An Assessment of MT1A (rs11076161), MT2A (rs28366003) and MT1L (rs10636) Gene Polymorphisms and MT2 Concentration in Women with Endometrial Pathologies.

Several studies have indicated a relationship between metallothionein (MT) polymorphisms and the development of different pathologies, including neoplastic diseases. However, no studies thus far have been conducted on the influence of MT polymorphisms and the development of endometrial lesions, including endometrial cancer. This study included 140 patients with normal endometrial tissue, endometrial polyps, uterine myomas and endometrial cancer. The tissue MT2 concentration was determined using the ELISA method. MT1A, MT2A and MT1L polymorphisms were analyzed using TaqMan real-time PCR genotyping assays. We found no statistical difference between the tissue MT2 concentration in patients with EC vs. benign endometrium (p = 0.579). However, tissue MT2 concentration was significantly different between uterine fibromas and normal endometrial tissue samples (p = 0.019). Menopause status did not influence the tissue MT2 concentration (p = 0.282). There were no significant associations between the prevalence of MT1A, MT2A and MT1L polymorphisms and MT2 concentration. The age, menopausal status, and diabetes status of patients were identified as EC risk factors.

Subacute safety assessment of recombinant Lactococcus lactis on the gut microbiota of male Sprague-Dawley rats.

BACKGROUND: Lactococcus lactis strain pGSMT/MG1363 is a genetically modified microorganism (GMM) that constitutively expresses human metallothionein-I fusion protein to combine with intracellular lead. Unlike traditional probiotics, pGSMT/MG1363 lacks a history of safe use in food. Administration of microorganism could influence the gut microbial community and consequently confer health benefits or cause disadvantages to the host. To date, little has been done to assess the influence of recombinant strain pGSMT/MG1363 on the stability of gut microbiota. RESULTS: Liver, testis and kidney sections of male Sprague-Dawley rats orally administered pGSMT/MG1363 for 6 weeks showed normal structure and no pathological damage. There were no adverse effects on the analyzed serum biochemical parameters between the pGSMT/MG1363 group and the MG1363 group. Principal coordinate analysis showed that, compared with the MG1363 group, the 6-week-old fecal gut microbiota of rats fed with pGSMT/MG1363 was not significantly different (Adonis, P = 0.802). pGSMT/MG1363 treatment for 6 weeks did not significantly change the relative abundance of gut microbiota at the phylum and genus levels in comparison with MG1363 treatment. CONCLUSION: Compared to the non-GM strain MG1363 group, administration of the recombinant strain pGSMT/MG1363 for 6 weeks showed no adverse effects on the analyzed physiological parameters and gut microbial compositions of male Sprague-Dawley rats. The results suggested that, in terms of gut microbiota stability, pGSMT/MG1363 could be considered as safe as MG1363, at least for short-term intake. Further toxicological evaluations still need to be considered before drawing a definite conclusion concerning the safe use of pGSMT/MG1363. © 2021 Society of Chemical Industry.

Growth performance, tissue precipitation, metallothionein and cytokine transcript expression and economics in response to different dietary zinc sources in growing rabbits.

The impact of different dietary zinc sources on the growth, serum metabolites, tissue zinc content, economics and relative expression of cytokine and metallothionein genes was evaluated in this study. A total of 120 35-day-old male New Zealand White (NZW) rabbits were randomly distributed into four dietary experimental groups with 10 replicates per group and 3 animals per replicate. The control group was fed basal diet with a Zn-free vitamin-mineral premix; the other three groups received control basal diet supplemented with 50 mg/kg level with zinc oxide (ZnO; as inorganic source), Zn-methionine (Zn-Met; as organic source) and zinc oxide nanoparticles (nano-ZnO). The results indicated that Zn-Met and nano-ZnO groups significantly improved body weight, daily weight gain (DWG), feed conversion ratio (FCR) and nutrient digestibility, as well as decreased mortality, compared to ZnO and control groups. Zn-Met and nano-ZnO significantly reduced serum total cholesterol but did not affect serum proteins and liver function. Nano-ZnO supplemented group also recorded the highest value of serum alkaline phosphatase (ALP), insulin-like growth factor (IGF-1) and lysozymes compared to other groups. Nano-ZnO supplementation had increased hepatic Zn and Cu content and decreased faecal Zn content. Also nano-ZnO group recorded higher expression levels of genes encoding for metallothionein I and metallothionein II, interleukin-2 and interferon-γ in the liver of rabbits. The findings of this study demonstrated zinc nanoparticles, and organic zinc supplementation had improved growth performance and health status of growing rabbits than inorganic zinc oxide.

