Melatonin counteracts cadmium-induced rat testicular toxicity via the mechanistic target rapamycin (mTOR) pathway.

The protective action of melatonin (MLT) against the harmful effects of cadmium (Cd) on testicular activity in rats has been documented previously; however, the involved molecular mechanisms have yet to be elucidated. Herein, we investigate the involvement of the mammalian target of rapamycin (mTOR) on the ability of MLT to counteract the damage induced by Cd on the rat testicular activity. Our study confirmed that Cd has harmful effects on the testes of rats and the protective action exerted by MLT. We reported, for the first time, that the addition of rapamycin (Rapa), a specific mTOR inhibitor, to animals co-treated with Cd and MLT completely abolished the beneficial effects exerted by MLT, indicating that the mTOR pathway partially modulates its helpful effects on Cd testicular toxicity. Interestingly, Rapa-alone treatment, provoking mTOR inhibition, produced altered morphological parameters, increased autophagy of germ and somatic cells, and reduced serum testosterone concentration. In addition, mTOR inhibition also reduced protein levels of markers of steroidogenesis (3ß-Hydroxysteroid dehydrogenase) and blood-testis barrier integrity (occludin and connexin 43). Finally, Rapa altered sperm parameters as well as the ability of mature spermatozoa to perform a proper acrosome reaction. Although further investigation is needed to better clarify the molecular pathway involved in MLT action, we confirm that MLT alleviating Cd effects can be used as a supplement to enhance testicular function and improve male gamete quality.

Interactions between cadmium and zinc on gene expression pattern of differentiation markers in MC3T3-E1 cell line.

We evaluated, in vitro, the interactions between cadmium (Cd) and zinc (Zn) during the proliferation and differentiation process using bone MC3T3-E1 cell line.Cells were treated with CdCl2 and/or ZnCl2 for 24 and 48 h and 5 µM CdCl2 was found as low cytotoxic dose and 25 µM ZnCl2 as the best Zn treatment for cell proliferation. Gene expression of some bone markers (Runx2, collagen α1 (Colα1), osteocalcin (Oc), alkaline phosphatase (ALP) and bone sialoprotein (BSP)) was studied at 24, 48 and 72 h.Treatment by CdCl2 depressed Runx2, Colα1, and BSP mRNA levels after 24 h. Oc and ALP gene expression was found to be decreased after 72 h.CdCl2 -exposure decreased ALP activity and Ca deposit in matrix. In concomitant treatment by CdCl2 and ZnCl2, gene expression of osteoblastic markers was found to be up-regulated (p < 0, 05) compared to CdCl2 treated cells, ALP staining and mineralization were increased.Our results show that Zn could prevent Cd-induced toxicity on MC3T3-E1 cells, probably through the restoration of Runx2, col α1, BSP, ALP and Oc and gene expression inhibited by Cd.

Disruption of Bone Zinc Metabolism during Postnatal Development of Rats after Early Life Exposure to Cadmium.

This current study was conducted to investigate whether bone tissue impairment induced by early life exposure to cadmium (Cd) during postnatal development could result from disruption to zinc (Zn) metabolism. For this reason, the offspring from mothers receiving either tap water, Cd, Zn or Cd + Zn during gestation and lactation periods were euthanized at PND21 and PND70. At the end of the lactation period (PND21), our results showed that exposure to Cd increased Cd accumulation and Zn depletion in the femur. Furthermore, calcium (Ca) level was reduced. At the molecular level, Cd induced an increase of MT-1 expression and caused an upregulation of ZIP2 accompanied with a down-regulation of ZnT5. Runx2, ALP, colα-1 and Oc mRNA levels were also decreased. In plasma, IGF-1 and osteocalcin concentrations were decreased. Further, Cd altered femoral growth by generating changes in the growth plate. Consequently, the toxic effect of Cd persisted at adult age (PND70) by decreasing bone volume (%BV/TV), bone mineral density (BMD) and Ca content and by increasing trabecular separation (Tb.Sp) in the distal femur. Interestingly, Zn supply provided total or partial corrections of several toxic effects of Cd. These data suggest that the increases of Zn bioavailability as well as the reduction of Cd accumulation in the femur following the changes in ZIP2 and ZnT5 expression are part of the mechanism involved in Zn protection against Cd toxicity on bone tissue.

