Studies On Endogenous NO Level, ENOS Activity And ENOS Phosphorylation By Arsenic Exposure

BackgroundChronic arsenic poisoning is now a major worldwide public health problem. Epidemiological studies link the excess intake of arsenic with arsenic-related vascular diseases such as peripheral and cardiovascular disease, arteriosclerosis, Raynaud's syndrome, hypertension, and Blackfoot disease. However, the exact mechanistic details remain unknown.NO is a messenger molecular playing important roles in neurotransmission, vasodilation, and immune response. Decreased availability of biologically active NO in the endothelium is implicated in the pathophysiology of several vascular diseases. The manifestations of vascular diseases caused by prolonged exposure to arsenic, such as peripheral arterial occlusive disease, hypertension, and atherosclerosis , are consistent with those induced by impaired production of endo-thelial NO. These associations have led us to hypothesize that systemic NO formation is reduced through prolonged exposure of humans to arsenic in the drinking water.On the other hand, the metabolism and biotransformation process of inorganic arsenic has been clarified recent years including reduction of iAsv to iAs~III with subsequent methylation yielding mono-, di-, and possibly trimethylated metabolites. MMA~III and DMA~III have been reported to be highly cytotoxic and genotoxic than iAs . And methlation of arsenic has been viewed as bioactivation process rather than detoxification process. Effects of MMA~III and DMA~III on arse-nic poisoning are now one of the important aspects of attention.ObjectivesOur field study was carried out in an endemic area of chronic arsenic poisoning. We compared blood levels of tAs, iAsv and its metabolites MMA and DMAV; serum levels of N02~ /N03" between subjects chronically exposed to arsenic and minimally exposed controls, as well as analytized the correlation of blood concentrations of iAsv, MMAV and DMAV with serum NO2"/NO3~levels, trying to evaluate the relation of systemic NO production with prolonged arsenic exposure.In the experimental studies, an in vitro study was first performed to examine a direct effect of MMA111 on eNOS activity with a crude eNOS enzyme prepared from bovine aortic endothelial cells ( BAEC). We also observed ROS generation and ROS-mediated eNOS phosphorylation by MMA111 in cultured BAEC.Methods1. Epidemiological investigation was carried out in Wuyuan, Inner Mongolia. 33 residents using tube wells containing high concentrations of tAs (0.41 ± 0.11 jxg/ml) , and 10 residents using tube wells containing minimal concentrations of tAs (0.02 ±0.01 u,g/ml) were selected as chronical arsenic exposure subjects and minimally exposed controls, respectively. Physical examinations including skin lesions were conducted, and the presence of cold weather-associated pain, numbness, and coldness in extremities of feet or hands, which were regarded as peripheral vascular dysfunction symptoms, were interviewed.2 . Determination of arsenic levels ; after addition of 2 M NaOH 2 ml to 0.5 ml blood or water samples, the mixture was heated at 95°C for 3 h. Levels of tAs, iAsv, MMAV and DMAvwere determined by AAS.3. Measurement of NO2"/NO3" : supernatant of deproteinized serum (1.5 ml) was mixed with equal volume of glycine-NaOH buffer (pH 9.7) and cadmium filings (2.5 g) pretreated with 5 mM CuS04 solution for 5 min. After heat-ing was done at 651 for 30 min to convert nitrate to nitrite, a portion (2 ml) of the mixture was reacted with the Griess reagent (1 ml) consisting of 1% sulfa-nilamide in 3 M HC1 and 0. 2% N-( 1 -naphtyl) -ethylenediamine HC1. The resulting sample was measured at 540 nm with NaN02 as a standard.4. Cell culture; BAEC was cultured in DMEM-F12 supplemented with 10% fetal calf serum, 10 U/ml penicillin and 100 |xg/ml streptomycin, 10 U/ ml heparin and 5 ng/ml fibroblast growth factor in a C02 incubator.5. eNOS enzyme preparation: membrane fractions were extracted from BAEC and used as the crude eNOS enzyme in the experiments.6. eNOS activity assay: crude eNOS enzyme was exposed to various arseni-cals (MMAm(0-15 jjlM) ; iAs111, iAs\ MMAV and DMAv10 uJM, respectively; or MMAm( 10 uJVf )/DTT( 100 uJVf) ). eNOS activity was determined as production of L-[3H] citrulline from L-[3H] arginine.7. Cell viability: measured by MTT assay.8. Western-blot analysis: BAEC was exposed to MMAm(0. 75 fxM, 0-15 min) without or with NAC pretreatment (1 raM, 2 h) , iAsm{ 100 fxM, 0-60 min) , or MMAni-DTT compound (0. 75 fxM, 0-15 min). Western blot was done with the specific antibodies for eNOS or phospho-eNOS^^c,.9 . Intracellular ROS generation ; assessed with the fluorescent probe , 2' ,7'-dichlorodihydrofluorescein diacetate (H2DCFDA) by fluorescent microscopy equipped with a digital imaging system.10. Measurement of intracellular arsenic concentration; BAEC was exposed to 0. 75 jxM of MMAIU, iAsm or MMAm-DTT compound for 15 min at 37 0. 05). What' s more, DTT (100 |xM) treatment markedly reversed the decreased eNOS activity by MMA exposure ( p <0. 05).3. Suppression of cell viability: we observed the changes of cell viability of BAEC exposed to MMA1" or iAs111 in low concentrations (0-2 u,M) for 24 h. As to MMA111, cell viability markedly decreased in a dose-dependent manner and cells were almost all died at 2 jxM. However, there was no effect on BAEC viability under such low levels of iAs . Only high doses of iAs were cytotoxic to BAEC. The IC50 values for MMA111 and iAsmwere determined to be approximately 1.7 jxM and 24 jxM, respectively.4. Induction of eNOS phosphorylation and ROS generation; exposure of BAEC to MMA111 (0.75 u-M ) caused a rapid increase in phosphorylated eNOSSerll79 (1.41-fold increase vs control) at 15 min after exposure ( p <0.05). On contrast, up to 100 jxM iAs111 caused the increase of phosphorylated eNOSs. erll79( 1. 32-fold increase vs control) at 30 min ( p <0. 05 ). NAC pretreatment ( 1 mM, 2 h) blocked the MMAm-induced eNOS phosphorylation; MMAm-stim-ulated ROS production was confirmed in live cells by detecting DCF fluorescence with fluorescent microscopy, which could also be suppressed by NAC; MMAm-DTT compound didn't induce the eNOS phosphorylation, and also didn't stimulate the ROS generation.5. Intracellular tAs concentrations; when BAEC was exposed to different arsenicals both at 0. 75 jxM for 15 min, intracellular tAs concentrations of MMA -exposed cells were about 13. 5 times higher than iAs111-exposed cells. Interestingly, MMAin-DTT exposure contained more intracellular tAs than MMA111.