Quantitative assessment of the association of polymorphisms in the metallothionein 2A gene with cancer risk.

OBJECTIVE: The aim of the study was to quantitatively assess the association of metallothionein 2A (MT2A) polymorphisms rs28366003 and rs1610216 with cancer risk. METHODS: Crude odd ratios (OR) with 95% confidence intervals (CI) were used to estimate associations of the polymorphisms with cancer risk. RESULTS: Six eligible case-control studies with 1899 cases and 2437 controls focused on rs28366003, and three of those six studies, with 548 cases and 926 controls, additionally focused on rs1610216. Pooled analysis showed that MT2A rs28366003 and rs1610216 were associated with cancer risk: (AG + GG) vs. AA, OR = 2.67; GG vs. (AG + AA), OR = 5.97; GG vs. AA, OR = 6.80; AG vs. AA, OR = 2.46; G vs. A, OR = 2.67 for rs28366003; and CC vs. (TC+TT), OR = 2.51; CC vs. TT, OR = 2.42 for rs1610216. Subgroup analysis based on ethnicity showed a significant association of rs28366003 with cancer risk in Asian and Caucasian populations. However, a significant association of rs1610216 with cancer risk was found only in the Asian population. CONCLUSION: MT2A rs28366003 and rs1610216 polymorphisms were associated with cancer risk and might serve as genetic biomarkers for predicting cancer risk. However, larger studies are needed to confirm these findings.

Metal contamination in quail meat: residues, sources, molecular biomarkers, and human health risk assessment.

Quail meat is an emerging source of high-quality animal protein. Quails are exposed to a wide range of xenobiotics such as heavy metals. In this study, residual concentrations of four toxic metals, of significant public health importance, including cadmium (Cd), lead (Pb), arsenic (As), and nickel (Ni), were determined in edible tissues of quails. In addition, metal loads were measured in water, feed, and litter samples collected from same quail farms as possible sources for quail exposure to heavy metals. The possible use of metallothionein (MT) and heat shock protein 70 (Hsp70) as molecular biomarkers of exposure to heavy metals was further investigated. Furthermore, the dietary intake and the potential risk assessment of the examined heavy metals among children and adults were calculated. The edible tissues of quails contained high concentrations of four heavy metals (contents (ppm/ww) ranging from 0.02 to 0.32 in Cd, 0.05 to 1.96 in Pb, 0.002 to 0.32 in As, and 1.17 to 3.94 in Ni), which corresponded to the high contents of these metals in the feeds, water, and litter. MT and Hsp70 mRNA expressions showed positive correlations with the concentrations of heavy metals in tissues indicating the possibility to use these proteins as biomarkers for quail's exposure to toxic metals. Dietary intake of quail meat and risk assessment revealed potential risks especially for children after prolonged exposure to the examined metals. Thus, legislations should be established and continuous screening of metal residues should be adopted in order to reduce the toxic metal concentrations in feeds and drinking water for quails. Reduction of exposure to heavy metals subsequently would lead to minimization of exposure of such toxicants through consumption of quail meat.

Assessment of complex water pollution with heavy metals and Pyrethroid pesticides on transcript levels of metallothionein and immune related genes.

Alteration of immunological function of an aquatic organism can be used as an indicator for evaluating the direct effect of exposure to pollutants. The aim of this work is to assess the impact of complex water pollution with special reference to Pyrethroid pesticides and heavy metals on mRNA transcript levels of Metallothionine and some immune related genes of Nile tilapia (Oreochromas Niloticus). Residues of six heavy metals and six Pyrethroid were assessed in water as well as fish tissues at three different sites of Lake Burullus, located at Northern Egypt. Variations of water physicochemical properties associated with different levels of heavy metals at the three different sections were recorded. Tissue residues of Fe, Mn and Zn, Cu, Ni exceed water levels in contrast to elevated water level of Pb. All assessed Pyrethroids are detected in fish tissue samples with higher concentration (3-42 folds) than that found in water samples especially Cypermethrin. Significant down-regulation of expression levels of metallothionein (MT) at the three sections of the lake was observed. The expression of immune related genes (IgM) and inflammatory cytokines (TNF, IL.8 and IL.1) were affected. IgM and TNF were significantly down-regulated at eastern and western section of the lake; meanwhile the expression of IL8 is down regulated at the three sections of the lack. IL1 was significantly up-regulated at eastern and middle sections. We conclude that, variable gene expression of MT and immune-related genes at the three sections of the lack impose different response to complex water pollution in relation to variable aquatic environment.

