Walid Rachidi

The toxicity redox mechanisms of cadmium alone or together with copper and zinc homeostasis alteration: its redox biomarkers.

Cadmium (Cd) is a toxic metal and can induce and/or promote diseases in humans (cancer, aging diseases, kidney and bone diseases, etc.). Its toxicity involves many mechanisms including the alteration of copper (Cu) and zinc (Zn) homeostasis leading to reactive oxygen species (ROS) production, either directly or through the inhibition of antioxidant activities. Importantly, ROS can induce oxidative damages in cells. Cadmium, Cu and Zn are also able to induce glutathione (GSH) and metallothioneins (MT) synthesis in a cell-type-dependent manner. As a consequence, the effects induced by these three metals result simultaneously from the inhibition of antioxidant activities and the induction of other factors such as GSH and MT synthesis. MT levels are regulated not only by the p53 protein in a cell-type-dependent manner, or by transcription factors such as metal-responsive transcription factor 1 (MTF-1) and cellular Zn levels but also by cellular GSH level. As described in the literature, DNA damage, GSH and MT levels are sensitive biomarkers used to identify Cd-induced toxicity alone or together with Cu and Zn homeostasis alteration.

Protective effect of selenium supplementation on the genotoxicity of di(2-ethylhexyl)phthalate and mono(2-ethylhexyl)phthalate treatment in LNCaP cells.

Selenium is an essential cofactor in the key enzymes involved in cellular antioxidant defense. It plays a critical role in testis and reproduction and regulates DNA damage within the prostate. Phthalates are ubiquitous environmental contaminants that cause alterations in endocrine and spermatogenic functions in animals. The objective of this study was to investigate the cytotoxicity and genotoxicity potentials of di(2-ethylhexyl)phthalate (DEHP), the most widely used phthalate and its primary toxic metabolite mono(2-ethylhexyl)phthalate (MEHP), and their effects on the antioxidant balance in the LNCaP human prostate adenocarcinoma cell line. Protection by selenium supplementation with either sodium selenite (SS, 30 nM) or selenomethionine (SM, 10 microM) was also investigated. Both DEHP (3mM) and MEHP (3 microM) caused significant decreases in cell viability; altered antioxidant status, particularly decreasing the GPx1 activity; and induced DNA damage as measured by the alkaline comet assay. Selenium supplementation was highly protective against cytotoxicity, partially prevented genotoxicity, and restored the antioxidant status. The results of this study suggested that the underlying mechanism of cytotoxicity and resulting disturbances produced by DEHP or MEHP was an an oxidative stress process and/or an effect on the expression of antioxidant enzymes, and accentuated the importance of selenium status, particularly with respect to the high probability of phthalate exposures and their adverse effects.

Overexpression of cellular prion protein induces an antioxidant environment altering T cell development in the thymus.

Cellular prion protein (PrPC) is an ubiquitously expressed glycoprotein whose roles are still widely discussed, particularly in the field of immunology. Using TgA20- and Tg33-transgenic mice overexpressing PrPC, we investigated the consequences of this overexpression on T cell development. In both models, overexpression of PrPC induces strong alterations at different steps of T cell maturation. On TgA20 mice, we observed that these alterations are cell autonomous and lead to a decrease of αβ T cells and a concomitant increase of γδ T cell numbers. PrPC has been shown to bind and chelate copper and, interestingly, under a copper supplementation diet, TgA20 mice presented a partial restoration of the αβ T cell development, suggesting that PrPC overexpression, by chelating copper, generates an antioxidant context differentially impacting on αβ and γδ T cell lineage.

Effects of di(2‐ethylhexyl)phthalate on testicular oxidant/antioxidant status in selenium‐deficient and selenium‐supplemented rats

Di(ethylhexyl)phthalate (DEHP), the most widely used plasticizer, was investigated to determine whether an oxidative stress process was one of the underlying mechanisms for its testicular toxicity potential. To evaluate the effects of selenium (Se), status on the toxicity of DEHP was further objective of this study, as Se is known to play a critical role in testis and in the modulation of intracellular redox equilibrium. Se deficiency was produced in 3‐weeks‐old Sprague–Dawley rats feeding them ≤0.05 mg Se /kg diet for 5 weeks, and Se‐supplementation group was on 1 mg Se/kg diet. DEHP‐treated groups received 1000 mg/kg dose by gavage during the last 10 days of the feeding period. Activities of antioxidant selenoenzymes [glutathione peroxidase 1 (GPx1), glutathione peroxidase 4 (GPx4), thioredoxin reductase (TrxR)], catalase (CAT), superoxide dismutase (SOD), and glutathione S‐transferase (GST); concentrations of reduced glutathione (GSH), oxidized glutathione (GSSG), and thus the GSH/GSSG redox ratio; and thiobarbituric acid reactive substance (TBARS) levels were measured. DEHP was found to induce oxidative stress in rat testis, as evidenced by significant decrease in GSH/GSSG redox ratio (>10‐fold) and marked increase in TBARS levels, and its effects were more pronounced in Se‐deficient rats with ∼18.5‐fold decrease in GSH/GSSG redox ratio and a significant decrease in GPx4 activity, whereas Se supplementation was protective by providing substantial elevation of redox ratio and reducing the lipid peroxidation. These findings emphasized the critical role of Se as an effective redox regulator and the importance of Se status in protecting testicular tissue from the oxidant stressor activity of DEHP.

