Effects Of Cadmium And Uranium Exposure Combined Gamma-irradiation On The Hematopoietic System And Renal Cell Damage And Protective Study For Uranium Internal Contamination

Combination exposure to heavy metal cadmium (Cd) toxicants and radiation is a quite common situation in both occupational and natural environments. Uranium (U) used for nuclear fuel and nuclear weapon charge was produced and recycled in the uranium industry worldwide. It has been shown that Cd, U and radiation are the important mutagenic factors. The combination effect of Cd and irradiation on health hazard has received considerable attention in recent years, but little is known about this interactive effect on health risk especially on hematopoietic system. On the other hand, the health hazard of uranium exposure and chelation therapy can be of concerned worldwide. Once uranium internal contamination for human occur in a radiation accident, no uranium decorporation drugs can be used in medical treatment until now.In the present study, the genotoxic and/or cytotoxic effects of chronic low dose Cd exposure and Cd exposure combined with gamma-irradiation on rat hematopoietic system and renal tissue were investigated with blood Cd concentration as Cd exposure index, in an attempt to provide experimental clue in assessing the health risk of the chronic interaction between environmental Cd and irradiation. Moreover, we investigated the effects of CBMIDA-CaNaT on uranium removal in vivo and in vitro, and the protective efficacy of CBMIDA-CaNa2 against uranium-induced human renal cell damages. It is necessary to develop a high effective and low toxic decorporation drugs for uranium poisoning treatment.Part I Effects of combined chronic cadmium exposure and gamma-irradiation on the hematopoietic system and kidney tissue of ratsObjective To investigate the effects of chronic cadmium exposure and cadmium exposure combined with y-ray irradiation on the hematopoietic system and kidney tissue of rats.Methods One hundred and thirty-two male SD rats were randomly divided into 6 groups with 22 rats each group:①normal saline (NS) control group,②0.1mg CdCl1.kg-1.d-1 low-dose exposure group (LD Cd group),③0.5mg CdCl2.kg-1.d-1 high-dose exposure group (HD Cd group),④2Gy radiation group,⑤0.1 mg CdCl2.kg-1.d-1 exposure plus 2 Gy radiation group (LD Cd+IR group), and⑥0.5mg CdClkg-1.d-1 exposure plus radiation group (HD Cd+IR group). Animals in all groups were either received intraperitoneal injection of NS or CdCl2 once daily for 8 weeks, and then irradiated with 2Gyγ-rays at a dose rate of 0.8Gy/min. Six rats/group were anesthetized 2-4h after irradiation, from which heart blood was drawn for Cd measurement using inductively coupled plasma mass spectrometry (ICP-MS), femoral bone marrow cells was collected for colony forming unit-granulocyte and macrophage (CFU-GM) culture, micronuclei formation of polychromatic erythrocytes (PCE) and the PCE/NCE (normochromatic erythrocyte) ratio assay. The 2nd eight rats/group were sacrificed 10 days after irradiation. The 3rd eight rats/group were exposed to Cd for additional 4 weeks after 8-week Cd exposure plus 2Gy irradiation. After rats were anesthetized, the heart blood was collected for Cd measurement, peripheral white blood cells (WBC) counting and the peripheral lymphocyte culture for testing micronuclei and hprt mutation detection using multinucleated cell assay. The pathological changes of renal tissue with H.E. staining were observed under a microscope. The total proteins were extracted from kidney tissue and the level of metallothionein (MT) protein expression was detected by Western Blot assay.Results After rats were exposed to 0.1 mg Cd.kg1.d-1 for 8 weeks through 12 weeks, Cd in blood reached to about 135ug.L-1 and had no significant effect on amount of WBC and micronuclei formation and hprt mutation frequency in peripheral blood lymphocytes. On the other hand, chronic exposure to O.lmg Cd/kg could significantly decrease the radiation-induced micronucleus formation and hprt mutant frequency, suggesting that exposure to low dose cadmium could induce antagonistic effect on radiation-induced genotoxicity. When Cd in blood reached to about 458-613ug.L-1 after exposure to 0.5mg Cd/(kg.d) for 8 weeks through 12 weeks, the amount of WBC was markedly increased, and the number of CFU-GM of marrow bone cells didn't change as compared to control rats, suggesting high dose Cd could stimulate an inflammatory response. Meanwhile, micronucleus formation and hprt mutant frequency of lymphocytes significantly increased compared to control rats. Moreover, an additive effect on micronuclei and hprt mutant induction was observed in HD Cd+IR group with an increase of up to 20%-70% and 10%-20% compared to rats exposed to high dose Cd and irradiated rats, respectively. For 2Gy irradiation, the number of WBC was significantly reduced after 10 days of irradiation, micronuclei formation of PCE was markedly increased and the ratio of PCE/NCE and the number of CFU-GM was significantly decreased after 2-4h of irradiation. The rats exposed to low dose Cd for 8 weeks showed significantly increased micronuclei formation of PCE and decreased ratio of PCE/NCE although the number of CFU-GM didn't change as compared with normal control, indicating that PCE of