Toxic Effects Of Arsenic And Sulfur Dioxide Co-exposure On Renal And Kidney In Mice

Arsenic?As?and sulfur dioxide?SO₂?are two environmental pollutants.As is a metal with strong toxicity,which has been identified as a carcinogen.Both short-term and long-term exposure to As can cause certain negative effects on human health.It is generally accepted that the non-occupational route of human exposure to As is mainly through drinking water.Up to now,epidemic areas of arsenicosis mainly involve 70 countries around the world,and in China more than 8 provinces and 37 counties were affected.More than 200 million people are exposed to As at concentrations higher than 10 ?g/L,and one hundred million sufferers were distributed in Asia.On the other hand,benefiting from the continuous optimization of China's energy structure and the enforcement policies of industrial waste gas treatment,the sulfur dioxide?SO₂?pollution has basically been controlled in large and medium-sized cities.However,with increasing consumption of coal,the pollution of SO₂ is still existed in many areas.Moreover,in some areas,like Guizhou and Yunnan province of China,the As pollution is often accompanied by SO₂ pollution because of high arsenic coal burning.Based on above reasons,humans are often exposed to SO₂,in addition to As.Previous study showed that inorganic arsenide was mainly methylated by liver after inhaling into human body,while kidney was regarded as the main storage and excretory organ of As.Thus,As could induce liver and kidney toxicity.Meanwhile,many studies show that SO₂ causes not only respiration system disease but also a systemic toxicity.Moreover,chronic exposure to As and SO₂ has been reported to be a causative factor in the initiation of many cancers,such as upper respiratory,skin,liver and bladder cancer.Therefore we presume As-SO₂ co-exposure could induce more serious liver and kidney injury.However,all previous studies focus on single exposure to As or SO₂,whereas possible synergistic effects due to the combination of As and SO₂ are largely disregarded.In view of the above questions,we proposed this topic:1? In this study we mimic the environmental conditions and treat C57BL/6 mice with 5 mg/L As for two months and 5mg/m3 SO₂?6 h/day?for a month alone or in combination and test their liver.We find that As or SO₂ exposure alone could decrease activities of antioxidase such as superoxide dismutase?SOD?,catalase?CAT?,while As-SO₂ co-exposure could further decrease antioxidase activities.On the other hand,As-SO₂ co-exposure induces massive accumulation of reactive oxygen species?ROSs?,which including O2·-and H2O2,and malondialdehyde?MDA?,the production of lipid peroxidation,and eventually lead to oxidative stress injury in liver.Moreover,beside the liver injury and inflammation cell infiltration exist in As or SO₂ group,As-SO₂ co-exposure could also induce balloon-like changes in liver cells,obvious hyperemia in part of central vein and adjacent hepatic sinus,and hepatic sinus deformation.Subsequently,with various technologies,such as western blotting and quantitative polymerase chain reaction,we found that compare with As or SO₂ exposure alone,As-SO₂ could increase the expression level of nuclear factor kappa B?NF-?B?and signal transducers and activators of transcription?STAT-3?.The variation trend of interleukin-6?IL-6?,interleukin-1??IL-1??and tumor necrosis factor?TNF-??,the downstream factors of STAT-3 and NF-?B signaling pathway,induced by As-SO₂ co-exposure are same with STAT-3 and NF-?B.Accordingly,we found that As-SO₂ co-exposure could increase oxidative stress injury and activate NF-?B signaling pathway,ultimately leading liver injury.2? Except the toxicity effects of mice liver,we also detect the status of mice kidney.We find that glomerular fibrosis happened to mice kidney of As-SO₂ co-exposure group.Moreover oxidative injury and inflammatory responses were found more serious in As-SO₂ group.To further study its mechanism,we detected the key indicator of autophagy,such as microtubule associated protein light chain 3?LC-3?and the mammalian target of rapamycin?m TOR?,results showed that there are no significant differences between As-SO₂ group and As/SO₂ group.On the contrary,members of the Cysteinyl aspartate specific proteinase?Caspase?family,which is an important family in the progression of apoptosis,were remarkably changed by As-SO₂ co-exposure.Based on above results,we presume that As-SO₂ co-exposure could aggravate liver and kidney injury through oxidative stress injury and inflammatory responses mediated apoptosis.To verify the above results,we established a 293 T cell model of As-SO₂ co-exposure.To determine whether oxidative stress injury,inflammatory responses and cell apoptosis play a role in As-SO₂ induced kidney injury,we apply N-Acetyl-L-cysteine?NAC,the inhibitor of ROS?,BAY11-7082?the inhibitor of NF-?B?and Z-VAD-FMK?the inhibitor of caspase family?.After inhibitors were applying,As-SO₂ co-exposure induced cell viability decrease were reversed.Accordingly,we found that As-SO₂ co-exposure could increase oxidative stress injury?activate NF-?B signaling pathway and make it transform into cell nuclear? activate the downstream factors of NF-?B,such as IL-1? and TNF-??induce caspase family activation?ultimately leading kidney injury.3? Previous studies have shown that As or SO₂ exposure is related with occurrence and development of hepatocellular carcinoma,however there are no study focus on possible synergistic effects due to the combination of low concentrations of As and SO₂.In our study,Hep G2 cells were treated with 5 10-5 mg/L SO₂ or 1 10-3 mg/L As for 120 hours.Wound healing assay results suggested that As or SO₂ exposure alone do not cause the obvious cell migration.On the other hand,co-exposure to As and SO₂ not only promoted Hep G2 cell migration but also induced more filopodia on the cell surface.Western blotting and quantitative polymerase chain reaction were used to detected whether Integrin family plays a role in As-SO₂ induced cell migration,results showed that this process is Integrin ?v?3 dependent.To verify the above results,Cilengitide,the inhibitor of Integrin ?v?3,was performed,result showed that it can significantly attenuated the As-SO₂ induced Hep G2 cell migration.In addition,we also found that interleukin-6?IL-8?,transforming growth factor-??TGF-??and matrix metalloproteinase?MMP?family are involved in this process.4? Since our study confirmed that As exposure can aggravate liver and kidney damage by inducing oxidative stress injury,we speculated that antioxidants could play a certain role in reversing liver and kidney damage caused by As exposure.Previously in this laboratory,we extracted anthocyanin,a natural strong antioxidant,from muscathumburg.And we already proved that this kind of anthocyanin has strong anticarcinogenic activity in Hep G2 cells in former study.Therefore we have a hypothesis,which is anthocyanin could reverse As induced toxicity.To determine whether anthocyanin protects against liver and kidney inflammation from As exposure,Kunming mouse were exposure to As and/or different concentrations of anthocyanin.Result showed that,both two concentrations of anthocyanin could significant reduce expression level of As increased MDA,and recover the antioxidative enzyme activity that were reduced by As exposure.Anthocyanin could also decrease the inflammatory responses related proteins,such as IL-1? and TNF-?.In this study,we found that As-SO₂ co-exposure could aggravate liver and kidney injury in vitro and in vivo,and discussed the possible mechanism by using 293 T cells.We also found that low concentrations of As-SO₂ co-exposure promote Hep G2 cells migration through Integrin ?v?3.Moreover,we found natural antioxidant anthocyanin could significantly reverse the toxicity effects of As.Our study not only could generate some novel mechanistic hypotheses of liver and kidney impairment caused by As and SO₂,but also provide experimental evidence for health protect effects of anthocyanin on As exposure.