Analysis On The Reproduction Toxicity And Proteomic Associated With Placental Barrier Of Rats Exposed To Low Level Cadmium

Cadmium (Cd), a heavy metal, is extensively used in the fields of chemical, electronic and nuclear industries. Since the notorious event of itai-itai disease happened in Toyama Prefecture, Japan in 1955, the attentions have been intensively paid worldwide on the adverse health effects of Cd and its prevention and treatment measures. United Nations Environment Programme and International Commission on Occupational Health cataloged Cd as an emphatically-studied environmental pollutant. The World Health Organization listed Cd as a preferential-studied food contaminant. In China, Cd as a highly toxic environmental pollutant, it causes harmful effects in the environmental areas through tobacco smoking, diet, drinking water and inhalation from the air. Especially, another major hazard of Cd comes from occupational exposure such as the working in batteries, dyes, coatings, electroplating, plastic stabilizers, alloys and control rods for nuclear reactors. Although recent years there are many technologies and policies restricting the use of Cd, since it has a long biological half-life of 15～30 years. Furthermore, most of the occupational Cd exposed workers are young, the reproduction toxicity and the long-term adverse effects on their offspring are the research focuses for the future.Cd can accumulate in a variety of organs, especially in bones, kidneys and liver as well as the reproductive organs including the placenta. Meanwhile, placenta as a barrier plays an important role in defending the reproduction toxicity of Cd, which is obviously different with other heavy metals like lead (Pb) and mercury (Hg) can easily penetrate the placenta barrier. Although the biological translation processes of Cd and Pb in the body are similar, their penetrativities to the placenta are totally different. There is an interesting phenomenon that the placenta barrier is flimsy to Pb. Some authors pointed out that nearly 90% of Pb can cross the placenta to the foetus, but for Cd it has been reported that cord blood was only about 10% of the maternal blood in humansThe penetrative limitation indicates placenta has obvious barrier effect to Cd. However, it is unclear which proteins specially involved in placental uptake and efflux of Cd.Metallothionein (MT) rich in cysteine is a significant detoxification on Cd. It can adjust the inner balance of the metal elements required, antioxide and protect DNA damage, which is related with angiogenesis, cell proliferation, apoptosis and survival. There are some abroad reports that Cd exposure can induce the expression of placental tissue MT, reduce the accumulation of Cd in animal embryonic brain tissue. The cysteine sulfhydryl group (SH) of MT can strongly chelate toxic metals, it not only can detoxicate through the combination of cadmium ion, but also combine with different affinity of zinc, copper and other metal ions releasing the other combined Cd ion and adjusting the balance of trace elements in the body. MT in the placenta has an important role protectiving the fetus’s growth and development. But the role of placental MT as a barrier for Cd is inconclusive. MT can nonspecifically bind to some valence+2 metals. Pb, Hg and some heavy metals can also induce the synthesis of MT in placenta to decrease their toxicities. It is unknown why the filtration rate is so different. In addition to recent studies speculated that MT in uterus and placenta did not play a significant role in preventing Cd transportation through placenta from uterus to fetus. They detected much Cd was transported as the chemical form of nonMT-Cd from mother rat to foetus. Moreover they also analyzed the metal transporters DMT1, ZIP, and ZnT, found besides MT-Ⅱ gene expression increased in the uterus and placenta, DMT1 and ZIP14 involved in the uptake of Cd into the cytosol in placenta. We suspected that maybe there are other proteins play more indirect roles in the placental storage of the metals especially of Cd. To analyze the molecular mechanism of Cd transport on placenta and the reproduction toxicity of low level Cd exposure, we simulate generation reproduction toxicity experiment, using the female SD rats consecutively exposed to Cd including two estrous cycles and the periods of pregnancy and lactation.The biological effect of low-level long-term Cd exposure is significantly different with those in vivo and in vitro experiments under the conditions of occupational or non-occupational high-dose short-term exposure. The reasonable dose setting is very important in animal experiment simulating the actual exposure level of people. In this test we consulted lots of references. Most animal studies in toxic and carcinogenic tests used Cd dosages in the range of 1-5 mg/kg·bw·d. In Japan, young female Wistar rats were exposed to 1 mg Cd/1 in drinking water for 24 months as a low-level lifetime exposure to Cd. The researchers determined that an orally administered dose of 1 mg/kg·bw·d to rats would represent a level similar to the daily Cd intake in human. Combining the previous researches and preliminary test of us, we used 1 mg/kg·bw·d as a low-level occupational exposure of Cd. The actual intake of Cd into the body was estimated 3.6 ug/kg·bw·d according the intestinal absorption rate of 0.36～0.54% intake, slightly higher than the dietary exposure according to the World Health Organization recommended Provisional Tolerable Weekly Intake of 7ug/kg·bw·d.As the Human Genomic Project (HGP) completed ahead schedule, proteomics theory and the technical method has become the main theme of the post genome era. As a large-scale, high throughput, high sensitivity method, proteomics can be effectively and objectively analyzed the overall intracellular proteins. Through the