Effects of glutathione on the in vivo metabolism and oxidative stress of arsenic in mice.

In this study, we investigated the in vivo effects of exogenous glutathione and buthionine sulfoximine on arsenic methylation and antioxidant capacity in mice exposed to arsenic via drinking water. Thirty-six female albino mice were randomly divided into six groups. All groups were given free access to drinking water that contained arsenic continuously except the control group. After ten days, mice were treated with different levels of glutathione or buthionine sulfoximine. The levels of the metabolites of arsenic were determined in the liver and urine. The levels of glutathione and total antioxidant capacity were determined in the whole blood and liver. Our results showed that the increase of arsenic species in the liver as well as the decrease of blood and hepatic glutathione and total antioxidant capacity, were all relieved by exogenous glutathione consistently. We also observed the involvement of glutathione in promoting arsenic methylation and urinary elimination in vivo. Increase of total arsenic in the urine was mainly due to the increase of dimethylated arsenic. Furthermore, administration of glutathione increased the first methylation ratio and secondary methylation ratio in the liver and urine, which resulted in the consequent increase of dimethylated arsenic percent and decrease of inorganic arsenic percent in the urine. Opposite effects appeared with the administration of buthionine sulfoximine, a scavenger of glutathione. Our study indicated that exogenous glutathione not only accelerated the methylation and the excretion of arsenic, but also relieve the arsenic-induced oxidative stress. This provides a potential useful chemopreventive dietary component for human populations being at risk of arsenic exposure.

Are MTHFR C677T and MTRR A66G Polymorphisms Associated with Overweight/Obesity Risk? From a Case-Control to a Meta-Analysis of 30,327 Subjects.

Several studies have examined the associations of methylenetetrahydrofolate reductase (MTHFR) C677T and methionine synthase reductase (MTRR) A66G polymorphisms with being overweight/obesity. However, the results are still controversial. We therefore conducted a case-control study (517 cases and 741 controls) in a Chinese Han population and then performed a meta-analysis by combining previous studies (5431 cases and 24,896 controls). In our case-control study, the MTHFR C677T polymorphism was not significantly associated with being overweight/obesity when examining homozygous codominant, heterozygous codominant, dominant, recessive and allelic genetic models. The following meta-analysis confirmed our case-control results. Heterogeneity was minimal in the overall analysis, and sensitivity analyses and publication bias tests indicated that the meta-analytic results were reliable. Similarly, both the case-control study and meta-analysis found no significant association between the MTRR A66G polymorphism and being overweight/obesity. However, sensitivity analyses showed that the associations between the MTRR A66G polymorphism and being overweight/obesity became significant in the dominant, heterozygous codominant and allelic models after excluding our case-control study. The results from our case-control study and meta-analysis suggest that both of the two polymorphisms are not associated with being overweight/obesity. Further large-scale population-based studies, especially for the MTRR A66G polymorphism, are still needed to confirm or refute our findings.

Excitotoxicity Induced by Realgar in the Rat Hippocampus: the Involvement of Learning Memory Injury, Dysfunction of Glutamate Metabolism and NMDA Receptors.

Realgar is a type of mineral drug containing arsenic. The nervous system toxicity of realgar has received extensive attention. However, the underlying mechanisms of realgar-induced neurotoxicity have not been clearly elucidated. To explore the mechanisms that contribute to realgar-induced neurotoxicity, weanling rats were exposed to realgar (0, 0.3, 0.9, 2.7 g/kg) for 6 weeks, and cognitive ability was tested using the Morris water maze (MWM) test and object recognition task (ORT). The levels of arsenic in the blood and hippocampus were monitored. The ultrastructures of hippocampal neurons were observed. The levels of glutamate (Glu) and glutamine (Gln) in the hippocampus and hippocampal CA1 region; the activities of glutamine synthetase (GS) and phosphate-activated glutaminase (PAG); the mRNA and protein expression of glutamate transporter 1 (GLT-1), glutamate/aspartate transporter (GLAST), and N-methyl-D-aspartate (NMDA) receptors; and the level of intracellular Ca(2+) were also investigated. The results indicate that the rats developed deficiencies in cognitive ability after a 6-week exposure to realgar. The arsenic contained in realgar and the arsenic metabolites passed through the blood-brain barrier (BBB) and accumulated in the hippocampus, which resulted in the excessive accumulation of Glu in the extracellular space. The excessive accumulation of Glu in the extracellular space induced excitotoxicity, which was shown by enhanced GS and PAG activities, inhibition of GLT-1 mRNA and protein expression, alterations in NMDA receptor mRNA and protein expression, disturbance of intracellular Ca(2+) homeostasis, and ultrastructural changes in hippocampal neurons. In conclusion, the findings from our study indicate that exposure to realgar induces excitotoxicity and that the mechanism by which this occurs may be associated with disturbances in Glu metabolism and transportation and alterations in NMDA receptor expression.

