| ObjectiveTT51is a new type of genetically modified Bt rice currently under development in China with our own intellectual property, created by inserting a synthetic fusion gene of Cry1Ab/Ac into parental rice MingHui63. Fields tests indicate that TT51showed excellent insect resistant characteristics to major Lepidopteran pests and was released a biosecurity certificate in late2009by the Ministry of Agriculture. In recent years, people pay special attention to safety of transgenic crops all over the world. Thus, safety evaluation conducted to ensure safety of this transgenic crop is necessary. In order to evaluate food safety of this transgenic rice, to provide more scientific data for food safey risk assessment and development of transgenic crop safety evaluation technology including special toxicity assessment like reproductive toxicity and developmental neurotoxicity, the influence of the transgenic CrylAb/Ac rice TT51on the growth and reproductive function of rats were observed through a two-generation reproduction toxicity study.Methods1A two-generation reproduction study with TT51in rats1.1Qualitative and quantitative detection of CrylAb/Ac protein and Nutrients compositional analysis of TT51The qualitative presence of CrylAb/Ac protein in TT51, MingHui63and control was measured by using a Quickstix kit for CryAb/Corn Leaf&seed, while quantitative detection of CrylAb/Ac protein was determined with an antibody specific enzyme linked immunosorbent assay (ELISA). Carbohydrates, fat, fiber, vitamins, minerals and other ingredients such as heavy metals, pesticides and aflatoxin B1of common control rice, parental rice MingHui63and transgenic CrylAb/Ac rice TT51were determined in accordance with Chinese Standard methods.1.2A two-generation reproduction study with TT51in ratsNinety female and forty five male Wistar rats were4weeks of age at the start of the study. All animals were assigned to acclimate to the the experimental housing condition for7days during which they were kept on basal diet. At the beginning of the study, animals were randomly assigned into three experimental groups and they were fed on a self-feeding with corresponding diets (Control, MingHui63and TT51) with thirty female and fifteen male rats in each group. Before each mating period, parental rats were treated with corresponding diets for10weeks. Male rats were housed together with female rats in the same group on a one-to-one basis at18:00pm through one night. The presence of sperm in the vaginal smear under an optical microscope or a vaginal plug was considered evident of successful mating and designated as day0of gestation (GDO) and the pregnant rat were housed in single cages. During the mating, intercourse of siblings was avoided. The male rats (F0) that were used for mating were sacrificed for relevant checks. On PND4, litters were randomly adjusted to eight pups comprising of four males and four females. No adjustment was made for total litters fewer than eight pups. The females were allowed to deliver spontaneously and nurse their pups until PND21(the day of weaning). At weaning day on PND21the successfully survive family number of dams and litters should no less than20. During mating, gestation and lactation until weaning of F1, F0females were also fed with corresponding diets. All animals were provided with unlimited tap water. After weaning, F1and F2rats were housed individually as previously described till F2rats were PND70.Observation Indexes:All rats were observed daily for clinical signs of mortality or morbidity. Body weight gain and food consumption were measured and recorded weekly. Estrous cycle of females was measured at puberty. Copulation index, fertility index, gestation length, live-birth rate, the numbers of live and dead pups, sex ratio of birth pups were recorded and calculated. In females exhibiting evidence of successful mating, body weight and food consumption of dams were recorded on days0,7,14, and20of pregnancy and days0,7,14, and21of lactation. Physical developmental parameters of the F1and F2pups such as opening of the eyes and the ears, the growth of the hair and the teeth, descent of the testes and the opening were all observed and evaluated. The day on which all the pups of a litter attached the criteria for indicators were recognized as the day of reach the standard day. All pups from no less than20dames from each group were observed. At the schedule day, the rats were killed under sodium pentobarbital anesthesia (60mg/kg, IP), fixed with10%formalin, stained with HE staining for histopathological examination. Hematology, serum chemistry parameters, serum sex hormone for both females and males for all adult, sperm head counts and deformity for males were measured at the end day of the study.1.3Effects exposure to TT51on the reproductive system in offspring rats1.3.1Uterotrophic Assay for Fl female offspringOn PND21eight F1female offspring rats in were randomly selected each group and treated with3μg/(kg·d) E2by intraperitoneal injection for3consecutive days.6h after the last injection uterine wet and blotted weigh were measured and Compared between groups to determine the estrogenic effect after exposure to TT51in offspring rats.1.3.2Effects of parental exposure to TT51on the reproductive system in Fl male ratsAfter weaning, eight F1male offspring rats were randomly selected in each group and fed with basal rodent diet for70days, during this period body weights gain weigh and feed consumption were measured weekly; animal growth and development performance were observed daily. At the end of the study, the rats were killed under sodium pentobarbital anesthesia (60mg/kg, IP), fixed with10%formalin, reproductive organs stained with HE for histopathological examination. Hematology, serum chemistry parameters, sperm head countt and deformity and relative organ/body weights of the reproduction organ were all measured.1.4Effects exposure to TT51on gene expression of Pituitary-testicular-axis in F1ratsAfter mating, eight