【精品论文】Effects of dibutyl phthalate on ovarian active substance of mice.doc

精品论文effects of dibutyl phthalate on ovarian active substance of micewang yu, wang shengqing, ye wengbin, he jiujun5(department of biology and chemistry, longnan teachers college, gansu chengxian 742500) abstract: with superoxide dismutase(sod), catalase(cat), alkaline phosphatase(akp), acid phosphatase(acp), malondialdehyde(mda) and bax protein expression as characteristic indexes, the effects of dibutyl phthalate(dbp) on ovarian active substance of mice were studied, and the toxic mechanisms were discussed. one hundred sixty mice were given intragastric administration of dbp (0,10100, 250, or 500 mg/(kgd), respectively, for 30 days. at 5, 10, 20, 30 days after dbp exposure, the ovarian examinations were taken. the results revealed that administration of dbp decreased theactivities of sod, cat, akp and acp, while mda content and bax protein expression increased. two-way analysis of variance indicated that dose contributed more to dbp-induced ovarian damage than treatment time. the present study demonstrated that dbp promoted apoptosis and inhibited15antioxidant capacity of the ovarian.keywords: dibutyl phthalate(dbp); ovarian; antioxidant capacity; bax protein; mice0introductionit has caused serious concern about dibutyl phthalate, being added to certain foods and20benerages manufactured in taiwan. dbp is used extensively in the manufacture of plastics (such as pvc piping and carpet backing), various paints, varnishes and lacquers, medical supplies (such as transfusion and denta l materials), safety glass for automobiles, food packaging, cosmetics (such as nail polishes and perfume oils), textiles (as a lubricant and as an insect repellant forimpregnation in clothing), and paper coatings1. although used as an insect repellant, dibutyl25phthalate is not considered to be as effective as dimethyl phthalate. because of this worldwide use, there is a high potential for human exposure through direct sources in the workplace and the home environment. in addition, numerous indirect sources exist in water, air, and foodstuffs due to the pervasiveness of dbp in the environment and by contamination through leachable products. the potential for long-term human exposure to environmental chemicals such as pesticides and30plasticizers, particularly in utero and in neonates, has raised additional concerns2.the antioxidant defense system is an important active oxygen scavenging system in organisms, and plays a crucial role in the protective defense response3-4. when an antioxidant defense reaction occurs in an organism in response to toxicants, superoxide dismutase (sod) is the first antioxidant to react with the reactive oxygen species, causing the superoxide anion35radical (o-2) to disproportionately form h2o2 and o2. subsequently, h2o2 is decomposed bycatalase (cat) into h2o and o2 to decrease the accumulation of h2o2 in organisms 5. malondialdehyde (mda) can be formed in the lipid peroxidation reaction. thus mda content can be used as an index of the extent of lipid peroxidation in the tissue, which indirectly represents the level of cell injury. it has been demonstrated that dbp can intensify the tissues40(liver, kidney, testis, lung) lipid peroxidation6, inducing damage to the antioxidant defensesystem and the histological structure of the mouse thymus, liver, kidney, testis and lung 7-13. however, the mechanisms of dbp toxicity remain unclear. most previous studies of dbp-related reproductive system damage have focused on the effects of dbp on the histological structure of the testis. the present study hypothesized that dbp inducesfoundations: the science fund of gansu province (no. 1107rjzk243) and gansu provincial educationcommittee(no.1128b-01).brief author introduction:wang yu, (1973-), master, associate professor, research direction: cell and developmental biology. e-mail: - 8 -45pathological damage in the ovarian of mice. we sought to investigate the effects of dbp on the antioxidant capacity of the ovarian with sod, cat, acp, akp and mda as indices of antioxidant function, observe cell apoptosis by bax protein levels.1materials and methods1.1 materials501.1.1animalsa total of 160 healthy, adult, kunming female mice, regardless of gender, weighing 20-22g, were provide by the experimental animal center of lanzhou university, china (certification no.14-006). the experimental procedures were conducted in accordance with the guidancesuggestions for the care and use of laboratory animals, formulated by the ministry of science55and technology of the peoples republic of china.1.1.2reagentsthe dibutyl phthalate(dbp) samples(a purity of 99%) were provided by hengxing chemical co., ltd., tianjin, china. rabbit anti-bax protein polyclonal antibody and normal goat serum were purchased form wuhan boster bioengineering institute, china. sod, cat, acp, akp and60mda determination kits were provided by nanjing jiancheng bioengineering institute, nanjing, china. all the other reagents were of analytical grade or guaranteed grade.1.2methods1.2.1establishing the animal modela total of 160 mice were equally and randomly assigned to four groups (40 mice per group):65control, low-dose dbp(100 mg/kg), middle-dose dbp (250 mg/kg), and