Jianhai Zhang

Proteomic analysis of brain proteins of rats exposed to high fluoride and low iodine.

Epidemiological investigations reveal that high fluoride and low iodine have strong adverse effects on the intelligence quotient (IQ) of children. Studies also report that in some high fluoride areas, iodine deficiency also exists, especially in China. Here, with the proteomic techniques, we first report on the proteomic changes in brain proteins in offspring rats at postnatal day 20 exposed to high fluoride and/or low iodine. To investigate molecular mechanisms of central neural system injury induced by the above two elements, proteins were isolated and profiled by two-dimensional gel electrophoresis (2DE). By the analysis of Image-Master 2D Elite software, 71 protein spots in 2DE gels of treatment groups were gained and up- or down-regulated by two folds, and 5 proteins were regulated by five folds, with the comparison to the control group. The proteins changed by five folds were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The identified proteins are mainly related with cellular signaling, energy metabolism, and protein metabolism and provide a valuable clue to explore the mechanism underlining the neurotoxicity of high fluoride and low iodine. Moreover, these results could provide potential biomarkers for hazards caused by excessive fluoride and low iodine.

Inflammatory responses induced by fluoride and arsenic at toxic concentration in rabbit aorta

Epidemiological and experimental studies have demonstrated the atherogenic effects of environmental toxicant arsenic and fluoride. Inflammatory mechanism plays an important role in the pathogenesis of atherosclerosis. The aim of the present study is to determine the effect of chronic exposure to arsenic and fluoride alone or combined on inflammatory response in rabbit aorta. We analyzed the expression of genes involved in leukocyte adhesion [P-selectin (P-sel) and vascular cell adhesion molecule-1(VCAM-1)], recruitment and transendothelial migration of leukocyte [interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1)] and those involved in pro-inflammatory cytokines [interleukin-6 (IL-6)]. We found that fluoride and arsenic alone or combined increased the expression of VCAM-1, P-sel, MCP-1, IL-8, and IL-6 at the RNA and protein levels. The gene expressions of inflammatory-related molecules were attenuated when co-exposure to the two toxicants compared with just one of them. We also examined the lipid profile of rabbits exposed to fluoride and (or) arsenic. The results showed that fluoride slightly increased the serum lipids but arsenic decreased serum triglyceride. We showed that inflammatory responses but not lipid metabolic disorder may play a crucial role in the mechanism of the cardiovascular toxicity of arsenic and fluoride.

Fluoride induced Autophagy via the regulation of mTOR phosphorylation in mice Leydig cells

Fluoride is known to impair testicular function and decrease testosterone levels, yet the underlying mechanisms remain inconclusive. The objective of this study is to investigate the roles of autophagy in fluoride-induced male reproductive toxicity using both in vivo and in vitro Leydig cell models. Using transmission electron microscopy and monodansylcadaverine staining, we observed increasing numbers of autophagosomes in testicular tissue, especially in Leydig cells of fluoride-exposed mice. Further study revealed that fluoride increased the levels of mRNA and protein expression of autophagy markers LC3, Beclin1, and Atg 5 in primary Leydig cells. Furthermore, fluoride inhibited the phosphorylation of mammalian targets of rapamycin and 4EBP1, which in turn resulted in a decrease in the levels of AKT and PI3K mRNA expression, as well as an elevation of the level of AMPK expression in both testes and primary Leydig cells. Additionally, fluoride exposure significantly changed the mRNA expression of the PDK1, TSC, and Atg13 regulator genes in primary Leydig cells but not in testicular cells. Taken together, our findings highlight the roles of autophagy in fluoride-induced testicular and Leydig cell damage and contribute to the elucidation of the underlying mechanisms of fluoride-induced male reproductive toxicity.Fluoride is known to impair testicular function and decrease testosterone levels, yet the underlying mechanisms remain inconclusive. The objective of this study is to investigate the roles of autophagy in fluoride-induced male reproductive toxicity using both in vivo and in vitro Leydig cell models. Using transmission electron microscopy and monodansylcadaverine staining, we observed increasing numbers of autophagosomes in testicular tissue, especially in Leydig cells of fluoride-exposed mice. Further study revealed that fluoride increased the levels of mRNA and protein expression of autophagy markers LC3, Beclin1, and Atg 5 in primary Leydig cells. Furthermore, fluoride inhibited the phosphorylation of mammalian targets of rapamycin and 4EBP1, which in turn resulted in a decrease in the levels of AKT and PI3K mRNA expression, as well as an elevation of the level of AMPK expression in both testes and primary Leydig cells. Additionally, fluoride exposure significantly changed the mRNA expression of the PDK1, TSC, and Atg13 regulator genes in primary Leydig cells but not in testicular cells. Taken together, our findings highlight the roles of autophagy in fluoride-induced testicular and Leydig cell damage and contribute to the elucidation of the underlying mechanisms of fluoride-induced male reproductive toxicity.