comparison of protein expression levels between different samples, differential expression proteins were identified, which can be used to solve various biological problems. At present there are many novel differential proteomic analysis methods, among which the difference gel electrophoresis (DIGE) technology becomes one of the most popular method, because it is not only has the high-resolution feature, inherited from two-dimensional gel electrophoresis(2-DE), but also has high reproducibility, high sensitivity, high throughput and high dynamic range.In conclusion, this study therefore conducted to study the role of the transporters in the placental barrier in regulating maternal Cd transferation to fetus. We simulated the animal test of reproduction toxicity, carried out a consecutive low dose Cd exposure on Sprague-Dawley (SD) female rats to observe the reproductive and developmental toxicity. We used a technology platform based on difference gel electrophoresis, combined with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Tandem Mass Spectroscopy (MALDI-TOF/TOF MS) and bioinformatics analysis to find the proteins associated with the barrier effect of placenta at low dose Cd.The statistical package SPSS (ver.13.0) was used for statistical analysis. Data were expressed as the mean value with standard deviation according with normal distribution, otherwise as the median with inter-quartilerange. Differences among experimental groups were examined by t-test or one-way analysis of variance (ANOVA), the method of X2-test was adapted to the qualitativedata. Values of P< 0.05 were considered of significance.The animal experimental results showed that in Cd-M and Cd-H groups, from Day 19 gavage, a slower increase of the body weights of the females was observed, showing a statistical difference with that of the control (P<0.05). Meanwhile, although the variation tendency of Cd-L and Pb groups were similar to other treated groups, there was no statistical significance with that of the control. The facts that, on Day 20 gavage in parental females, blood Cd median concentration in Cd-L group was 12.09(10.42-14.57)μg/L, blood Pb median concentration was 119.93 (105.83-130.45)μg/L, and that on Day 44 gavage in parental females, urine Cd median concentration was 5.48(4.03-9.23)μg/L, urine Pb median concentration 127.38(97.00-160.22)μg/L. The results presented evidences that the low dose is reasonable according to the national occupational health standards in human. The results also displayed on Day 20 gestation, the means of Cd and Pb concentration in the parental blood were respectively 16.70±4.77 and 284.95±149.49μg/L, significantly higher than those of the control (respectively 1.56±1.09 and 44.04±16.69μg/L). Contrarily Cd and Pb concentrations in umbilical cord were very different, the ratio of umbilical cord to maternal blood in Cd group was only 8.38%, which has no significant difference with that of the control group. On the other hand, the ratio in Pb group was reached up to 58.2%, significantly higher than that of the control (P<0.05). The data indicated that the barrier effect of placenta is much strongly against Cd but not Pb. Thereby placenta may play an important role in preventing Cd transportation from parental females to foetuses.The reproduction index of parental females in Cd treated groups was not different with that of the control on Day 20 gestation andDay 28 lactation. The facts indicated there was no significant reproductive toxicity at 1 mg/kg·bw·d dose Cd. The body weights and placenta weights of foetuses in Cd-L group on Day 20 gestation were significantly higher than those of the control. Litter size was not affectedbecause in Cd-L group (10.00±3.46) it was not different with that of the control (10.71±2.93). On Day 28 lactation, the litter size in control, Cd-L, Cd-M and Cd-H group was 12.33±2.66,7.60±4.04,9.50±3.07and 10.83±1.94 respectively. Furthermore, we noticed that the body weights of the pups in Cd-L and Cd-M groups during lactation were also higher than that of the control. Even this high-body-weight could be resulted from the fact of low litter size in Cd-L group(P<0.05), Cd-M group still had this fact after eliminating the related confounding factors. Low body weight in Cd-H group was because of the obvious developmental toxicity of Cd despite of placental barrier effect. We speculate that the high-body-weight in pups maybe related with relatively low dose Cd exposure because it is an environmental contaminant that mimic the effects of estrogen. In our study, although we did not test the time of vaginal opening and analyze the level of hormone, the weight gain of Cd exposure might hint that we should not ignore the hormone effect on low dose conditions in the human surveillance.According to the analysis of differential protein spots combined with different maps of DIGE and Decyder software, we set these spots which were up-regulated or down-regulated 1.5 times as the differential protein spots. Compared the Cd-L group with that of the control,11 differential protein spots were found, the numbers of up-regulated ones were 2 and on the contrary the down-regulated were 9. Compared the Cd-H group with that of the control,6 differential protein spots were found, the numbers of up-regulated were 4 and the down-regulated 2. Compared the Pb group with that of the control,28 differential protein spots were found, the numbers of up-regulated were 19 and the down-regulated 9. Some differential protein spots between groups were changeable, we finally analysed 32 differential protein spots. Protein spots of interest were excised by automatic Spot picker, and then the picked spots were carried out with in-gel digestion, followed by ABI 4800 MALDI TOF/TOF mass spectrometry. Retrieved SWISSPROT database through the mascot software, gotten rid of the