high-dose dbp (500 mg/kg). the control group was treated with deionized water. the other three groups were treated respectively with different concentrations (100, 250, or 500 mg/(kgd) of dbp6-7. each group received intragastric administration for 50 consecutive days, once per day, 0.5 ml once. all 160 mice were included in the final analysis.701.2.2immunocytochemistryat 5, 10, 20, 30 days after dbp exposure, five mice were randomly taken from each group. tissue samples from the ovarian were obtained after opening the peritoneal. the tissues were fixed with 20% formalin, followed by dehydration, waxing, embedding, and sectioning (6 m thick). the slices were dewaxed with xylene, hydrated in gradient alcohol, boiled in citrate buffer75solution (ph 6.00.1) in a heated pressure cooker for 1.5 minutes to retrieve antigen, andincubated for 10 minutes at room temperature with 3% hydrogen peroxide to block endogenous peroxidase activity. the slices were incubated with rabbit anti-bax protein polyclonal antibody (1:200) at 4 overnight, followed by incubation in quick maxvisiontm test kit at room temperaturefor 15 minutes and dab coloration. the reaction time was controlled under microscopy. after the80reaction was terminated with distilled water, the slices were counterstained with hematoxylin for30 seconds, rinsed until blue, dehydrated in an alcohol gradient, cleared with xylene, mounted with neutral gum, and observed by microscopy. the negative control was treated with pbs rather than primary antibodies. a total of 5 visual fields from the cerebral cortex (400) were randomly selected to quantify the total number of cells and positive cells. the ratio of bax protein-positive85cells was calculated according to positive cells/total cells 100%. the average absorbance valueof bax protein was measured using a pathological image analysis system.90951001051.2.3pretreatment of the ovarian samples and determination of the antioxidant defense indicesthe ovarian tissue was immediately put into an ice bath immediately after the blood was soaked up by filter paper. samples were then homogenized with a glass homogenizer. the obtained homogenates were centrifuged for 10 minutes at 3 000 r/min, and the supernatant was collected and stored at -20c for determining the antioxidant defense indices described below. sod, cat, acp, akp activities and mda content were determined with a method established by nanjing jiancheng bioengineering institute, china. the measured sample was compared with the light absorption of a blank control liquid, and the sod, cat, acp, akp activities and mda content were calculated on the basis of the protein. the enzyme (sod, cat, acp, akp) activities were expressed in units per milligram protein. the mda content was expressed in units per milligram protein.1.2.4statistical analysisall data were analyzed with spss 16.0 software (spss, chicago, il, usa) using two-way analyses of variance, and were expressed as mean sd. the t-test was used for comparison between two groups. p 0.05 was considered statistically significant.2results2.1effects of dbp on sod, cat, acp, akp activity and mda content in ovarian tissueto examine the effects of dbp on activity related to the antioxidant capacity of the ovarian after dbp exposure, we measured changes in sod, cat, acp, akp activities and mda content (fig. 1-a e).200180sod activity (u/mg)160140120100806040200aaaaaaaacontrol grouplow-dose dbp groupamiddle-dose dbp groupahigh-dose dbp groupa1105 10 20 30fig.1-a changes of sod activity at different doses160140cat activity (u/mg)120100806040200aaaaaaaacontrol group low-dose group middle-dose groupahigh-dose groupaa5 10 20 30fig. 1-b changes of cat activity at different doses2018control group16acp activity (u/mg)14a12a1086420aaa aaalow-dose groupmiddle-dose groupahigh-dose groupaa1155102030fig. 1-c changes of acp activity at different doses1614acontrol groupakp activity (u/mg)12a a1086420low-dose groupaaamiddle-dose groupaaahigh-dose groupaa1205102030fig. 1-d changes of akp activity at different doses35acontrol groupmda content (nmol/mg)302520a a151050aa aaaa aaalow-dose group middle-dose group high-dose group1255102030fig. 1-e changes of mda content at different dosesfig. 1-ae effects of ole on ovarian sod, cat, acp, akp activity and mda content after dbp exposure in mice. values are expressed as mean sd (n = 16). a p 0.05), while sod, cat, acp, and akp activities increased markedly at 10-30 days (p 0.05) after dbp exposure. the sod, cat, acp, akp activities in both the middle-dose dbp and high-dose dbp groups at 5-30 days were significantly lower than those in the control group (p 0.05).the mda content in the ovarian was determined at 5-30 days after dbp exposure. the results revealed that the mda content in the ovarian in each group increased significantly compared with the control group (p 0.05), but the effect of dose was significant (p 0.01 or p 0.05), as was the interaction between them (p0.05). according the partial 2 value, dose contributed more to sod, cat, acp, and akpactivity and mda content than time (the partial 2 values for dose were 0.944, 0.891, 0.873, 0.684, and 0.965; 2 values for time were 0.009, 0.013, 0.031, 0.055, and 0.008).2.2 expression of bax protein in the ovarianbax