Investigation On Mechanisms For Reproductive Toxicology And Cytotoxicity Of Acrylamide In Male Animals And Adult Stem Cells

It is known that acrylamide is a carcinogen (category 2) which has neurotoxicity, genetoxicity and reproductive toxicology. Public health concerns have been raised by Swedish studies showing that relatively high levels of acrylamide were formed during the frying, roasting, or baking of a variety of foods for a decade.The occupational exposure to acrylamide is mainly by the synthesis process of polycrylamide. Acrylamide is one intermediate chemical substance that formed in the production process of the polycrylamide, which is widely used in tip water treatment, municipal wastewater disposal, well technology, architecture field, paper industry, feed processing, soil-solidified-agent, cosmetics, daily chemical additives, bioengineering test, and so on. At the same time, it also exists in the cigarette smoke, suggesting that it can be formed in the heating process of the biological raw.However, acrylamide is present in cosmetic, cigarette smoke, certain foods that have been cooked at high-temperature, and drinking water in our daily life. In addition, acrylamide can enter into organism through skin, oral cavity and respiratory tract, then it distributes to the tissues of whole body rapidly. If pregnant women are exposed to acrylamide, the fetus can be affected. Furthermore, the reproductive capacity of the male animal may be affected if they intaked certain dose of acrylamide. Therefore, the European Chemicals Agency has added acrylamide to the Candidate List of Substances Very High Concern (SVHC) for Authorisation in March 2010.Recent years, toxical mechanisms of acrylamide on the reproduction have attracted the attention of scientists. In the present experiment, I use rats as a model animal to examine its in-vivo reproductive toxicology. I also use the mesenchymal stem cells of rats and mini-pigs of Wuzhishan as cell models to study its in-vitro cell toxicology and mechanisms of its reproductive and cytotoxicity.1. Oral acrylamide affects the development and reproductive performance of male ratsThe present experiment is conducted to investigate the toxicity of acrylamide on the reproductive system in male rats. Thirty SD male rats weaned at 21-day-old were divided into three groups randomly. Acrylamide solutions at doses of 5 and 10 mg/kg/d were fed in treatment groupsⅠandⅡconsecutively for 8 weeks by free drinking, respectively, while distilled water was fed to control group. In the 4th and 8th weeks of the experiment, the daily gain, body weight, organ weights and other indexes of experimental animals were detected. In addition, rat testis, epididymis and all kinds of organs were taken for histomorphological observation. At the same time, the sperm density and morphology in the cauda of epididymis were also examined at the 8th week. The results indicated that body weight gain of the rats of treatment groups was significantly lower than that of control group (P<0.05). The development of testis and epididymis were affected. In addition, the sperm density in the cauda of epididymis of the groupⅡwas significantly lower than that of the control group (P<0.05). However, there was no obvious difference between the groupⅠand the control group (P>0.05). The histopathologic lesions were present in the testes of treated rats, while the spermatogenic cells in seminiferous tubules were partially exhausted. The results of my experiment suggested that acrylamide may retard the growth of the rats and affect their sexual organ development, and have toxic effects on seminiferous tubules which lead to the decrease of sperm production in the male rats.2. Investagation on the reproductive toxicity mechanisms of oral intake of acrylamide in ratsIn order to further explore the male reproductive toxicity mechanism of acrylamide, the serum total testosterone of sub-chronic exposure treatment groupⅠ(5 mg/kg/d) and treated groupⅡ(10mg/kg/d) rats were measured, the histopathology and immunohistochemistry of testis and epididymis were analysed, and the unit area number of Leydig cells were computed between the treatment and control groups in the present experiment. Moreover, quantitative analysis of immunohistochemical images in pathological tissues were conducted by TissueGnostics Image Analysis Systems. The expression status of sGCα1 protein in epididymis was scanned organization-widely both in treatment group and control group. The results suggested that, compared with control group, the content of total serum testosterone of acrylamide-chronically-treated groupⅡwas increased significantly (P<0.05), and the hyperplasia of Leydig cells around seminiferous Myelodysplastic abnormally (P<0.05), while there was not significantly different between the treatment groupⅠand the other two. In treatment groupⅡ, the expression of sGCα1 protein in interstitial cells of histopathology testis was lower than that of control group, while the expression of sGCβ1 were not different between the two groups. The image analysis of epididymis indicated that the expression of sGCα1 and sGCβ1 was not changed among