Adverse Effect Of Copper On Intestinal Epithelial Cells And Subacute Toxicity Of Copper On Rats

Since copper is an essential element, both defiency and excess of copperhave adverse effect on human health. For healthy humans who are not occupationallyexposed the major route of exposure to copper is oral. Human and animal studiesindicated that gastrointestinal (GI) tract is the target organ of copper; but themechanism of copper toxicity on GI tract remains unclear. The effects of acute andchronic copper toxicosis by gavage and via feed are well known; however the effectsof ingestion of copper through drinking water are less well characterized. We used invitro test and animal study to investigate intestinal epithelial toxicity of copper andsubacute toxicity of copper administered in drinking water on rats. In in vitro study, Caco-2 cell monolayers was used to study the cytotoxicityof copper, effects of copper on functions of intestinal epithelial cells and involvedmechanisms. The results showed: 1. A time- and dose-dependent decrease of cellviability, significant increase of ROS production, decreased cloning efficiency andproliferation in Cu-treated cells. 2. The apical side of Caco-2 cell was more resistantto copper than the basolateral side of Caco-2 cells, because of the polarizedcharacterization of Caco-2 cell monolayers. 3. Apical treatment with copper (30 -100μM, Hanks'buffered salt solution, up to 3 h) induced a time- andconcentration-dependent increase in permeability reflected by progressive decrease ofTEER and increased mannitol passage of Caco-2 cell monolayers, accompanied bydeorganization of F actin, but without significant effect on tight junctional proteinZO-1. 4. At a dose without any adverse effects on viability and permeability ofCaco-2 cell monolayers, copper treatment (300 μM, complete medium, 24 h)enhanced apical (AP) to basolateral (BL) and decreased BL to AP transport, andincreased accumulation of Rho-123 in Caco-2 cells. 5. A time- andconcentration-dependent cellular accumulation of Rho-123 in Caco-2 cells. 6. Copperinduced MT-2A transcription demonstrated by a concentration-dependent increase inMT-2A mRNA levels in copper treated cells. In summary, copper exposure coulddecrease the viability and proliferation of Caco-2 cells; copper could decrease the 3复旦大学博士学位论文 铜的肠道上皮细胞毒性和对大鼠的亚急性毒性研究barrier functions of Caco-2 cell monolayers through increasing paracellularpermeability and decreasing P-glycoprotein activities; copper could increasemetallothionein (MT) expression through inducing MT gene transcription, whichmight play important roles in copper detoxification and absorption. The resultssuggested that changes in paracellular permeability and P-gp activity could reflect theepithelial toxicity of copper, which might be used as sensitive endpoints to reflectrelevant xenobiotics on GI epithelius. In animal study, a subacute toxicity study (14-day exposure) was conductedin male Sprague-Dawley rats by the drinking water route. Animals were examined forhistopathology, clinical pathology, tissue copper and zinc levels, tissue MT expressionlevels and MT mRNA levels in related target organs. The cupric sulfate concentrationin drinking water of 4 groups was 0, 100, 300 and 1000 ppm respectively. Theestimated dose of 4 groups was 0, 6.4, 18.8 and 49.9 mg Cu/kg bw/day respectively.The results showed: 1. Cupric sulfate in drinking water caused decreased waterconsumption and decreased final body weight gain (75.2% of that of controls) atconcentration of 1000 ppm. The decreased body weight gain in 1000 ppm group werelikewise attributed to decreased feed consumption and decreased water consumptionrather than to specific copper toxicity. 2. In the 300 ppm and 1000 ppm groups, thecopper concentrations in the liver were significantly increased compared with that ofcontrols (2.45±0.23 mg/kg wet weight). In the 1000 ppm group, the liver copperconcentration was 18.12±9.19 mg/kg wet we...