protein-positive cells were observed by immunohistochemical staining, and were primarily located in ovarian granulosa cells. positive particles were located in the cell membrane and cytoplasm. very small numbers of bax protein-positive cells were observed in the ovarian of the control group and positive particles were lightly stained. bax protein-positive cells exhibitedstrong expression in the cytoplasm in the ovarian of the dbp-treated groups (fig.2).20 m20 ma20 mb c20 m20 md e150fig. 2 bax protein expression in ovarian at 30 days (immunohistochemical staining, optical microscope, bars:20m). bax protein was expressed predominantly in the cell membrane and cytoplasm. in the control group (a), bax protein positive products were stained light yellow. in the low-dose dbp group (b), the middle-dose dbp group (c) and the high-dose dbp group (d), the positive products were brown, and their levels were greater than in the control group. (e) negative control group.155statistical results showed that, compared with the contol group, the average absorbance value and rate of bax protein-positive cells significantly increased in the ovarian of the dbp-treated groups (p 0.01 or p 0.05). in particular, the high-dose dbp group exhibited the greatestincrease in the number of bax protein-positive cells (p 0.01, tab. 1).tab. 1 bax protein expression in the ovarian at 30 days after dbp exposure (xs, n=10)groupaverage absorbancerate of positive cells (%)control0.270.0516.342.03low-dose dbp0.290.07 a35.065.73 bmiddle-dose dbp0.390.09 b43.425.27 bhigh-dose dbp0.450.08 b75.618.82 ba p0.05, b p0.01 vs. normal group; dbp: dibutyl phthalate.1601651701751801851903discussiondbp is an important chemical material widely applied in the industries of plastic (pvc) piping, various varnishes and lacquers, safety glass, nail polishes, paper coatings, dental materials, pharmaceuticals, and plastic food wrap. concomitant with this extensive worldwide use is the high potential for human exposure to dibutyl phthalate in the workplace and the home environment through direct sources as well as indirectly, through contamination of water, air, and foodstuffs1-2. many studies have reported that dbp inhibits telomerase and sod activity, and expression ofcd36, telomerase and telomerase reverse transcriptase6-7. although the results of the studies mentioned above provide evidence for the notion that dbp causes damage to the tissues, this should be confirmed in other populations. in the current study, the activities of sod, cat, acp and akp were significantly lower than those of the control group after administration of dbp with 3 different concentrations (100, 250, or 500 mg/(kgd). these findings indicate that dbp might inhibit the activities of sod, cat, acp and akp. it has also been documented that lower levels of dbp exposure could significantly decrease the activity of sod in testis, liver, kidney andlung 6,13. one possible explanation for the mechanisms is that oxidative dbp can antagonize22 2metal ions (such as cu2+, zn2+, fe2+, mg2+) that are essential for the activity of antioxidant enzymes, resulting in a loss or decrease of the activities of sod, cat, acp and akp. in the present study, mda content in the ovarian increased significantly after administration of dbp. previous studies have demonstrated that the level of mda in liver and lung was elevated while sod activity decreased with increased dbp exposure 6,13. two possible explanations exist for these findings: (1) dbp may induce cells to produce superoxide anion (o -) and h o among others; this results in an accumulation of the lipid peroxides in the cell membranes of organisms,which is responsible for increasing mda content. (2) dbp inhibits the activities of sod, cat, acp and akp, thus increasing the peroxidation reaction.previous studies have indicated that dbp can impair the liver, kidney, testis and lung, in a time- and dose-dependent manner6,11-13. our results are consistent with these previous findings. however, the present study demonstrated that dose contributed more to dbp-induced ovarian damage than treatment time. this may be because dbp is easily transported through the blood- membrane barrier, and is involved in damage to the organs (liver, kidney, testis and lung) 6,11-13. therefore, the dose of dbp exposure is also an important factor that modulates the toxic impact of dbp on the ovarian.the bcl-2 family genes, including pro-apoptotic (e.g., bax, bcl-xs, and bak), and anti-apoptotic (e.g., bcl-2, bcl-xl, and bcl-w) members, regulate a distal step in an evolutionarily conserved pathway for programmed cell death. bax and bcl-2 genes primarily regulate apoptosis in body. the bax/bcl-2 ratio determines the survival or death of cells following an apoptotic insult,195200205210215220225230235240and an elevated bax/bcl-2 ratio can trigger apoptosis and target the cells for death14. in the present study, increased level of bax protein expression was observed in dbp-treated groups.inhibition of sod, cat, acp and akp activities with 3 different concentrations (100, 250, or500 mg/(kgd) of dbp indicated that a large number of superoxide anions (o2-) and h2o2 were produced through redox metabolism by nitrobenzene in the ovarian. therefore, the antioxidant defens