The effects of di(2-ethylhexyl)phthalate on rat liver in relation to selenium status.

This study was performed to determine the hepatotoxicity of di(2‐ethylhexyl)phthalate (DEHP) in relation to selenium status. In 3‐week‐old Sprague‐Dawley rats, selenium deficiency was induced by a ≤0.05 selenium mg/kg. A selenium supplementation group was given 1 mg selenium/kg diet for 5 weeks. Di(2‐ethylhexyl)phthalate‐treated groups received 1000 mg/kg dose by gavage during the last 10 days of the experiment. Histopathology, peroxisome proliferation, catalase (CAT) immunoreactivity and activity and apoptosis were assessed. Activities of antioxidant selenoenzymes [glutathione peroxidase 1 (GPx1), glutathione peroxidase 4 (GPx4), thioredoxin reductase (TrxR1)], superoxide dismutase (SOD), and glutathione S‐transferase (GST); aminotransferase, total glutathione (tGSH), and lipid peroxidation (LP) levels were measured. Di(2‐ethylhexyl)phthalate caused cellular disorganization while necrosis and inflammatory cell infiltration were observed in Se‐deficient DEHP group (DEHP/SeD). Catalase activity and immunoreactivity were increased in all DEHP‐treated groups. Glutathione peroxidase 1 and GPx4 activities decreased significantly in DEHP and DEHP/SeD groups, while GST activities decreased in all DEHP‐exposed groups. Thioredoxin reductase activity increased in DEHP and DEHP/SeS, while total SOD activities increased in all DEHP‐treated groups. Lipid peroxidation levels increased significantly in SeD (26%), DEHP (38%) and DEHP/SeD (71%) groups. Selenium supplementation partially ameliorated DEHP‐induced hepatotoxicity; while in DEHP/SeD group, drastic changes in hepatic histopathology and oxidative stress parameters were observed.

Di(2-ethylhexyl)phthalate-induced renal oxidative stress in rats and protective effect of selenium

This study was designed to examine the oxidative stress potential of di(2-ethylhexyl)phthalate (DEHP) on rat kidney and to evaluate possible protective effect of selenium (Se) status. Se deficiency (SeD) was produced in 3-week old Sprague−Dawley rats by feeding them ≤ 0.05 Se mg/kg diet for 5 weeks; Se supplementation group (SeS) was on 1 mg Se/kg diet. DEHP treated groups received 1000 mg/kg dose by gavage during the last 10 days of the feeding period. Activities of antioxidant selenoenzymes [glutathione peroxidase 1 (GPx1), glutathione peroxidase 4 (GPx4), thioredoxin reductase (TrxR)], catalase (CAT), superoxide dismutase (SOD), and glutathione S-transferase (GST); concentrations of total glutathione (GSH), thiols and thiobarbituric acid reactive substance (TBARS) levels were measured. DEHP treatment was found to induce oxidative stress in rat kidney, as evidenced by significant decreases in GPx1 (~20%) and SOD (~30%) activities and GSH levels (~20%), along with marked decrease in thiol content (~40%) and increase in TBARS (~30%) levels. The effects of DEHP was more pronounced in SeD rats, whereas Se supplementation was protective by providing substantial elevations of GPx1 and GPx4 activities and GSH levels. These findings emphasized the critical role of Se as an effective redox regulator and the importance of Se status in protecting renal tissue from the oxidant stressor activity of DEHP.

Metallothionein expression in HaCaT and C6 cell lines exposed to cadmium

Metallothioneins (MT) are low-molecular weight, cysteine-rich metal-binding proteins. MT play a role in the homeostasis of essential metals such as zinc (Zn) and copper (Cu), detoxification of toxic metals such as cadmium (Cd) and protection against oxidative stress. In this study, we examined the expression of MT in HaCaT and C6 cells as a strategy to enhance protection against Cd-mediated toxicity. At basal level, HaCaT cells showed higher MT level than C6 cells which could explain the resistance of HaCaT cells. Western blot showed that C6 cells treated with 20micromol/L Cd for 24h did not express any MT. MT were initially expressed in the cytoplasmic or periplasmic compartment and were then translocated in the nucleus after 24h treatment by Cd both in HaCaT and C6 cells. In addition, the cell treatment with Cd was followed by an increase in the cellular zinc level but the electrophoretic mobility shift assay (EMSA) experiment did not show any translocation of metal-responsive transcription factor-1 (MTF-1) to the nucleus of HaCaT cells. These absence of translocation could be due to the presence of MT in these cells at the basal state. The translocation study in HaCaT cells suggested that the MT translocation in the nucleus was greater than observed in C6 cells. The latter observation could explain HaCaT cells resistance to Cd concentrations up to 50micromol/L. Our results suggested that the C6 cell sensitivity was correlated with the decrease in MT level at 20micromol/L Cd occurring after the transcription of MT gene.