[Effect of subchronic realgar exposure on Glu and Gln in infant rat brain].

OBJECTIVE: To study the effect of realgar on Glu and Gln on rat brain tissues. METHODS: Forty-eight Wistar rats were divided into 4 groups randomly:control group,low dosage group, moderate dosage group and high dosage group. The treatment groups were treated with realgar by gastric perfusion at a dosage of 0.3 g/kg, 0.9 g/kg, 2.7 g/kg and the control group ones were orally given the same volume of 0.5% sodium carboxymethylcellulose (CMC-Na) for 6 weeks. The contents of inorganic arsenic, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) in brain tissues were measured by hydride generation-atomic absorption (HG-AAS) method. The contents of amino acid neurotransmitters in brain tissues of rats were determined by means of high performance liquid chromatography with precolumn derivatization. RESULTS: The levels of MMA and DMA in brain increased as the dosage of realgar increased, while the second methylation index declined. Compared with control group,the levels of Glu was significantly decreased in realgar treated group (P < 0.05); Gln also tended to decrease and that of low dosage group was obviously decreased compared with controls. CONCLUSION: Realgar exposure can cause accumulation of MMA and DMA,declination of second methylation index and the reduction of Glu and Gln in brain tissue.

[Study on water processing conditions of Realgar].

OBJECTIVE: To optimize the water processing (shui-fei) condition of Realgar. METHODS: The processing conditions were optimized by L9 (3)4 orthogonal design with three factors as water dose, process times and dryness temperature. The content of dissolved arsenic As(III) in Realgar was tested by High Performance Liquid Chromatography-hydride generation on-line coupled with atomic fluorescence spectrometry (HPLC-HG-AFS). RESULTS: The optimized conditions were 15 times quantity of water, 8 times for processing and dryness temperature of 40 degrees C. CONCLUSION: The method is accurate and effective,which can be used to evaluate the quality of processed products of Realgar.

Variations in arsenic methylation capacity and oxidative DNA lesions over a 2-year period in a high arsenic-exposed population.

OBJECTIVE: To assess long-term variations in arsenic methylation and oxidative DNA lesions of chronic high arsenic-exposed populations. METHODS: A follow-up study was conducted in 64 chronic high arsenic-exposed subjects from 2004 to 2006. Urinary arsenic species and 8-hydroxydeoxyguanine were measured. RESULTS: Percentages of urinary inorganic arsenic, monomethylarsonate and urinary 8-hydroxydeoxyguanine (8-OHdG) level were significantly higher, but the percentage of dimethylarsinate, the primary methylation index (PMI) and secondary methylation index (SMI) was lower in the ninth year of arsenic exposure compared with the seventh year. Substantial differences in relative arsenic methylation capacity were observed between the seventh and ninth year. Percentages of arsenic species, PMI and SMI were significantly correlated between siblings, and between parents and children. CONCLUSIONS: Arsenic methylation may decrease, but oxidative DNA lesions may increase with the increase of cumulative arsenic exposure level. Both genetic and environmental factors may contribute to variability in arsenic methylation.

[Effect of glutathione and sodium selenite on the metabolism of arsenic in mice exposed to arsenic through drinking water].