Sulfur dioxide inhalation lowers sperm quality and alters testicular histology via increasing expression of CREM and ACT proteins in rat testes

Sulfur dioxide (SO2) is one of the main atmospheric pollutants worldwide, and is reported to be responsible for the formation of severe haze in China. Some studies have demonstrated a potential harmful effect of SO2 on the male reproductive system; however the underlying mechanism is still unknown. The purpose of this study is to investigate the roles of cytochrome P450 (P450), cAMP-responsive element modulator (CREM), and activator of CREM (ACT) in SO2-induced toxicity. Forty-eight male Wistar rats were randomly divided into an experimental and control group. The experiment group was exposed to SO2 in ambient air (10ppm, 4h/day), and the control group was treated with filtered air in the same conditions. After 2 weeks, the results showed a significant decrease in body weight and sperm motility, and an increase in the testis weight-to-body weight ratio as compared to the control group. Histological investigation suggested that SO2 exposure led to loose arrangement of the spermatogenic cells and local structural damage in the seminiferous tubules. Moreover, the expressions of P450, CREM and ACT proteins increased in the testes by 0.22%, 47.26% and 23.38%, respectively. Taken together, SO2 inhalation lowered sperm quality, altered testicular histology, and increased expressions of CREM and ACT proteins in the testes of rats. Overall, these results could contribute to a better understanding of SO2-induced male reproductive toxicity.

Analysis of the roles of dietary protein and calcium in fluoride‐induced changes in T‐lymphocyte subsets in rat

The roles of dietary protein (Pr) and calcium (Ca) levels on the changes in T‐lymphocyte subsets induced by excessive fluoride (F) intake were assessed using rats that were malnourished for 120 days as a model. The CD4+ and CD8+ T‐lymphocytes in the spleen tissue were determined by flow cytometry and immunofluorescence assay. The percentages of CD3+, CD4+, and CD8+ T‐lymphocytes were reduced in the spleen of rats exposed to excessive F, and malnutrition aggravated these changes in the T‐lymphocytes. In addition, the mRNA expression levels of IL‐1β, IL‐2, IL‐6, TNF‐α, and IFN‐γ in the spleen were downregulated significantly. We also reported herein the increased apoptosis ratio following caspase‐9 and caspase‐3 upregulation in the spleen of rats exposed to excessive amount of F. Light and transmisison electron microscopy revealed the irregularly arranged lymphocytes, few lymph nodules and the apoptotic characteristic of lymphocytes, which are caused by the increased expression of caspase. In addition, Pr and Ca supplementation reversed the morphologic and T‐lymphocytic changes in spleen under malnutrition. Taken together, our results revealed an endogenous caspase‐mediated mechanism of regulating the apoptosis of the T‐lymphocyte subsets, as well as the immune‐related cytokine secretion, which reduces the immune function in F‐induced rats.

Choline supplementation alleviates fluoride-induced testicular toxicity by restoring the NGF and MEK expression in mice.

