Inhibitory effect of cadmium on estrogen signaling in zebrafish brain and protection by zinc.

The present study was conducted to assess the effects of Cd exposure on estrogen signaling in the zebrafish brain, as well as the potential protective role of Zn against Cd-induced toxicity. For this purpose, the effects on transcriptional activation of the estrogen receptors (ERs), aromatase B (Aro-B) protein expression and molecular expression of related genes were examined in vivo using wild-type and transgenic zebrafish embryos. For in vitro studies, an ER-negative glial cell line (U251MG) transfected with different zebrafish ER subtypes (ERα, ERß1 and ERß2) was also used. Embryos were exposed either to estradiol (E2 ), Cd, E2 +Cd or E2 +Cd+Zn for 72 h and cells were exposed to the same treatments for 30 h. Our results show that E2 treatment promoted the transcriptional activation of ERs and increased Aro-B expression, at both the protein and mRNA levels. Although exposure to Cd, does not affect the studied parameters when administered alone, it significantly abolished the E2 -stimulated transcriptional response of the reporter gene for the three ER subtypes in U251-MG cells, and clearly inhibited the E2 induction of Aro-B in radial glial cells of zebrafish embryos. These inhibitory effects were accompanied by a significant downregulation of the expression of esr1, esr2a, esr2b and cyp19a1b genes compared to the E2 -treated group used as a positive control. Zn administration during simultaneous exposure to E2 and Cd strongly stimulated zebrafish ERs transactivation and increased Aro-B protein expression, whereas mRNA levels of the three ERs as well as the cyp19a1b remained unchanged in comparison with Cd-treated embryos. In conclusion, our results clearly demonstrate that Cd acts as a potent anti-estrogen in vivo and in vitro, and that Cd-induced E2 antagonism can be reversed, at the protein level, by Zn supplement. Copyright © 2016 John Wiley & Sons, Ltd.

Development of real-time PCR method for the detection and the quantification of a new endogenous reference gene in sugar beet "Beta vulgaris L.": GMO application.

KEY MESSAGE : Here, we describe a new developed quantitative real-time PCR method for the detection and quantification of a new specific endogenous reference gene used in GMO analysis. The key requirement of this study was the identification of a new reference gene used for the differentiation of the four genomic sections of the sugar beet (Beta vulgaris L.) (Beta, Corrollinae, Nanae and Procumbentes) suitable for quantification of genetically modified sugar beet. A specific qualitative polymerase chain reaction (PCR) assay was designed to detect the sugar beet amplifying a region of the adenylate transporter (ant) gene only from the species of the genomic section I of the genus Beta (cultivated and wild relatives) and showing negative PCR results for 7 species of the 3 other sections, 8 related species and 20 non-sugar beet plants. The sensitivity of the assay was 15 haploid genome copies (HGC). A quantitative real-time polymerase chain reaction (QRT-PCR) assay was also performed, having high linearity (R (2) > 0.994) over sugar beet standard concentrations ranging from 20,000 to 10 HGC of the sugar beet DNA per PCR. The QRT-PCR assay described in this study was specific and more sensitive for sugar beet quantification compared to the validated test previously reported in the European Reference Laboratory. This assay is suitable for GMO quantification in routine analysis from a wide variety of matrices.

Chemical composition and antibacterial activity of a hexane extract of Tunisian caprifig latex from the unripe fruit of Ficus carica.

CONTEXT: The plant kingdom has become a target in the search for new drugs and biologically active lead compounds. The common Jrani Tunisian caprifig Ficus carica L. (Moraceae) is one of the large number of plant species that are used in folklore medicine yet to be investigated for the treatment of many diseases, including those of infectious nature. OBJECTIVE: Hexane extract of the Tunisian common Jrani caprifig latex was assayed for antibacterial activity against several Gram-positive and Gram-negative bacteria. Chemical composition of the extract was also investigated. MATERIALS AND METHODS: The hexane extract was obtained from Tunisian Jrani caprifig latex by maceration, and then analyzed by gas chromatography-mass spectrometry. The extract was tested in vitro for antibacterial activity by the disc diffusion method and minimal inhibitory concentration (MIC) was also determined for all the test cultures. RESULTS: Thirty-six compounds of the extract were identified, 90.56% of the total area of peaks were coumarins. A strong bactericidal effect was demonstrated. The most sensitive bacteria were Staphylococcus saprophyticus clinical isolate, and Staphylococcus aureus ATCC 25923, with a MIC of 19 µg/mL. DISCUSSION AND CONCLUSION: These findings demonstrate an effective in vitro antibacterial activity of the hexane extract of caprifig latex.