same protein within appraisal result,17 differential proteins were identified. We carried out the bioinformatics analysis of the function of all 17 identified differential proteins. Firstly the identified differential proteins were processed with subcellular localization software WoLF PSORT. After it, the protein functional analysis was done through protein domain and motif database. The result showed that there were 5 differential proteins exised at protein interaction network simultaneously, which indicated that these differential proteins had functional relation and might associated with each other. Among these proteins, ATP-binding cassette sub-family B member 4(Abcb4) and ATP-binding cassette sub-family G member 2(Abcg2) are known to couple the energy derived from ATP hydrolysis essentially to translocate, among various substrates, toxic compounds including heavy metals across the cell membrane. Heat shock protein 90 kDa beta member 1(Hsp90β1) and heat shock 70 kDa protein 5 (Hspa5), the two proteins existed direct interaction and they always composed the multimeric protein complexes with eukaryotic translation initiation factor 2(eIF2). Prohibitin (Phb) was a pleiotropic protein in the cell located in mitochondrial inner membrane, nuclear and plasma membrane. So we chose the 5 differential proteins to do functional experiments.In order to verify Abcb4, Abcg2, Hsp90β1, Hspa5 and Phb changes of expression and location in placental tissuse, we used Western blot and immunohistochemistry methods to demonstrate the result of DIGE. The experimental results indicated that the levels of Abcb4 and Abcg2 expression were negatively correlated with Cd exposure, being significantly down-regulated in the treated groups (P<0.05). Interestingly, we found a dose-response relationship between Cd exposure and Abcb4 or Abcg2 protein expression. Different doses of Cd exposure had effects on the expression and location of Phb in placental tissue. The expression of Phb was increased according to the Cd exposure, especially in lost-trophoblast. The another differential protein spot founded by DIGE was numeber 1443, it was identified as Phb protein as well. The trend had no significant change with the control. The results of Abcg2 on Western blot were not fit with DIGE. We analyzed the target might be associated with the modification of protein, because the aim of Western blot detection with the antibody was total protein, and the difference of DIGE testing found that protein points might be the proteins after modification, so the changes of protein expression trend appeared inconsistent.After all, to mimic the Cd exposure level more closely to the occupational poputlation, we set a low level of Cd exposure in animal test of reproductive toxicity. Based on differential proteomics analysis techniques with difference gel electrophoresis, MALDI TOF/TOF mass spectrometry and Bioinformatics, we investigated the barrier effects of low level Cd exposure on placenta to gain insight into associated toxicological mechanisms and functions. Throughout the research, three conclusions were drawn as follows:1. We had established an animal model of low dose of Cd exposure. The results of blood/urine Cd levels in different periods presented evidences that the low dose was reasonable according to the national occupational health standards in human. The results indicated there was no significant reproductive toxicity at 1,3,9 mg/kg·bw·d dose Cd. The body weights were increased in Cd-L and Cd-M group, the high-body-weight in pups with relatively low dose Cd exposure maybe related with the estrogen-like effects. On the contrary the body weights were decreased in Cd-H group because of the direct toxicity. The weight gain of Cd exposure might hint that we should not ignore the hormone effect on low dose conditions in the human health surveillance.2. The results of animal test demonstrated the evidence that the placental barrier worked strongly against low level Cd exposure. We set a different map of DIGE on low level Cd exposure and analysed 32 differential protein spots. We carried out the bioinformatics analysis of the function for all 17 identified differential proteins. Combined with subcellular localization analysis, the protein functional analysis by protein domain and motif database, we identified 5 differential proteins. The result showed that these proteins maybe regulate Cd across through placenta and related with oxidative stress during Cd reacted with placenta.3. We used Western blot and immunohistochemistry methods to demonstrate Abcb4, Abcg2, Hsp90β1, Hspa5 and Phb changes of expression and location in placental tissues. The levels of Abcb4 and Abcg2 expression were negatively correlated with Cd exposure, showing a dose-response relationship between Cd exposure and Abcb4 or Abcg2 protein expression. The expression of Phb was increased according to the Cd exposure, especially in lost-trophoblast. The results of Hsp90β1 and Hspa5 on Western blot were fit with DIGE. We speculated that Abcb4 and/or Abcg2 might be responsible for the removal of Cd out of the placenta and thus affect the in vivo toxic effect of Cd to fetus. One of the mechanisms responsible for Phb’s antioxidant function was its positive regulation of the antioxidant factor erythroid 2-related factor 2(Nrf-2), the other reason might be its inhibition on the signal transduction and gene transcription mediated by Cd with estrogen receptor. The mechanism needs to further study with different designs of experiments to verify.This study not only provided the dynamic experimental data for low dose of Cd exposure in reproductive and developmental toxicity researches, but also discussed the mechanism of down-regulation of Abcb4 or Abcg2 associated with Cd exposure in placenta and the protein of Phb reacted with Cd oxidative stress in placenta.