The effects of di(2-ethylhexyl) phthalate and/or selenium on trace element levels in different organs of rats.

Di(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer for synthetic polymers, is known to have endocrine disruptive potential, reproductive toxicity, and induces hepatic carcinogenesis in rodents. Selenium (Se) is a component of several selenoenzymes which are essential for cellular antioxidant defense and for the functions of mammalian reproductive system. The present study was designed to investigate the effects of DEHP exposure on trace element distribution in liver, testis, and kidney tissues and plasma of Se-deficient and Se-supplemented rats. Se deficiency was produced by feeding 3-week old Sprague-Dawley rats with ≤0.05mg Se/kg diet for 5 weeks, and supplementation group were on 1mg Se/kg diet. DEHP treated groups received 1000mg/kg dose by gavage during the last 10 days of feeding period. Se, zinc (Zn), copper (Cu), iron (Fe) and manganese (Mn) levels were measured by inductively coupled plasma mass spectrometry (ICP-MS). Se supplementation caused significant increases in hepatic, renal, and testicular Se levels. With DEHP exposure, plasma Se and Zn, kidney Se, Cu and Mn levels were significantly decreased. Besides, liver Fe decreased markedly in all the DEHP-treated groups. Liver and kidney Mn levels decreased significantly in DEHP/SeD group compared to both DEHP and SeD groups. These results showed the potential of DEHP exposure and/or different Se status to modify the distribution pattern of essential trace elements in various tissues, the importance of which needs to be further evaluated.

Prion protein protects against zinc-mediated cytotoxicity by modifying intracellular exchangeable zinc and inducing metallothionein expression

PrPC contains several octapeptide repeats sequences toward the N-terminus which have binding affinity for divalent metals such as copper, zinc, nickel and manganese. However, the link between PrPC expression and zinc metabolism remains elusive. Here we studied the relationship between PrPC and zinc ions intracellular homeostasis using a cell line expressing a doxycycline-inducible PrPC gene. No significant difference in 65Zn2+ uptake was observed in cells expressing PrPC when compared with control cells. However, PrPC-expressing cells were more resistant to zinc-induced toxicity, suggesting an adaptative mechanism induced by PrPC. Using zinquin-ethyl-ester, a specific fluorophore for vesicular free zinc, we observed a significant re-localization of intracellular exchangeable zinc in vesicles after PrPC expression. Finally, we demonstrated that PrPC expression induces metallothionein (MT) expression, a zinc-upregulated zinc-binding protein. Taken together, these results suggest that PrPC modifies the intracellular localization of zinc rather than the cellular content and induces MT upregulation. These findings are of major importance since zinc deregulation is implicated in several neurodegenerative disorders. It is postulated that in prion diseases the conversion of PrPC to PrPSc may deregulate zinc homeostasis mediated by metallothionein.

α6 Integrin (α6high)/Transferrin Receptor (CD71)low Keratinocyte Stem Cells Are More Potent for Generating Reconstructed Skin Epidermis Than Rapid Adherent Cells

The epidermis basal layer is composed of two keratinocyte populations: Keratinocyte Stem cells (KSC) and Transitory Amplifying (TA) cells that arise from KSC division. Unfortunately, no specific marker exists to differ between KSC and TA cells. Here, we aimed at comparing two different methods that pretended to isolate these two populations: (i) the rapid adhesion method on coated substrate and (ii) the flow cytometry method, which is based on the difference in cell surface expressions of the α6 integrin and transferrin receptor (CD71). Then, we compared different parameters that are known to discriminate KSC and TA populations. Interestingly, we showed that both methods allow enrichment in stem cells. However, cell sorting by flow cytometry (α6high/CD71low) phenotype leads to a better enrichment of KSC since the colony forming efficiency is five times increased versus total cell suspension, whereas it is only 1.4 times for the adhesion method. Moreover, α6high/CD71low cells give rise to a thicker pluristratified epithelium with lower seeding density and display a low Ki67 positive cells number, showing that they have reached the balance between proliferation and differentiation. We clearly demonstrated that cells isolated by a rapid adherent method are not the same population as KSC isolated by flow cytometry following α6high/CD71low phenotype.