OBJECTIVE: To explore the effect of glutathione (GSH) and sodium selenite on the metabolism of arsenic in the liver, kidney and blood of mice exposed to iAsIII through drinking water. METHODS: The mice were randomly divided into control, arsenic, GSH and sodium selenite group, respectively. And each group had eight mice and the mice were exposed to 50 mg/L arsenite by drinking water for 4 weeks. Mice were intraperitoneally injected with GSH (600 mg/kg) and sodium selenite (1 mg/kg) for seven days from the beginning of the fourth week. At the end of the fourth week, liver, kidney and blood were sampled to assess the concentrations of inorganic arsenic (iAs), monomethylarsenic acid (MMA), dimethylarsenic acid (DMA) by hydride generation trapping by ultra-hypothermia coupled with atomic absorption spectrometry. RESULTS: The liver DMA (233.76 +/- 60.63 ng/g) concentration in GSH group was significantly higher than the arsenic group (218.36 +/- 42.71 ng/g). The concentration of DMA (88.52 +/- 30.86 ng/g) and total arsenic (TAs) (162.32 +/- 49.45 ng/g) in blood of GSH group was significantly higher than those [(45.32 +/- 12.19 ng/g), (108.51 +/- 18.00 ng/g), respectively] of arsenic groups(q values were 3.06, 6.40, 10.72 respectively, P < 0.05). The primary methylated index (PMI) (0.65 +/- 0.050) and secondary methylated index (SMI) (0.55 +/- 0.050) in liver sample of GSH group were significantly higher than those (0.58 +/- 0.056, 0.44 +/- 0. 093) in arsenic group. In blood samples, the PMI (0.85 +/- 0.066) in GSH group was significantly higher than that (0.54 +/- 0.113) in arsenic group (q values were 3.75, 5.26, 4.21 respectively, P < 0.05). However, no significant difference was identified between sodium selenite and arsenic groups in liver, kidney or blood samples. And no significant difference was detected in kidney samples among all arsenic exposing groups. CONCLUSION: Exogenous GSH could promote the methylated metabolism of iAsIII, but sodium selenite showed no significant effects.

[Effects of sodium arsenite on catalase in human keratinocytes].

OBJECTIVE: To evaluate the effects of sodium arsenite on the activity, the mRNA and the protein expression of CAT in human keratinocyte cell line (HaCaT). METHODS: The activity of catalase (CAT) was detected by ultraviolet direct velocity assay. RT-PCR was used to detect the mRNA expression of CAT and Western blotting was conducted to detect the protein expression of CAT. RESULTS: If the cells were treated with higher than 5.0 micromol/L sodium arsenite, the activity, mRNA and protein expression of CAT were decreased significantly and in a dosage dependent fashion (P < 0.05). CONCLUSION: CAT is inhibited by sodium arsenite in the transcription, translation and activity levels.

[Cytotoxicity study of vascular endothelial cells by different kinds of arsenicals].

OBJECTIVE: To observe the cytotoxicity of bovine aortic endothelial cells (BAEC) by different kinds of arsenicals. METHODS: Cultured BAEC was exposed to arsenite (iAsIII , 0 - 50 micromol/L), arsenate (iAsV ,0 - 10mmol/L) or monomethylarsonous acid (MMAH III, 0 - 2micromol/L) for 24h, respectively. MTT assay and ICP-MS method were used to evaluate the cell viability and the total arsenic concentration (tAs) of BAEC, respectively. RESULTS: iAsm , iAsV and MMAIII could all decrease the cell viability in a dose-dependent manner (P <0.01). The IC50 values for iAsm , iAsV and MMAIII were determined to be approximately 24.1 micromol/L, 155.9 micromol/L and 1.71 micromol/L, respectively. The order of cytoxicity in BAEC was MMAIII > iAsIII > iAsV . The take-up rate of MMA' by BAEC was significantly higher than iAsR exposure (P < 0.01). CONCLUSION: MMAIII , one of the active intermediate in inorganic arsenic methylation, is highly toxic in BAEC. Methylation of arsenic could be more like a bioactivation process rather than a detoxification process.

[Effect of fluoride on activities of enzyme and ultrastructure in primary cultured rat hepatocytes].

OBJECTIVE: To study the cell viability, activities of enzyme and ultrastructure changes induced by sodium fluoride in primary cultured rat hepatocytes. METHODS: Hepatocytes were isolated using half-in situ collagenase digestion method. Cellular viability was determined by MTT method. The activities of ALT and AST were determined by spectrophotography method. The ultrastructure changes of hepatocyte were observed under transmission electron microscope. RESULTS: After cultured with various concentrations of fluoride for 24 hours, a dose-dependent decrease of cell viability was detected in the hepatocytes. The activities of AST and ALT in the 2 mmol/L and 4 mmol/L groups were significantly higher than the control group (P < 0.05). Transmission electron microscope study showed that in fluoride treated hepatocytes the changes included swollen mitochondria and disordered, disrupted endoplasm reticulum. CONCLUSION: Excessive fluoride induced significant toxicity in primary cultured hepatocytes which manifested the injuries of membrane and organell plasma membrane.