Fluoride is known to cause male reproductive toxicity, and the elucidation of its underlying mechanisms is an ongoing research focus in reproductive toxicology and epidemiology. Choline, an essential nutrient, has been extensively studied for its benefits in nervous system yet was rarely discussed for its prospective effect in male reproductive system. This study aims to explore the potential protective role of choline against NaF-induced male reproductive toxicity via MAPK pathway. The male mice were administrated by 150mg/L NaF in drinking water, 5.75g/kg choline in diet, and their combination respectively from maternal gestation to postnatal 15weeks. The results showed that fluoride exposure reduced body weight growth, lowered sperm count and survival percentages, altered testicular histology, down-regulated the mRNA expressions of NGF, Ras, Raf, and MEK genes in testes, as well as significantly decreased the expressions of both NGF and phosphor-MEK proteins in testes. Examination of data from choline-treated mice revealed that choline supplementation ameliorated these fluoride-induced changes. Taken together, our findings suggest that choline supplementation alleviates fluoride-induced testicular toxicity by restoring the NGF and phosphor-MEK expression. The suitable dosage and supplementation periods of choline await further exploration.

Co‐exposure to fluoride and sulfur dioxide on histological alteration and DNA damage in rat brain

Fluoride (F) and sulfur dioxide (SO2) are the two common environmental contaminants that are associated with neurotoxicity. The present study was conducted to explore individual and combined exposure effects of F and SO2 on histological alteration and DNA damage in rat brain. For this, male Wistar albino rats were exposed to sodium fluoride (100 mg/L NaF) and sulfur dioxide (39.3 mg/m3) individually and in combination for 8 weeks. Histological alteration in brain is evaluated by hematoxylin–eosin staining, showed shrunken neurons, darkly stained small nucleus and decreased cell numbers in F and SO2 exposed groups. The effect of F and SO2 on DNA damage was assessed by comet assay. The results showed an increase in ratio of tailing and tail length in F or/and SO2 administered rats. In addition, the proportion of grade II and III were also increased in individual and combined exposed groups. Compared with the individual exposure, the proportion the grade III was significantly high in combined exposure, suggesting a synergistic effect of F and SO2. These results indicate that the brain was more susceptible to the toxic effects of F and SO2. And combined exposure to these pollutants can lead more pronounced toxic effects on brain.

Arsenic-Induced Autophagy in the Developing Mouse Cerebellum: Involvement of the Blood–Brain Barrier’s Tight-Junction Proteins and the PI3K–Akt–mTOR Signaling Pathway

This study was designed to determine whether the tight-junction (TJ) proteins of the blood-brain barrier (BBB) and the PI3K-Akt-mTOR signaling pathway are involved during arsenic (As)-induced autophagy in developing mouse cerebella after exposure to different As concentrations (0, 0.15, 1.5, and 15 mg/L As(III)) during gestational and lactational periods. The dosage was continually given to the pups until postnatal day (PND) 42. Studies conducted at different developmental age points, like PND21, 28, 35, and 42, showed that exposure to As led to a significant decrease in the mRNA-expression levels of TJ proteins (occludin, claudin, ZO-1, and ZO-2), PI3K, Akt, mTOR, and p62, with concomitant increases in Beclin1, LC3I, LC3II, Atg5, and Atg12. Also, As significantly downregulated occludin and mTOR protein-expression levels with concomitant upregulation of Beclin1, LC3, and Atg12 at all the developmental age points. However, no significant alterations were observed in low- and medium-dose-exposed groups at PND42. Histopathological analysis revealed the irregular arrangement of the Purkinje cell layer in the As-exposed mice. Ultrastructural analysis by transmission electron microscopy (TEM) revealed the occurrence of autophagosomes and vacuolated axons in the cerebella of the mice exposed to high doses of As at PND21 and 42, respectively. Finally, we conclude that developmental As exposure significantly alters TJ proteins, resulting an increase in BBB permeability, facilitating the ability of As to cross the BBB and induce autophagy, which might be partly the result of inhibition of the PI3K-Akt-mTOR signaling pathway, in an age-dependent manner (i.e., PND21 mice were found to be more vulnerable to As-induced neurotoxicity), which could be due to the immature BBB allowing As to cross through it. However, the effect was not significant in PND42, which could be due to the developed BBB.