Assessment of the Mitigative Capacity of Dietary Zinc on PCB126 Hepatotoxicity and the Contribution of Zinc to Toxicity.

Hepatic levels of the essential micronutrient, zinc, are diminished by several hepatotoxicants, and the dietary supplementation of zinc has proven protective in those cases. 3,3',4,4',5-Pentachlorobiphenyl (PCB126), a liver toxicant, alters hepatic nutrient homeostasis and lowers hepatic zinc levels. The current study was designed to determine the mitigative potential of dietary zinc in the toxicity associated with PCB126 and the role of zinc in that toxicity. Male Sprague-Dawley rats were divided into three dietary groups and fed diets deficient in zinc (7 ppm Zn), adequate in zinc (30 ppm Zn), and supplemented in zinc (300 ppm). The animals were maintained for 3 weeks on these diets, then given a single IP injection of vehicle or 1 or 5 µmol/kg PCB126. After 2 weeks, the animals were euthanized. Dietary zinc increased the level of ROS, the activity of CuZnSOD, and the expression of metallothionein but decreased the levels of hepatic manganese. PCB126 exposed rats exhibited classic signs of exposure, including hepatomegaly, increased hepatic lipids, increased ROS and CYP induction. Liver histology suggests some mild ameliorative properties of both zinc deficiency and zinc supplementation. Other metrics of toxicity (relative liver and thymus weights, hepatic lipids, and hepatic ROS) did not support this trend. Interestingly, the zinc supplemented high dose PCB126 group had mildly improved histology and less efficacious induction of investigated genes than did the low dose PCB126 group. Overall, decreases in zinc caused by PCB126 likely contribute little to the ongoing toxicity, and the mitigative/preventive capacity of zinc against PCB126 exposure seems limited.

Ecotoxicological assessment of TiO2 byproducts on the earthworm Eisenia fetida.

The increasing production of nanomaterials will in turn increase the release of nanosized byproducts to the environment. The aim of this study was to evaluate the behaviour, uptake and ecotoxicity of TiO(2) byproducts in the earthworm Eisenia fetida. Worms were exposed to suspensions containing 0.1, 1 and 10 mg/L of byproducts for 24 h. Size of TiO(2) byproducts showed aggregation of particles up to 700 µm with laser diffraction. Only worms exposed at 10 mg/L showed bioaccumulation of titanium (ICP-AES), increasing expression of metallothionein and superoxide dismutase mRNA (Real-time PCR) and induction of apoptotic activity (Apostain and TUNEL). TiO(2) byproducts did not induce cytotoxicity on cœlomocytes, but a significant decrease of phagocytosis was observed starting from 0.1 mg/L. In conclusion, bioaccumulation of byproducts and their production of reactive oxygen species could be responsible for the alteration of the antioxidant system in worms.

Multiple platform assessment of the EGF dependent transcriptome by microarray and deep tag sequencing analysis.

BACKGROUND: Epidermal Growth Factor (EGF) is a key regulatory growth factor activating many processes relevant to normal development and disease, affecting cell proliferation and survival. Here we use a combined approach to study the EGF dependent transcriptome of HeLa cells by using multiple long oligonucleotide based microarray platforms (from Agilent, Operon, and Illumina) in combination with digital gene expression profiling (DGE) with the Illumina Genome Analyzer. RESULTS: By applying a procedure for cross-platform data meta-analysis based on RankProd and GlobalAncova tests, we establish a well validated gene set with transcript levels altered after EGF treatment. We use this robust gene list to build higher order networks of gene interaction by interconnecting associated networks, supporting and extending the important role of the EGF signaling pathway in cancer. In addition, we find an entirely new set of genes previously unrelated to the currently accepted EGF associated cellular functions. CONCLUSIONS: We propose that the use of global genomic cross-validation derived from high content technologies (microarrays or deep sequencing) can be used to generate more reliable datasets. This approach should help to improve the confidence of downstream in silico functional inference analyses based on high content data.

Metallothionein induction by Cu, Cd and Hg in Dicentrarchus labrax liver: assessment by RP-HPLC with fluorescence detection and spectrophotometry.