the groups. However, the expression of sGCα1 in the caput and cauda of epididymis was higher than the corpus epididymis. The result implied that the reproductive toxicity mechanism of acrylamide may be through the sGC protein of NO/sGC/cGM pathway to disturb the tight junction of seminiferous tubules, leading to spermatogenic cells exhausted. Meanwhile, the differential expression of sGC heterodimers within the testis and epididymis suggested that they may play a different physiological role in the synthesis of steroid hormones.3. Effect of acrylamide on the osteogenic differentiation of bone marrow mesenchymal stem cells in ratsThe present experiment was conducted to investigate the cell toxicity effect of acrylamide on bone marrow mesenchymal stem cell in rats. The bone marrow mesenchymal stem cells were isolated and purified from femurs and tibias rats at 1 month-old, and identified by osteogenic differentiation and cell surface antigens.The groups were designed as 0,5,100,300 and 1000μM concentrations of acrylamide in the basic medium, respectively. After consecutive treating for 8 days, the effect of acrylamide on morphological change of cells was observed. The inhibition rate of cell proliferation was detected by CCK-8, and the cell proliferation rate was calculated. A 0,5,25,100,200 and 300μM concentrations of acrylamide were added during the 21-day osteoblast differentiation course. At last, the effects of acrylamide in osteogenic differentiation were detected by alizarin red and alkaline phosphatase staining. The results showed that acrylamide lead the morphological changes of the bone mesenchymal stem cells. The rates of cell growth inhibition in 5,100,300 and 1000μM acrylamide treatment groups were 7.69%,30.77%,53.85% and 100%, respectively, while the doubling time of MSC cells of control,5,100 and 300μM acrylamide treatment groups were 72,79.7,88.1 and 111.2 hours respectively. With the increase of acrylamide concentration, the osteogenic differentiation of cells was significantly inhibited. The expression of alkaline phosphatase was significantly reduced (P<0.01). These findings suggested that acrylamide inhibit bone marrow mesenchymal stem cell proliferation and osteogenic differentiation in rats.4. Investagation on the cytotoxical mechanisms of acrylamide on the bone marrow mesenchymal stem cells of Wuzhishan mini-pigsThe present experiment was conducted to investigate the toxicity mechanism of acrylamide on the osteogenic differentiation of bone marrow mesenchymal stem cell. The bone marrow mesenchymal stem cells were isolated and purified from femurs and tibias of 6-day-old Wuzhishan pigs, and identified by osteogenic and fat-genic differentiation. The acrylamide-treated groups were added 0,50,100,500 and 1000μM concentrations of acrylamide in the basic medium respectively. After 1 hour treatment, the content of reactive oxygen species (ROS) and total glutathione peroxidase in cells were examined. After consecutive treating for 3 days, the cell morphology was observed, and the change of cytoskeleton detected by Actin-Tracker Green. At the same time, the RNA was extracted and the expression of actin gene assayed. The results revealed that, in the acrylamide-treated groups, the content of reactive oxygen species levels increased dependent with the dose of acrylamide in cells, and there was significant difference (P<0.05) between the 500, 1000μM treatment groups and the control group. In addition, compared to the control group, the content of total glutathione peroxidase was reduced significantly (P<0.05) in the 500, 1000μM treatment groups whose cytoskeleton micro filaments rearranged, cell shape become round, and the microfilaments shortened. The Quantitative PCR results showed that the actin gene of cytoskeleton in 1000μM treatment group was significantly (P<0.05) reduced, while there were no obvious (P>0.05) differences in the above indicators between the 50,100μM treatment groups and control group. In short, the experiment findings indicated that high concentration acrylamide could cause the cell morphology of bone marrow mesenchymal stem cell changed, affect the metabolism of reactive oxygen species, induce cytoskeleton shortened, and therefore influence the cell proliferation capacity in Wuzhishan pigs.In conclusion, my in-invo experiment indicated that acrylamide could retard the growth of SD rats resulting in the decrease of the daily gain. Furthermore, acrylamide decreased the sperm density in the cauda of epididymis, which may induce via the NO/sGC/cGM pathway, then affect/influence the tight junction of seminiferous tubules, leading to spermatogenic cells exhausted. In addition, the interstitial cell hyperplasia and increased hormone level may be caused by the compensatory regulation in spermatogenesis. While in-vitro results showed intracellular reactive oxygen accumulated and cytoskeletal filament shorten of the mediate and high dose treated groups, leading to cell proliferation capacity decreased. At last, the alkaline phosphatase activity in the bone marrow mesenchymal stem cells decreased, cell morphology changed and osteoblast differentiation was inhibited.