The Erythrocyte Damage And Its Mechanism In Rats Exposed Low Levels Arsenic

Objective1. To analyze and compare the changes of the erythrocyte and RBC membrane proteins in ratsexposed low levels arsenic and to explore the potential biomarkers of chronic arsenicpoisoning.2. To study the molecular mechanism of injury on rats erythrocyte membrane proteins in lowlevels arsenic exposure and to open up new avenues for the mechanism of arsenic toxicity.3. To establish the animal model exposed the low levels arsenic in drinking water, to determinethe general toxicity and the RBC membrane damage indices and to provide a basis for thesafety evaluation of national standard for arsenic in drinking water.Methods40 male Sprague- Dawley rats (140g-160g) were divided randomly into 4 groups: controlgroup, 10μg/L As treatment group, 60μg/L As treatment group and 360μg/L As treatment group.The As treatment groups were exposed to arsenic dissolved in drinking water. And the rats ofcontrol group drink ordinary clean tap water. After 30 days of exposure, all animals wereanesthetized and collected blood samples from the abdominal aorta. The major organs wereremoved and weighted. Analysised and compared rats general toxicity indicators including bodyweight, organ weight coefficient in each group. The RBC toxic damage indices were detected indifferent groups, specifically including: red blood cell parameters, eryptosis rate, erythrocytemorphology, erythrocyte deformability and aggregation. The erythrocyte membrane proteinswere seprated and compared by sodium dedecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Immunofluorescence Assay (IFA).Results1. Rats general toxicity indicatorsAt the same time, the weights of rats in different groups have no significant differences(P>0.05). Compared with the control group, in 360μg/L arsenic exposure group spleencoefficients increased significantly, the difference was statistically significant (P<0.05); the restof the brain, heart, lung, liver and kidney coefficients have no significant difference among thegroups (P>0.05). 2. The RBC toxic damage indices2.1 Red Blood Cell ParametersCompared with the control group, the abnormal changes have not occurred in red blood cellparameters of the exposed groups (P>0.05).2.2 Eryptosis RateFlow cytometry results showed that, compared with the control group, eryptosis rateincreased significantly in 360μg/L arsenic exposure group (P<0.05).2.3 Erythrocyte MorphologyThe red blood cell morphology was examined by scanning electron microscopy (SEM). Inthe As treatment groups abnormal shape red blood cells were observed. And the changes in360μg/L As treatment group were particularly obvious, including more irregular-shaped redblood cells and spheroechinocytes.2.4 Erythrocyte Deformability and AggregationErythrocyte rheology test results showed that, compared with the control group, in 360μg/Larsenic exposure group, the low shear whole blood apparent viscosity, relative viscosity andreduced viscosity were significantly decreased (P<0.05). In the 360μg/L As exposure group, theintermediate shear whole blood apparent viscosity was significantly lower (P<0.05). And theerythrocyte aggregation index in 360μg/L As exposure group was significantly smaller than theindex in the control group (P<0.01). The high shear whole blood apparent viscosity, relativeviscosity and reduced viscosity have no significant change in the different groups (P>0.05). Theerythrocyte rigidity index and deformation index also have no significant changes (P>0.05).3. The damage of erythrocyte membrane proteinsSix different proteins, including spectrin-α, spectrin-β, ankyrin, band 3 protein, band 4.2protein and actin, were isolated from the erythrocyte membrane of rats by SDS-PAGE.Compared with the control group, ankyrin content has decreased in the 360μg/L dose group, andthe difference had statistical signification (P<0.01). With exposure levels rising, band 3 proteinexpression presented an increase trend in the treatment groups. And the result showed that theincrease of band 3 was statistically significant in the 360μg/L dose group (P<0.05). Theexpression levels of the remaining four proteins were not significantly changed (P>0.05).Immunofluorescence assay revealed that band 3 protein and CD35 increased and aggregated tothe RBC membrane in rats exposed to arsenic.Conclusions1. Low levels of arsenic exposure can produce toxic effects on rat erythrocyte, resulting inincreased eryptosis rate, abnormal RBC morphology and decreased RBC aggregation. These changes may be related to erythrocyte membrane proteins abnormalities caused by arsenicexposure, which specifically included band 3 and ankyrin levels abnormalities and band 3and CD35 clustering on the erythrocyte membrane.2. The injury of RBC membrane protein appeared earlier than the abnormal changes of RBCnumber and volume in rat of arsenic exposure. Erythrocyte membrane protein may be theearly biological marker in patients of chronic arsenic poisoning. Compared with other organs,spleen may be the early target organ in rats exposed to low levels arsenic.