Metallothionein was quantified in sea bass Dicentrarchus labrax intraperitoneally (i.p.) injected with different Cu, Cd and Hg doses (50-250 microg kg(-1) wet wt) after 48 h exposure. A distinct peak with 16.8 min retention time was obtained by reversed-phase high performance liquid chromatography coupled to fluorescence detection (RP-HPLC-FD) with the three metals. Total metallothionein levels assayed in unheated liver extracts by RP-HPLC-FD were significantly higher (1.3-1.95-fold) than those obtained by the well-established spectrophotometric method. In the RP-HPLC-FD method, metallothionein increased linearly with Cu and Hg doses, being saturated beyond 100 mug kg(-1) Cd. Maximum induction was obtained at 100 microg kg(-1) Cd (5.3-fold), and 250 microg kg(-1) Cu or Hg (8- and 5.1-fold, respectively). At low doses no metallothionein induction was shown by the less sensitive spectrophotometric assay.

Assessment of heavy metal contamination using real-time PCR analysis of mussel metallothionein mt10 and mt20 expression: a validation along the Tunisian coast.

In mussel Mytilus galloprovincialis tissues, metallothionein belongs to two different gene classes, mt10 and mt20, showing differential expression at both basal conditions and under heavy metal challenge. In this study, a new more highly sensitive technique, expression analysis of mt10 and mt20 mRNA levels by quantitative reverse transcription polymerase chain reaction, was used to assess the effects of heavy metal contamination in the digestive glands of mussels caged along the Tunisian coast. To validate the new assay, total metallothionein protein, amount of heavy metals (zinc, copper, cadmium), and a biomarker of oxidative stress such as malondialdehyde content, were assessed in the same tissues. At the investigated sites, the molecular assay showed variations of mt20 relative gene expression levels within one or two orders of magnitude, with maximum values at two sites severely polluted with cadmium, Mahres (100-fold) and Menzel Jemile (165-fold). Changes in mt10 expression were recorded at all sites where copper had significantly accumulated, although fold induction levels were less pronounced than those of mt20. In this paper, gene expression data are discussed in relation to the studied biomarkers, demonstrating that the molecular technique based on the differential expression of mt10 and mt20 genes represents (i) a useful and robust tool for studying and monitoring heavy metal pollution under field conditions, and (ii) an improvement in the application of metallothionein as a biomarker of response to exposure to heavy metals in marine mussels.

An improved method for analyzing coenzyme Q homologues and multiple detection of rare biological samples.

We have developed a simple method for the estimation of coenzyme Q homologues, neurotransmitters, metal ions, lipid peroxidation, gene expression, and DNA fragmentation simultaneously from genetically engineered mice brain regions and cultured neurons. The primary objective of this study was to improve conventional time-consuming, cumbersome, and less efficient procedures, and reduce the cost of conducting kinetic studies in rare biological samples. The improved method is novel, precise, efficient, accurate, sensitive, economical, versatile, and highly reproducible. The recovery and shelf life of coenzyme Q homologues was significantly increased and the chromatograms exhibited reduced background and retention times. It is envisaged that in addition to coenzyme Q homologues, the improved method could be utilized for the multiple analyses of DNA, RNA and proteins from clinically significant biopsy and autopsy samples.

Serial analysis of gene expression identifies metallothionein-II as major neuroprotective gene in mouse focal cerebral ischemia.

We applied serial analysis of gene expression (SAGE) to study differentially expressed genes in mouse brain 14 hr after the induction of focal cerebral ischemia. Analysis of >60,000 transcripts revealed 83 upregulated and 94 downregulated transcripts (more than or equal to eightfold). Reproducibility was demonstrated by performing SAGE in duplicate on the same starting material. Metallothionein-II (MT-II) was the most significantly upregulated transcript in the ischemic hemisphere. MT-I and MT-II are assumed to be induced by metals, glucocorticoids, and inflammatory signals in a coordinated manner, yet their function remains elusive. Upregulation of both MT-I and MT-II was confirmed by Northern blotting. MT-I and MT-II mRNA expression increased as early as 2 hr after 2 hr of transient ischemia, with a maximum after 16 hr. Western blotting and immunohistochemistry revealed MT-I/-II upregulation in the ischemic hemisphere, whereas double labeling demonstrated the colocalization of MT with markers for astrocytes as well as for monocytes/macrophages. MT-I- and MT-II-deficient mice developed approximately threefold larger infarcts than wild-type mice and a significantly worse neurological outcome. For the first time we make available a comprehensive data set on brain ischemic gene expression and underscore the important protective role of metallothioneins in ischemic damage of the brain. Our results demonstrate the usefulness of SAGE to screen functionally relevant genes and the power of knock-out models in linking function to expression data generated by high throughput techniques.

Selected contribution: a three-dimensional model for assessment of in vitro toxicity in balaena mysticetus renal tissue.