Protective role of zinc against the neurotoxicity induced by exposure to cadmium during gestation and lactation periods on hippocampal volume of pups tested in early adulthood.

The present study was conducted to assess the possible effect of cadmium (Cd) throughout gestation and lactation on the volume of the subregion of the hippocampus as well as the potential protective role of zinc (Zn) against Cd neurotoxicity. For this purpose, female rats received either tap water, Cd, Zn or Cd + Zn in their drinking water during gestation and lactation. At postnatal day 35 (PND35), the male pups were sacrificed, and their brains were taken for histologic, chemical, and biochemical analysis. Hippocampal volume was measured in histologic brain slices using Cavalieri's principle. Zn depletion was observed in the brains of pups issued from mothers exposed to Cd. Biochemical analysis further revealed that Cd exposure significantly increases the superoxide dismutase (SOD) activity, as well as the metallothionein (MT) level. During histologic investigation, our results showed that gestational and lactational exposure to Cd significantly altered and decreased the volume of CA1, CA3 pyramidal cell layer and the dentate gyrus. However, there were no marked differences shown in CA2 subfield. Compared to Cd group, co-treatment with Cd and Zn provided correction of the changes induced by the Cd exposure. These results highlight the protective role of Zn against Cd-induced alteration in the hippocampus which is a crucial structure implicated in learning and memory processes.

Disruption of the zinc metabolism in rat fœtal brain after prenatal exposure to cadmium.

This study was carried out to investigate the effects of maternal Cd and/or Zn exposure on some parameters of Zn metabolism in fetal brain of Wistar rats. Thus, female controls and other exposed by the oral route during the gestation period to Cd (50 mg CdCl2/L) and/or Zn (ZnCl2 60 mg/L) were used. The male fetuses at age 20 days of gestation (GD20) were sacrificed and their brains were taken for histological, chemical and molecular analysis. Zn depletion was observed in the brains of fetuses issued from mothers exposed to Cd. Histological analysis showed that Cd exposure induces pyknosis in cortical region and CA1 region of the hippocampus compared to controls. Under Cd exposure, we noted an overexpression of the genes coding for membrane transporter involved in the intracellular incorporation of Zn (ZIP6) associated with inhibition of that encoding the transporters involved in the output of the Zn into the extracellular medium (ZnT1 and ZnT3). A decrease in the expression of the gene encoding the neuro-trophic factor (BDNF) associated with overexpression of the encoding the metal regulatory transcription factor 1 (MTF1), factor involved in the homeostasis of Zn, was also noted in Cd group. Interestingly, Zn supply provided a total or partial restauration of the changes induced by the Cd exposure. The depletion of brain Zn contents as well as the modification of the profile of expression of genes encoding membrane Zn transporters, suggest that the toxicity of Cd observed in fetal brain level are mediated, in part, by impairment of Zn metabolism.

Early-Life Exposure to Cadmium Triggers Distinct Zn-Dependent Protein Expression Patterns and Impairs Brain Development.

The objective of this study was to determine if the brain development impairment induced by early-life exposure to cadmium (Cd) could result from changes in the expression pattern of distinct zinc (Zn)-dependent proteins. For this purpose, adult female rats receiving either tap water, Cd, Zn, or Cd + Zn in their drinking water during gestation and lactation periods were used. After birth, the male offspring were screened for locomotors and sensorial defects. At postnatal day 21 (PND 21), the male pups were sacrificed and their brains, liver, and plasma were taken for chemical, biochemical, and molecular analyses. Our results show that exposure to Cd significantly increased the metal accumulation and decreased Zn concentrations in the brain of male pups from Cd-treated mothers. Besides, Cd exposure reduced significantly the locomotor activity of the offspring in open-field test, the body weight, and the cranio-caudal length at PND21. Insulin-like growth factor-I (IGF-1) levels in the plasma and liver were also decreased in male pups from Cd-treated mothers. Cd-induced brain development disruption was accompanied by a significant increase of the superoxide dismutase (SOD) activity, induction of the metallothionein (MT) synthesis, and, at the molecular level, by an upregulation of Zrt-,Irt-related protein 6 (ZIP6) gene and a significant downregulation of the expression of the Zn transporter 3 (ZnT3) and brain-derived neurotrophic factor (BDNF) genes in the brain. No significant changes on the expression of genes encoding other Zn-dependent proteins and factors such as ZnT1, ZIP12, NF-κB, and Zif268. Interestingly, Zn supplementation provided a total or partial correction of the changes induced by the Cd exposure. These data indicated that changes in expression of ZnT3 and ZIP6 as well as alteration of other transcription factors, such as BDNF, or Zn-dependent proteins, such as SOD and MTs, in response to Cd exposure might be an underlying mechanism of Cd-induced brain development impairment.