[Oxidative stress induced by NaAsO2 in HaCaT cells].

OBJECTIVE: To study the level of oxidative stress induced by sodium arsenite (NaAsO2) in HaCaT cells. METHODS: The AlamarBlue assay was used to evaluate the viability of HaCaT. The level of ROS was detected by staining. cells with DCFH-DA. The contents of reduced (GSH) and oxidated (GSSG) glutathione were detected with the fluorescent method. The apoptosis and necrosis rates were counted based on the PI staining. RESULTS: The reduction of AlamarBlue increased in the cells treated at dose of 0.001 -1 micromol/L NaAsO2 and decreased in the cells treated at doses of over 10 micromol/L NaAsO2. The fluorescent density of DCF significantly increased in all experimental groups, the contents of GSH and GSSG increased in groups higher than that of 1 micromol/L and higher than that of 5 micromol/L respectively. The apoptosis and necrosis rates were increased markedly at dose of 20 micromol/L. CONCLUSION: Arsenic could induce enhancive ROS in HaCaT. At low levels of arsenic the proliferation of HaCaT was stimulated while at high levels of arsenic was inhibited. The increase in contents of GSH and GSSG could be associated with the excessive ROS and with detoxification in HaCaT.

[Effects of fluoride on proliferation and differentiation of rat osteoblasts in vitro and the antagonistic action of vitamin C].

OBJECTIVE: To study the effects of fluoride on the proliferation and differentiation of osteoblasts in sucking rats and the antagonism of vitamin Cin vitro. METHODS: The enzyme digesting method was used to isolate the rat osteoblasts; the proliferative response was determined by the percents of reduced alamarBlue; the activity of alkaline phosphatase (ALP) was measured by ELISA method. RESULTS: The proliferation of sucking rat osteoblasts was increased at 0.10 - 1.00 mmol/L of NaF, whereas inhibited at >or= 2.00 mmol/L. ALP activity was increased at 0.01 - 0.05 mmol/L of NaF, and decreased at >or= 0.10 mmol/L. The inhibition on proliferation and differentiation at 2 mmol/L NaF was antagonized by vitamin C. CONCLUSION: Fluoride had a two-phase effect on osteoblasts, vitamin C could antagonize the inhibitory effect of higher concentration of fluoride on proliferation and differentiation of osteoblasts.

2,4,6-trinitrotoluene-induced reproductive toxicity via oxidative DNA damage by its metabolite.

Several epidemiological studies and animal experiments showed that 2,4,6-trinitrotoluene (TNT), a commonly used explosive, induced reproductive toxicity. To clarify whether the toxicity results from the interference of endocrine systems or direct damage to reproductive organs, we examined the effects of TNT on the male reproductive system in Fischer 344 rats. TNT administration induced germ cell degeneration, the disappearance of spermatozoa in seminiferous tubules, and a dramatic decrease in the sperm number in both the testis and epididymis. TNT increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in sperm whereas plasma testosterone levels did not decrease. These results suggest that TNT-induced toxicity is derived from direct damage to spermatozoa rather than testosterone-dependent mechanisms. To determine the mechanism of 8-oxodG formation in vivo, we examined DNA damage induced by TNT and its metabolic products in vitro. 4-Hydroxylamino-2,6-dinitrotoluene, a TNT metabolite, induced Cu(II)-mediated damage to 32P-labeled DNA fragments and increased 8-oxodG formation in calf thymus DNA, although TNT itself did not. DNA damage was enhanced by NADH, suggesting that NADH-mediated redox reactions involving TNT metabolites enhanced toxicity. Catalase and bathocuproine inhibited DNA damage, indicating the involvement of H2O2 and Cu(I). These findings suggest that TNT induces reproductive toxicity through oxidative DNA damage mediated by its metabolite. We propose that oxidative DNA damage in the testis plays a role in reproductive toxicity induced by TNT and other nitroaromatic compounds.