Partial characterization of a novel amylase activity isolated from Tunisian Ficus carica latex.

CONTEXT: A large number of plants still need to be investigated through screening of amylases suitable for industry. In the present study, and for the first time, we describe the amylolytic activity of Saint Pedro Ficus carica L. (Moraceae) crude latex of Kahli and Bidhi varieties. OBJECTIVE: Effects of temperature, pH, metal ions, and inhibitors and compatibility with some commercial detergents were investigated for amylase activity. MATERIALS AND METHODS: Amylase activity was screened in crude latex using the DNS method and potato starch as a substrate. Analyses of amylolytic reaction products by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) were performed. RESULTS: Bidhi and Kahli amylases were active in optimal pH of 6.5 and 7 at 45°C, respectively, displaying a half life of 85 and 60 min, respectively, at 80°C, and they were very stable in a wide range of pH (4-12). Bidhi amylase activity increased to 260% by addition of 10(-3) mM Fe(2+) or 10(-2) mM Cu(2+), and was strongly inhibited by Mg(2+) and EDTA. In the presence of Ca(2+) and Mg(2+), Kahli amylase activity was dramatically enhanced by 220 and 260%, respectively. The compatibility of both amylases with certain commercial detergents was also shown to be good as enzymes retained up to 98% of their activities after 30 min of incubation at 80°C. DISCUSSION AND CONCLUSION: Analysis of amylolytic reaction products by TLC and HPLC suggested that Kahli amylase was an amyloglucosidase and Bidhi amylase was ß-fructose, α(1-4) glucose. Bidhi amylase is a good choice for application in starch, food, detergents and medical industries.

Cadmium-induced ovarian pathophysiology is mediated by change in gene expression pattern of zinc transporters in zebrafish (Danio rerio).

This study explored the potential for expression pattern of genes encoding zinc (Zn) transporters to be involved in the cadmium (Cd)-induced reproductive toxicity in female of zebrafish. For this purpose, oocytes maturity and ovarian histology as well as Cd, Zn and metallothioneins (MTs) accumulation and expression of genes encoding Zrt-,Irt-related protein 10 (ZIP10), Zn transporter 1 (ZnT1) and zebrafish metallothionein (zMT) were examined in ovaries of adult zebrafish exposed to 0.4 mg/L Cd in water and supplemented with Zn (5 mgkg(-1)) in their diet for 21 days. Cd-exposure decreased the expression of ZnT1 and caused up-regulation of ZIP10 and zMT gene expression. These changes were accompanied by increased Cd and MTs accumulation, decreased Zn contents as well as by histopathological damages in ovarian tissues. The co-exposure of fish to Cd and Zn abolished ZnT1 down-regulation and rendered a persistently increased ZIP10 mRNA level. This treatment also decreased Cd and MTs accumulation, reversed Cd-induced Zn depletion and partially restored Cd-induced histological changes in ovarian tissues. These results imply that the downregulation of ZnT1 as well as the overexpression of ZIP10, in responses to the ovarian Zn depletion induced by Cd, play a major role in Cd accumulation and consequently in its toxicity. The protective effect of dietary Zn supplementation against Cd-induced toxicity is mediated, at least in part, by the increase of Zn availability and subsequently the induction of ZnT1 gene expression.

Mechanisms underlying the protective effect of zinc and selenium against cadmium-induced oxidative stress in zebrafish Danio rerio.

The present study was designed to elucidate the protective effect mechanism of Zinc (Zn) and Selenium (Se) on cadmium (Cd)-induced oxidative stress in zebrafish. For this purpose we investigate the response of oxidative stress markers, metallothionein accumulation and gene expression in liver and ovary of female zebrafish exposed to 0,4 mg/l Cd in water and supplemented with Zn (5 mg kg(-1)) and/or Se (2 mg kg(-1)) for 21 days in their diet. Liver and ovary Cd uptake was evaluated after the exposure period. Cd exposure significantly inhibited the antioxidant enzyme activities termed as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxydase (GPx) and caused a pronounced malondialdehyde (MDA) accumulation in both organs. Co-administration of Zn and Se reversed the Cd-induced toxicity in liver and ovary measured as MDA accumulation. Interestingly, gene expression patterns of Cat, CuZnSod and Gpx were up-regulated when related enzymatic activities were altered. Zebrafish metallothionein transcripts (zMt) significantly decreased in tissues of fish supplemented with Zn and/or Se when compared to Cd-exposed fish. Our data would suggest that Zn and Se protective mechanism against Cd-induced oxidative stress is more depending on the correction of the proteins biological activities rather than on the transcriptional level of related genes.