Combination of Fluoride and SO 2 Induce DNA Damage and Morphological Alterations in Male Rat Kidney

Background/Aims: We investigated the combined toxic effect of sodium fluoride (NaF) and sulfur dioxide (SO2) on kidney morphological changes and DNA damage in male Wistar rats. Methods: In this study we selected totally 96 male Wistar rats (12-week-old) then randomly group-housed them into four cages, treated with deionized water, NaF, SO2 and co-treatment of NaF and SO2 respectively. Morphological changes of kidney were detected by hematoxylin and eosin (H&E) staining at 2, 4, 6 and 8 weeks. Correspondingly, tailing ratio and comet length were measured by BAB Bs Comet Assay System, including DNA damage special unit were calculated to evaluate the grades of kidney DNA damage at the same time. Results: Treated groups showed a body weight decrease when compared to control group. However, no significant difference in the relative weight of kidney was found in all four groups. It is noteworthy that at 2, 4, 6 and 8 weeks after exposure, the morphological alteration of renal tubules were observed in all treated groups, especially in group-IV. Also, at 4 and 6 weeks, notable DNA damage was found in all treated groups, as assessed by significantly increasing trend of comet length tailing ratio. Conclusion: The study manifests that presence of NaF and SO2 will not only induce renal tissue lesions but also impact DNA integrity. In addition, this combined exposure exhibits a synergistic effect, characterizing a dose-dependence and time correlation. These findings may provide novel insights regarding perturbations of DNA damage and its functions as a potential new mechanism, by which cautious interpretation of NaF and SO2 co-exposure evolved in both animals and human beings is necessary.

Perfluorooctanoic acid exposure disturbs glucose metabolism in mouse liver

ABSTRACT Environmental pollutants such as perfluorooctanoic acid (PFOA) can influence human metabolism processes and are associated with certain metabolic diseases. To investigate the effect of PFOA on liver glucose homeostasis, adult male Balb/c mice were orally administered 1.25 mg/kg of PFOA for 28 d consecutively. Compared with the control mice, the body weights of the PFOA‐treated mice were unchanged following exposure. However, PFOA exposure increased fasting blood glucose levels and decreased glycogen and glucose content in the liver of treated mice, but did not influence blood insulin significantly. The increased blood glucagon might contribute to the hyperglycemia observed in the PFOA‐treated group compared with the control group. In addition, pyruvate tolerance tests supported enhanced glucose production ability in PFOA‐exposed mice. Consistent with the increase in blood glucose and decrease in hepatic glucose and glycogen, PFOA exposure decreased the protein level of glycogen synthase in the mouse liver, but increased the level of glucokinase. Furthermore, liver pyruvate, as well as mRNA levels of enzymes involved in the Krebs cycle, such as citrate synthase, isocitrate dehydrogenase, and alpha‐ketoglutarate dehydrogenase, increased in the PFOA‐treated group. PFOA exposure did not affect muscle glucose or glycogen levels. Indirect calorimetry showed higher VO2 consumption and respiratory quotient values in the PFOA‐treated group compared with the control group, implying that PFOA treatment might promote energy consumption in mice, with a reliance on carbohydrates as a primary source of energy. Thus, our findings indicate that subacute exposure to PFOA might enhance glycogenolysis and gluconeogenesis and promote carbohydrate consumption. HighlightsPFOA exposure induced high fasting blood glucose.PFOA exposure decreased glycogen and glucose content in the liver.PFOA exposure enhanced glycogenolysis and gluconeogenesis.PFOA exposure promoted carbohydrate consumption.