marrow bone was sensitive to Cd exposure. But exposure to low dose Cd for 8 weeks could decrease the radiation-induced micronuclei formation of PCE, showing the adaptive response to following irradiation. For high dose Cd exposure for 8 weeks, the ratio of PCE/NCE was significantly decreased and micronuclei formation pf PCE was markedly increased as compared with that of LD Cd group although number of CFU-GM didn't change. Moreover, exposure to high dose Cd could enhance the severity of radiation-induced marrow bone cells damages. The pathological observation of kidney tissue showed that 2 Gy irradiation cause mild renal cortex damage 10d or 4 weeks post-irradiation, exhibiting mild degeneration and necrosis of renal glomerulus capillary endothelium and renal tubular epithelial cells and minor interstitial edema. Though exposure to low dose Cd for 8 and 12 weeks also resulted in minor renal glomerulus and tubule damage, they were more obvious than that of 2Gy irradiation group 4 weeks post-irradiation. However, combined low dose Cd exposure and irradiation didn't aggravate kidney tissue damage. Exposure to high dose Cd for 8 and 12 weeks led to more serious renal tissue damage than that of LD Cd group, showing bleeding and congestion of renal glomerulus, necrosis and exfoliation of renal tubular epithelial cell, tubular ectasia and vacuolation and lots of inflammatory cell infiltration in renal cortex and medulla. Furthermore, combined high dose Cd exposure and irradiation caused more serious tissue damage than that of HD Cd group.Conclusion Chronic exposure to low dose Cd induces the adaptive response of peripheral blood lymphocytes and PCE of marrow bone to radiation. Chronic exposure to high dose cadmium could stimulate an inflammatory response and increase the radiation-induced cytotoxicity and/or genotoxicity of peripheral blood lymphocytes and PCE and granular progenitor cell of marrow bone. Exposure to low dose Cd causes the minor damage of kidney tissue, which becomes more serious with Cd dose and exposure time. Combined low dose Cd exposure and irradiation didn't affect the severity of kidney tissue damage, while combined high dose Cd exposure and irradiation led to synergistic effect on kidney tissue damage.Part II:Efficacy of chelator CBMIDA-CaNai for removing uranium and protecting uranium-induced human kidney cell damage in vivo and in vitroObjective To assess the efficacy of C-BMIDA-CaNai for removing U(Ⅵ) and protecting uranium-induced cell damage in rats and human proximal tubular epithelial cells (HK-2), and explore the mechanism by which CBMIDA-CaNa2 protecting uranium-induced cell damage.Methods The cytotoxicity of U02(CH3COO)2 to HK-2 cells for 48h was assayed by viable cell counting. After HK-2 cells were exposed to different concentration of UO2(CH3COO)2 immediately or for 24h or 48h followed by 10,50 or 250μmol/kg CBMIDA-CaNai treatment for another 24h or 48h, the uranium contents in HK-2 cells were measured by ICP-MS, the cell survival was assayed by cell counting kit-8 assay, micronuclei formation by the cytokinesis-block technique and the production of intracellular reactive oxygen species (ROS) by 2',7'-dichlorofluorescin diacetate (DCFH-DA) oxidation, and the level of MT protein expression was detected by Western Blot assay. CBMIDA-CaNa2 injection at a dose of 120μmol/kg was administrated by intramuscularly to male SD rats immediately after a single intraperitoneal injection of UO2(CH3COO)2. Twenty-four hours later uranium contents in urine, kidneys and femurs were measured by ICP-MS. DTPA-CaNa3 was used for control for these experiments.Results The IC50 value of UO2(CH3COO)2 to HK-2 cells for 48h was 629μmol/L from that the concentration of UO2(CH3COO)2 for uranium removal and uranium-induced cell damage assay was determined. After HK-2 cells that had been pre-treated with UO2(CH3COO)2 for 24h were treated with the chelators for another 24h,49-51% of the intracellular uranium was mobilized by 50 and 250μmol/L of CBMIDA-CaNa2. When HK-2 cells were pre-treated with UO2(CH3COO)2 for 48h followed by CBMIDA-CaNa2 treatment for another 24h,37-38% of intracellular uranium were removed from UO2(CH3COO)2-treated cells. Furthermore, treatment of UO2(CH3COO)2-treated HK-2 cells with CBMIDA-CaNa2 significantly enhanced the cell survival, decreased the micronuclei formation and inhibited the production of intracellular ROS. In addition, uranium induced the increased level of MT protein expression in HK-2 cells, while CBMIDA-CaNa2 didn't affect the uranium-induced MT protein expression. On the other hand, CBMIDA-CaNa? significantly reduced the amount of 24h urinary uranium excretion to 49% of the injected doses, and decreased the amount of uranium retention in kidney and bone of rats to 24% and 72% of uranium-treated group, respectively. Although DTPA-CaNa3 markedly reduced the uranium retention in kidney of rats and HK-2 cells, it was lower than that of CBMIDA-CaNa2 and couldn't protect uranium-induced cell damage.Conclusions CBMIDA-CaNa? effectively decorporates the uranium from UO2(CH3COO)2-treated rats and HK-2 cells, which is better than that of DTPA-CaNa3. Moreover, it could scavenge the uranium-induced intracellular ROS and protect the uranium-induced cell damage. CBMIDA-CaNa2 is worth further investigation.