In vitro cytotoxic and antiviral activities of Ficus carica latex extracts.

The latex of fig fruit (Ficus carica) is used in traditional medicine for the treatment of skin infections such as warts and also diseases of possible viral origin. Five extracts (methanolic, hexanic, ethyl acetate, hexane-ethyl acetate (v/v) and chloroformic) of this species were investigated in vitro for their antiviral potential activity against herpes simplex type 1 (HSV-1), echovirus type 11 (ECV-11) and adenovirus (ADV). To evaluate the capacity of the extracts to inhibit the replication of viruses, the following assays were performed: adsorption and penetration, intracellular inhibition and virucidal activity. Observation of cytopathic effects was used to determine the antiviral action. The hexanic and hexane-ethyl acetate (v/v) extracts inhibited multiplication of viruses by tested techniques at concentrations of 78 µg mL(-1). These two extracts were possible candidates as herbal medicines for herpes virus, echovirus and adenovirus infectious diseases. All extracts had no cytotoxic effect on Vero cells at all tested concentrations.

Influence of combined treatment with zinc and selenium on cadmium induced testicular pathophysiology in rat.

The present study was conducted to evaluate the potential benefit of combined treatment with zinc (Zn) and selenium (Se) in reversing cadmium (Cd)-induced testicular pathophysiology compared to Se or Zn treatment alone in rats. For this purpose, male rats received either tap water, Cd, Cd+Zn, Cd+Se or Cd+Zn+Se in their drinking water, for 35 days. Cd exposure caused a significant decrease in plasma and testicular concentrations of Se and Zn which was accompanied by decreased plasma testosterone level, sperm count and motility, enzymatic activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) as well as by increased lipid peroxidation (as malondialdehyde, MDA). With Se or Zn administration, during exposure to Cd, only partial corrective effects on depletion of testicular and plasma Se and Zn levels, sperm characteristics and oxidative stress have been observed. The combined treatment of Cd-exposed animals with Se and Zn assured a more significant decrease in plasma and testicular Cd concentrations and a more efficient protection against the observed testicular damage as evidenced by the total prevention of both Se and Zn deprivation and by the entire restoration of the sperm motility and the testicular antioxidant status.

Metallothionein gene expression in liver of rats exposed to cadmium and supplemented with zinc and selenium.

Cadmium (Cd), one of the most widely distributed heavy metals, is highly toxic to humans and animals. It is well known that zinc (Zn) and selenium (Se) administration reduce the Cd-induced toxicity and that metallothioneins can have a protective effect to mitigate Cd toxicity in biological systems. In this study we report the expression analysis of the two metallothioneines gene classes MT-1 and MT-2 as well as the total metalloprotein content in the liver of rats exposed to Cd (200 ppm), Cd + Zn (200 ppm + 500 ppm), Cd + Se (200 ppm + 0.1 ppm) or Cd + Zn + Se (200 ppm + 500 ppm + 0.1 ppm) in their drinking water for 35 days. Metals accumulation was quantified in rat liver. Cd decreased significantly the hepatic concentrations of Se and increased those of Zn. The treatment of Cd-exposed rats with Se alone or combined with Zn reversed the Cd-induced depletion of Se concentrations in the liver. However, Zn or Zn + Se administration significantly increased the liver Cd uptake and had no effect on the Cd-induced increase in hepatic concentrations of Zn. The molecular assay showed a decreasing trend of MT-1 relative gene expression levels in animals supplemented with Zn (6.87-fold), Se (3.58-fold), and their combination (1.69-fold) when compared to Cd-treated animals (16.22-fold). Upregulation of the MT-2 expression were recorded in all conditions, although fold induction levels were less pronounced than MT-1 expressions. Our data suggest that the well-established protective effect of Zn and Se against Cd-induced toxicity passes through non-MT gene expression mechanisms, being more dependent on the oxidative stress status of the cell.

In vitro antimicrobial activity of four Ficus carica latex fractions against resistant human pathogens (antimicrobial activity of Ficus carica latex).

