) Low stocking density (L, 9 birds per cage); (b) High stocking density (H, 12 birds per cage); (c) H + 50 ppm NAM; (d) H + 500 ppm BA; (e) H + 50 ppm NAM + 500 ppm BA (COMB). The results showed that high stocking density significantly reduced the feed intake and body weight gain of broilers, while COMB improved the growth performance at high stocking density. High stocking density significantly reduced the liver metallothionein content, liver mitochondrial membrane potential and the activities of Na+K+-ATPase and Ca2+Mg2+-ATPase. In contrast, the liver metallothionein contents in the NAM, BA and COMB fed group were higher than those in the H group. COMB increased the activity of ATPase as well, but it failed to enhance the mitochondrial membrane potential. Stocking density also affected gut microbiota of broilers. The high-density group increased the relative abundance of Blautia. Supplementation of BA and NAM increased the abundance of Lactobacillus and Bifidobacteria, respectively. In conclusion, a combination of NAM and BA can improve the performance, antioxidant capacity, mitochondrial function and intestinal microbiota of broilers at high stocking density.

Involvement of metallothionein, homocysteine and B-vitamins in the attenuation of arsenic-induced uterine disorders in response to the oral application of hydro-ethanolic extract of Moringa oleifera seed: a preliminary study

The painful invasive chelation therapy makes it challenging to continue the prolonged treatment against arsenic toxicity. Hence, the significance of the present preliminary investigation was to explore a noninvasive treatment strategy against sodium arsenite (As3+) by the use of a hydroethanolic extract of Moringa oleifera (MO) seed. Arsenic treatment (10 mg/kg body-weight) in animals showed significant level of oxidative stress as evidenced by increased serum levels of malondialdehyde (MDA), conjugated dienes (CD) and reduced level of non-protein thiol (NPSH). A significant diminution in the activities of enzymatic antioxidants was noted in As3+-treated rats. As3+ treatment showed a lengthy phase of metestrous in animals followed by significantly diminished ovarian steroidogenesis, increased ovarian follicular degeneration and distortion of uterine tissue histomorphology. In addition, there was a significant depletion of Vitamin-B9 (folate) and B12 following As3+ ingestion. The levels of circulating TNF-α, homocysteine (Hcy), uterine-IL-6, and liver metallothionein (MT-1) were significantly elevated in arsenic treated rats. MO at a dose of 100 mg/kg body-weight could successfully mitigate the uterine ROS generation by maintaining the uterine antioxidant status in As3+- treated rats. This seed extract prevented the deterioration of As3+-mediated ovarian-steroidogenesis and ovarian and uterine histoarchitecture significantly. B9 and B12 levels were also improved following the ingestion of the MO extract in arsenicated animals. Elevation of Hcy, TNF-α and IL-6 was also prevented by this MO seed extract in As3+-treated rats. A further increase of MT-1 level was achieved after MO ingestion in As3+-treated rats. Here, the alleviation of arsenic toxicity might involve via the regulation of the components of S-adenosine methionine (SAM) pool and MT-1.

Biliary and hepatic metallothionein, metals and trace elements in environmentally exposed neotropical cichlids Geophagus brasiliensis

One of the metal detoxifying mechanisms that occurs in fish is metallothionein (MT) induction and metal binding. Hepatic MT induction has been well described, but biliary MT metal detoxification has only recently been described in fish. In this scenario, metal-metal interactions have been increasingly evaluated to further understand the behavior of these contaminants regarding homeostasis and biological functions, as well as their toxic effects. Studies, however, have been mainly conducted concerning the elemental pair Se-Hg, and scarce reports are available concerning other metal pairs. Therefore, this study aimed to evaluate biliary and hepatic MT metal detoxification mechanisms in a territorial neotropical cichlid, Geophagus brasiliensis. Fish were sampled from the anthropogenically impacted estuarine Rodrigo de Freitas Lagoon, located in Southern Rio de Janeiro, and trace elements and MT were determined by inductively coupled plasma mass spectrometry (ICP-MS) and UV-Vis spectrophotometry, respectively, in fish liver and bile. MT in bile were significantly lower than in liver. Significant differences between bile and liver were observed for many trace elements, and, although most were higher in liver, Cd and Ni were significantly higher in bile, indicating efficient excretion from the body via the biliary route. A significant correlation was observed between MT and Fe in bile, and between MT in liver and Cu and Zn in bile. Molar ratio calculations demonstrated protective elements effects against Al, As, Cd, Hg, Pb and V in both bile and liver, as well as some novel interrelationships, indicating the importance of these investigations regarding the elucidation of element detoxifying mechanisms. Furthermore, investigation of other elemental associations may aid in decision-making processes regarding environmental contamination scenarios linked to public health.

Metallothionein is elevated in liver and duodenum of Atp7b(\u2212/\u2212) mice

Different mutations in the copper transporter gene Atp7b are identified as the primary cause of Wilson’s disease. These changes result in high copper concentrations especially in the liver and brain, and consequently lead to a dysfunction of these organs. The Atp7(−/−) mouse is an established animal model for Wilson’s disease and characterized by an abnormal copper accumulation, a low serum oxidase activity and an increased copper excretion in urine. Metallothionein (MT) proteins are low molecular weight metal-binding proteins and essential for the zinc homeostasis but also play a role for the regulation of other metals, e.g. copper. However the molecular mechanisms of MT in regard to Atp7b remain still elusive. In this study we investigate the expression of MT in the liver and duodenum of Atp7b(−/−) mice and wildtype mice. Hepatic and duodenal expression of MT was measured by real-time reverse transcription-polymerase chain reaction and post-translational expression was analyzed by immunoblot and immunofluorescence. Expression of MT in liver und duodenum was significantly higher in Atp7b(−/−) mice than in controls. Hepatic and duodenal copper, iron and zinc content were also studied. Compared to control hepatic copper and iron content was significantly higher while hepatic zinc content was significantly lower in Atp7b(−/−) mice. In the duodenum copper and zinc content of Atp7b(−/−) mice was significantly lower than in controls. Duodenal iron content was also lower in Atp7b(−/−) mice, but did not reach statistical significance. The results of our study suggest that metallothionein is elevated in the liver and duodenum of Atp7b(−/−) mice.