This study established two- and three-dimensional renal proximal tubular cell cultures of the endangered species bowhead whale (Balaena mysticetus), developed SV40-transfected cultures, and cloned the 61-amino acid open reading frame for the metallothionein protein, the primary binding site for heavy metal contamination in mammals. Microgravity research, modulations in mechanical culture conditions (modeled microgravity), and shear stress have spawned innovative approaches to understanding the dynamics of cellular interactions, gene expression, and differentiation in several cellular systems. These investigations have led to the creation of ex vivo tissue models capable of serving as physiological research analogs for three-dimensional cellular interactions. These models are enabling studies in immune function, tissue modeling for basic research, and neoplasia. Three-dimensional cellular models emulate aspects of in vivo cellular architecture and physiology and may facilitate environmental toxicological studies aimed at elucidating biological functions and responses at the cellular level. Marine mammals occupy a significant ecological niche (72% of the Earth's surface is water) in terms of the potential for information on bioaccumulation and transport of terrestrial and marine environmental toxins in high-order vertebrates. Few ex vivo models of marine mammal physiology exist in vitro to accomplish the aforementioned studies. Techniques developed in this investigation, based on previous tissue modeling successes, may serve to facilitate similar research in other marine mammals.

Ecotoxicological assessment of persistent organic and heavy metal contamination in Hong Kong coastal sediment.

The aim of the present project is to determine the feasibility of measuring hepatic cytochrome P4501A1 (CYP1A1) and metallothionein (MT) mRNA in fish as an integrative measurement of persistent organic pollutants (POPs) and heavy metal contamination in sediment arising in Hong Kong. Sediment samples were collected from different sites, including Victoria Harbour (VS6), Yim Tin Tsai (YTT) at Tolo Harbour, Mai Po marshes (MPM) at Deep Bay, and Southern Waters (SS6) of coastal waters. The samples were analyzed for total and extractable concentrations of Cd, Cu, Ni, Zn, and Pb, as well as PCBs and PAHs. In addition, biomarker responses were studied in tilapia exposed experimentally to coastal sediment for 7 days. Using RT-PCR technique, hepatic CYP1A1 and MT mRNA were measured. Three control groups were used, including one negative control group maintained in sea water only; the second and third positive control groups were in sea water but were intraperitoneally injected with either beta-naphthoflavone (40 microg/g body weight) or cadmium chloride (10 microg/g body weight), respectively. The chemical data showed that VS6, YTT, and MPM were classified as Class C sediment according to the sediment quality criteria defined by the Hong Kong Environmental Protection Department, indicating the sites were heavily polluted. The exposure of tilapia to the sediment induced hepatic CYP1A1 (VS6 > YTT > MPM > SS6) and MT (VS6 > MPM > YTT > SS6) levels. The induction patterns were comparable to the levels of POPs and metal contamination in the sediment, indicating that the biomarker responses could be used to differentiate low to high levels of contamination among sediment.

Strain dependence in mice of resistance and susceptibility to the testicular effects of cadmium: assessment of the role of testicular cadmium-binding proteins.

The nature of the cadmium (Cd)-binding proteins in the mouse testes is unknown, although some studies have implied metallothionein (MT) is responsible for the marked strain dependence of Cd-induced testicular necrosis in mice. This study attempted to define the role of MT in strain-dependent Cd resistance using NFS (susceptible) and BALB/c (resistant) mice. In all cases, testicular proteins were compared to hepatic MT isolated after treatment with zinc (Zn). A low-molecular-weight (Mr) Zn-, Cd-binding protein was detected in testicular and hepatic cytosol from both strains after gel filtration. These proteins were extractable by heat treatment and sequential acetone precipitation. When such extracts were further purified with reverse-phase HPLC, two forms of authentic MT were shown by amino acid analysis from both NFS and BALB/c livers. However, of two testicular forms separated by HPLC from NFS and BALB/c mice, neither could be classified as MT based on amino acid composition. Methylation of the MT-I gene was also studied in testicular and hepatic DNAs isolated from control mice or from mice made resistant to Cd-induced testicular necrosis by Zn treatment. Major differences in methylation between tissues were seen, as the testicular gene of both NFS and BALB/c mice was highly methylated, a condition often linked to genetic quiescence. Zn treatment had no effect on MT-I gene methylation in testes of either strain, although rendering the NFS strain resistant to Cd. Zn treatment did not alter levels of this testicular protein in either strain while causing a marked induction of hepatic MT in both. These results indicate the low-Mr Cd-, Zn-binding proteins present in the testes of both resistant and susceptible mice are not MTs and further that the MT gene may not be expressed in either strain.