Methanolic, hexanoïc, chloroformic and ethyl acetate extracts of Ficus carica latex were investigated for their in vitro antimicrobial proprieties against five bacteria species and seven strains of fungi. The green fruit latex was collected from Chott Mariam Souse, Middle East coast of Tunisia. The antimicrobial activity of the extracts was evaluated and based respectively on the inhibition zone using the disc-diffusion assay, minimal inhibition concentration (MIC) for bacterial testing and the method by calculating inhibition percentage (I%) for fungi-inhibiting activities. The methanolic extract had no effect against bacteria except for Proteus mirabilis while the ethyl acetate extract had inhibition effect on the multiplication of five bacteria species (Enterococcus fecalis, Citobacter freundei, Pseudomonas aeruginosa, Echerchia coli and Proteus mirabilis). For the opportunist pathogenic yeasts, ethyl acetate and chlorophormic fractions showed a very strong inhibition (100%); methanolic fraction had a total inhibition against Candida albicans (100%) at a concentration of 500 microg/ml and a negative effect against Cryptococcus neoformans. Microsporum canis was strongly inhibited with methanolic extract (75%) and totally with ethyl acetate extract at a concentration of 750 microg/ml. Hexanoïc extract showed medium results.

Protective effects of selenium, zinc, or their combination on cadmium-induced oxidative stress in rat kidney.

The present study was conducted to investigate whether the combined treatment with Se and Zn offers more beneficial effects than that provided by either of them alone in reversing Cd-induced oxidative stress in the kidney of rat. For this purpose, 30 adult male Wistar albino rats, equally divided into control and four treated groups, received either 200 ppm Cd (as CdCl(2)), 200 ppm Cd + 500 ppm Zn (as ZnCl(2)), 200 ppm Cd + 0.1 ppm Se (as Na(2)SeO(3)), or 200 ppm Cd + 500 ppm Zn + 0.1 ppm Se in their drinking water for 35 days. The results showed that Cd treatment decreased significantly the catalase (CAT) and glutathione peroxidase (GSH-Px) activities, whereas the superoxide dismutase (SOD) activity and the renal levels of lipid peroxidation (as malondialdehyde, MDA) were increased compared to control rats. The treatment of Cd-exposed rats with Se alone had no significant effect on the Cd-induced increase in the MDA concentrations but increased significantly the CAT activities and reversed Cd-induced increase in SOD activity. It also partially prevented Cd-induced decrease in GSH-Px activity. The treatment of Cd-exposed animals with Zn alone increased significantly the CAT activity and partially protected against Cd-induced increase in the MDA concentrations, whereas it had no significant effect on the Cd-induced increase in SOD activity and decrease in GSH-Px activity. The combined treatment of Cd-exposed animals with Se and Zn was more effective than that with either of them alone in reversing Cd-induced decrease in CAT and GSH-Px activities and Cd-induced increase in MDA concentrations. Results demonstrated beneficial effects of combined Se and Zn treatment in Cd-induced oxidative stress in kidney and suggest that Se and Zn can have a synergistic role against Cd toxicity.

Reversal of cadmium-induced thyroid dysfunction by selenium, zinc, or their combination in rat.

The aim of this study was to evaluate the potential benefit of combined treatment with zinc (Zn) and selenium (Se) in reversing cadmium (Cd)-induced thyroid dysfunction compared to Se or Zn treatment alone in rats exposed to Cd. For this purpose, 30 adult male Wistar albino rats were equally divided into control and four treated groups receiving either 200 ppm Cd (as CdCl2), 200 ppm Cd + 500 ppm Zn (as ZnCl2), 200 ppm Cd + 0.1 ppm Se (as Na2SeO3), or 200 ppm Cd + 500 ppm Zn + 0.1 ppm Se in their drinking water for 35 days. The results showed that Cd exposure increased significantly the relative thyroid weight (RTW), the thyroid Cd concentration, and the serum thyroid stimulating hormone (TSH) level, whereas the serum thyroxine (T4) level was decreased compared to control rats. The treatment of Cd-exposed rats with Se alone only partially protected from the Cd-induced decrease in serum T4 level. The treatment of Cd-exposed animals with Zn alone partially protected against Cd-induced thyroid dysfunction by maintaining normal RTW and by decreasing Cd concentration in the thyroid. It also partially prevents Cd-induced decrease in serum T4 level. The combined treatment of Cd-exposed animals with Se and Zn induced a more significant decrease in the thyroid Cd concentration than the Zn supplement and a total correction of the RTW. This treatment was also more effective than that with Se or Zn alone in reversing Cd-induced decrease in serum T4 level and Cd-induced increase in serum TSH level. Se and Zn can have a synergistic role against Cd-induced thyroid dysfunction.