Comparative Study Of Nanosized And Microsized Silicon Dioxide On Spermatogenesis Function Of Male Rats

IntroductionNanomaterials are particles with an aerodynamic diameter of 0. 1 ~ 100 nanometer range, which have large surface areas per unit mass and much exceptional physico - chemical properties. A large - sized production of nanomaterials with formations and surface properties to meet novel demands has afforded much more opportunities for people to contact them. However, potential hazards related to nanomaterials have not yet reported until now. Because the size of the nanoparticles allows them to pass freely throughout the organism and penetrate the nuclei of cells, exposure to nanoparticles causes more severe damages than that of larger particles of the same substance. Inhaled nanoparticles lead to a greater inflammatory response than that of fine particles, such as pulmonary granuloma formation, interstitial fibrosis, endomyocarditis, cardiac muscle degeneration and lung tumors in long - term.Nanosized silicon dioxide with predominant properties such as stability, dispersing and confluence is applied to rubber, dope, medicine and paper making. Any new nanomaterial before introducing it to the marketplace and into the product chain needs careful evaluation with regard to its toxicological and environmental risk perception. At present, a number of studies have shown an association between the increased environmental pollutants and reduction of human fertility. Thus, the potential effects on reproductive systems caused by nanomaterials should not be ignored. Two types of nanosized silicon dioxide particles were employed in present study comparing the effects induced by inhalation of nanosized and fine particles through evaluating sperm quality and quantity, testicular marker enzymes, lipid peroxidation and histopathology. We also investi-gated the mechanism underlying the different results.Materials and Methods1. Animals and treatmentForty - five male Wistar rats were divided into five groups exposed by 100mg/m3 or 300mg/m nm - SiO2 and μm - SiO2 in inhalation chambers two hours every other day for sixty - five days. Age - matched rats were exposed to room air with the same condition and served as controls.2. Methods2.1 Testicular and epididymal viscera coefficients viscera coefficients = Organ weight(mg)/Body weight(g)2.2 Sperm analysisThe number, motility and deformity of sperm were determined by routine procedures. The cauda epididymis was sliced in 4 ml 0. 9% saline and filtered through double layers of muslin cloth. The filtrate was mixed 1 % aqueous eosin and kept for 30 min for staining. An aliuot of the epididymal sperm suspension was used for sperm counts using Neubauer hemocytometer. Smears were prepared on glass slides and observed under a microscope [ 1000 for the head and tail abnormalities (1000 spermatozoa per animal) ].2. 3 Detection of marker enzymes and lipid peroxidation (LPO) in testes2.3.1 Lactate dehydrogenase isoenzyme - C4 ( LDH - C4 ) activity was determined by 2, 4 -dinitrophenylhydrazine (DNPH) method.2.3.2 Succinate dehydrogenase (SDH) activity was assayed by nitroblue tetrazolium (NBT) method.2.3.3 Superoxide dismutase (SOD) activity was assayed as described in the procedure enclosed with the kit of SOD.2.3.4 Products of lipid peroxidation were measured by the method of thio-barbituric acid reactive substance (TBARS).2.4 8 - hydroxydeoxyguanosine( 8 - OHdG) concentration in serum Serum 8 - OHdG levels were determined with a competitive ELISA kit ( Japan Institute for Contol of Aging, Japan). The determination range was 0. 5 -200 ng/ml. The 8 - OHdG monoclonal antibody and serum sample were loaded at 50 μl on a microtiter plate which has been coated with 8 - OHdG, and incubated at 37 ℃ for 1 hour, in accordance with the instructions of the manufacturer. After washing, the antibodies that remained bound to the 8 - OHdG in the sample were further bound with the horseradish peroxidase - conjugated secondary antibody. Subsequent addition of 3,3',5,5'-tetramethylbenzidine resulted in the development of color intensity proportional to the amount of antibody bound to the plate. The color reaction was terminated by stio solution ( phosphoric acid) and the absorbance was measured using a spectrophtometricplate reader at 450 nm wavelength.2.5 lipid peroxidation (LPO) in serum determination2. 5. 1 Products of lipid peroxidation were measured by the method of thio-barbituric acid reactive substance ( TBARS) .2.5.2 Superoxide dismutase (SOD) activity was assayed as described in the procedure enclosed with the kit of SOD.2. 