Protective role of zinc against the toxicity induced by exposure to cadmium during gestation and lactation on testis development.

To assess the effects of exposure to Cd and Zn on rat testicular development, offspring, from mothers receiving either tap water, Cd, Zn or Cd+Zn during gestation and lactation periods, were observed on gestational day (GD) 20 and on postnatal days (PND) 12, 21 and 35. During gestation, Cd induced maternal hypozincemia and less transfer of Zn to the fetus. During lactation, progressive Cd accumulation and Zn depletion in testis at PND12 and PND21 were noted. An increase of abnormal seminiferous tubules and a decrease in testis weight and plasmatic testosterone concentration were also observed at PND21 and PND35 respectively. Interestingly, Zn supply induced a significant protection against Cd toxicity. These results suggest that the toxic effects of Cd observed during development are mediated by the disruption of Zn metabolism, which is established in mothers during pregnancy causing Zn deficiency in fetuses and continues to become more pronounced during lactation.

Changes of the mRNA expression pattern of Zn transporters: a probable mechanism for cadmium retention and zinc redistribution in the suckling rat tissues.

The present study was conducted to provide potential mechanism that may be responsible for Cd retention and Cd-induced Zn redistribution in tissues of suckling rat. For this purpose, suckling rats from mother receiving either tap water, Cd, or Cd + Zn during lactation period were sacrificed on postnatal day (PND) 14 and PND 21 for performing chemical and molecular analysis. Our results show that Cd exposure, although it does not affect the milk consumption, it clearly alters the lactational transfer, absorption, and distribution of Zn in the suckling rat organism. At the molecular level, Cd caused upregulation of ZIP 3, ZIP 4, and ZIP 8 gene expressions in the mammary gland of mothers rats and in the intestine of their pups but decreased the expression of ZnT 2 and ZnT 4 only in the mammary tissue at PND 14 and PND 21. Zn supply reversed the Cd-induced decrease in the neonatal Zn apparent absorption and restores the gastrointestinal, brain, and plasma levels of this essential element in the suckling rat organism at PND 14 and PND 21. Also, with this treatment, the gene expressions of ZnT 1 in the mammary gland and ZnT 4 in the neonatal intestine were found to be upregulated at PND 21. Furthermore, our results show that Cd or Cd + Zn treatment increase the neonatal hepatic MTs accumulation at PND 14 only. These results imply that the downregulation of ZnT as well as the overexpression of ZIP transporters, in responses to the Zn depletion induced by Cd in the tissues of lactating rat and their offspring, play a major role in Cd accumulation and Zn redistribution in tissues of suckling rat.

Effects of cadmium and high temperature on some parameters of calcium metabolism in the killifish (Aphanius fasciatus).

This study aims to investigate the influence of high temperature on cadmium (Cd) toxicity in Aphanius fasciatus (Pisces: Cyprinodontidae). For this reason, Cd, mineral, and organic content in the vertebral column as well as the histological structure of gills and bone were compared in fishes exposed for 30 days to Cd (2 mg/L CdCl2) and/or high temperature (26 °C). Cd exposure caused a negative correlation between Cd and Ca concentrations (r = 0.98, p < 0.05), as well as a significant decrease in inorganic components (p < 0.05) and ash weight/dry weight ratio (p < 0.05) in the vertebral column. These changes were accompanied by an increased frequency of histological alterations in gills and bone. Concomitant treatment with Cd and high temperature increases Cd accumulation and Ca depletion in the skeletal tissue and increases the frequency and the severity of histological alterations. These results confirm that temperature increases Cd toxicity and needs to be taken into account for the accurate prediction and assessment of Cd-induced spinal deformities in fish.