6 Histopathological examination of testesTissues were fixed in 10% formalin, passed through ascending series of ethanol and then through xylene and embedded in paraffin. Tissues were sectioned at 5 μm and stained with haematoxylin and eosin for the discrimination of the stages of spermatogenesis. The slides were examined under the light - microscope and seminiferous tubular diameter was taken. Ten measurements were made per section using a MeraMorph/DP10/BX41 ( U. S. A). The means of measurements of parameter in each section were recorded for each animal.3. Data analysisData were expressed in means ± SEM . Comparison between nanosized and microsized or for the particles of different dose levels was made by t - test. Statistical analysis between experimental groups and control group was done by one -way analysis of variance ( ANOVA) followed by SPSS packages, post SNK -q test, and P < 0. 05 was considered significant.Results1. Body weights, testicular and epididymal coefficientsThe body weights were reduced in nm - SiO2 treated group from the fifth day of exposure to the end in comparison to the control ( P < 0. 05 ). A decrease in the testicular coefficients was only observed in nm - SiO2100 mg/m3 group compared with those of the controls ( P < 0. 05 ). The epididymal coefficients in nm - SiO2100 mg/m3 group and 300mg/m3 group were lower than those of the controls (P <0.05). The testicular coefficients in nanomaterials exposed group were lower than microsized particles exposed group of the same concentration at 100 mg/m ( P < 0.05 ). The comparison of testicular coefficients between nm - SiO2100 mg/m3 group and 300mg/m3 group showed that the latter were higher (P<0.05).2. Sperm analysisStudies have shown a reduction in sperm counts in cauda epididymides of all the administered groups in comparison to the controls (P <0.05). Comparison between different dose levels in nanomaterials exposed groups as well as microsized particles exposed groups demonstrated that sperm counts in 300 mg/m3 group were lower than 100 mg/m3 group, and the sperm density influenced by nanomaterials are far more lower than by microsized particls of the same concentration at 300 mg/m . An increase in the percentage of abnormal spermatozoa was observed in the whole administered groups compared with the control group (P <0.05) , while the sperm motility in all the experimental groups was significantly decreased compared with that in the control group (P <0.05).3. Activities of marker enzymes and lipid peroxidation ( LPO) in testisA marked decrease in activities of LDH - C4 was observed in all the exposed groups ( P < 0. 05 ) compared with the control group. The activities of LDH - C4 were significantly lower in nm - SiO2300 mg/m group than those in nm - SiO2 100 mg/m3 group ( P < 0. 05 ) , and LDH - C4 activities influenced by nanomaterials are far lower than by microsized particles of the same concentration at 300 mg/m3. The LDH - C4 activities were lower in μm - SiO2300mg/m3 group than those in μm - SiO2 100mg/m3 group ( P < 0. 05 ). The activities of SDH in all the exposed groups were reduced obviously compared with the controls (P <0.05). The results showed a general elevation in MDA levels in the treated groups compared with the control group (P < 0.05) , and MDA levels induced by nanomaterials were higher than by microsized particles of the same concentration at 100 mg/m and 300 mg/m3 ( P < 0. 05 ). Generation of SOD showed a significant decrease in testes of almost all the exposed groups ( P < 0. 05) expect nm -SiO2 100 mg/m3 group (P >0. 05) compared with the control group. The activities of SOD were significant lower in nm - SiO2300 mg/m group than nm -SiO2100 mg/m3 group (P < 0. 05) , and SOD activities in nm- SiO2 group are more higher than those in μm - SiO2 group of the same concentration at 100 mg/m3.4. 8 -hydroxydeoxyguanosine(8 - OHdG) concentration in serumThe results demonstrated a marked increase in the concentration of 8 -OHdG in all the experimental groups compared with the control group (P < 0. 05).5. Lipid peroxidation (LPO) in serumMDA levels and generation of SOD in serum remained unchanged in all the exposed groups ( P > 0.05 ).6. Histophathological studies in testes 6. 1 Photomicrographs of testesPhotomicrographs showed that no particular histopathological changes were observed in the control group. In the experimental rats, histological observation under light microscope showed different changes in all the administrated group, among which the lesions in nm - SiO2 exposed groups were the most severe, nm- SiO2300 mg/m3 group showed extensive sloughing of germinal cells and only the basement membrane was left. Some tubules also showed process of degeneration with cellular disharmony. A decrease in the number of the Sertoli cells and Leydig cells were seen, the margin of the interstice was enlarged and extravasate was exuded. The number of germinal cells in nm -SiO2100 mg/m group was a little more than that of nm - SiO2300 mg/m3group, and no tubular fracture and...