F1male rats were randomly selected from each group and killed under sodium pentobarbital anesthesia (60mg/kg, IP), then testicles, hypothalamus and pituitary were quickly separated and stored at-20℃until analysis. Testis enzyme ACP, LD and SDH activity were measure and relative expression of GnRH, FSH, LH and Ar mRNA were determined by real-time quantitative PCR analysis.2Effects exposure to TT51on neurobehavioral, learning and memory in offspring rats2.1Early neural development indicators of the offspring rats Early neural developmental parameters of the F1and F2pups such as cliff avoidance, surface righting, air righting, forelimb hanging and acoustic startle were all observed and evaluated. The day on which all the pups of a litter attached the criteria for indicators were recognized as the day of reach the standard day. All pups from no less than20dames from each group were observed for parameters above mentioned.2.2Sensory-motor function combination analysis in F2ratsOn PND2812F2male and female rats randomly selected from each group were conducted sensory-motor function analysis combination, including, hot plate test, rotarod test, grip strength test, measurements of hind limb landing foot splay test and motor activity test can be seen as a screening test combination for Sensory-Motor function. All of the above tests were measured for3times with an interval of30min; take the average as the result.2.3Learning-memory behavior combination analysis in F2ratsTo evaluate effects of exposure to TT51on learning and memory behavior ability in rats,12F2male and female rats randomly were selected from each group prepared for learning-memory behavior combination analysis. The effects on learning and memory behavior, such as passive avoidance response in step-through avoidance response and step-down avoidance response, locomotor activity in the open field test and, spatial learning ability in the Morris water maze were examined. Step-through avoidance response and step-down avoidance response test were arranged start on PND28while open field test and Morris water maze on PND70.Results1A two-generation reproduction study with TT51in rats1.1Qualitative and quantitative detection of CrylAb/Ac protein and Nutrients compositional analysis of TT51The CrylAb/Ac protein was detected with an antibody specific enzyme linked immunosorbent assay (ELISA), which is a semi-quantitative detection method. The TT51rice was found to be positive for the presence of CrylAb/Ac protein with a concentration of0.03187μg/g, while the MingHui63rice was found to be negative. The results of nutrients compositional analysis indicated that primary nutrients (protein, fat, carbohydrate, most minerals and vitamins) of all the transgenic rice and its parental rice are similar but the content of dietary fiber in MingHui63was higher than that of TT51. In addition, heavy metals, pesticides residues and aflatoxin concentrations of the three kinds of rice-based rodent diets were all measured in order to eliminate the influence of them.1.2A two-generation reproduction study with TT51in ratsIn this study, both clinical performance variables and histopathological responses were examined and Compared between groups. There were no significant differences between groups on body weights, food consumption, estrous cycle of females> copulation index, fertility index, gestation length, live-birth rate, the numbers of live and dead pups, sex ratio of birth pups, sperm count and deformity for males and relative organ/body weights. There were some statistically significant differences in hematology and serum chemistry parameters, but no histological abnormalities were seen in the brain, heart, liver, spleen, kidneys, stomach, small intestine, thymus. There were no significant difference between groups on serum hormone parameters for both females and males.1.3Effects exposure to TT51on the reproductive system in offspring rats1.3.1Uterotrophic Assay for Fl female offspringCompared with MingHui63rice and control group, parental exposure to TT51didn’t affect weights of uterus in F1female offspring rats.1.3.2Effects of exposure to TT51on the reproductive system in Fl male ratsThere were no significant difference on body weights gain, food intake, hematology, serum chemistry, serum hormones, sperm head countt and deformity and relative organ/body weights of reproductive organs between groups (p>0.05).No abnormalities were observed on gross as well as microscopic pathology analysis.1.4Effects exposure to TT51on gene expression of Pituitary-testicular-axis in Fl male ratsNo significant differences observed among groups on testis enzyme ACP, LD and SDH activity and expression levels of GnRH, FSH, LH and Ar mRNA (p>0.05). 2Effects exposure to TT51on neurobehavioral, learning and memory in offspring rats2.1Early neural development indicators of the offspring ratsNeural developmental parameters of the F1and F2pups such as cliff avoidance, surface righting, air righting, forelimb hanging and acoustic startle were all observed and evaluated, but there were no significant differences observed (p>0.05)2.2Sensory-motor function combination analysis in F2ratsF2rats were tested for Sensory-motor function by a sensory-motor function combination analysis, including, hotplate test, rotarod test, grip strength test, measurements of hind limb landing foot splay test and motor activity test, but there were no significant differences observed (p>0.05)2.3Learning-memory behavior combination analysis in F2ratsThe learning and memory behavior ability of F2rats were measured by Learning-memory behavior combination analysis method, such as passive avoidance response in step-through avoidance response and step-down avoidance response, locomotor activity in the open field test and, spatial learning ability in the Morris water maze were all includided in the learning-memory behavior combination analysis. There were no significant differences observed in this learning-memory behavior combination analysis (p>0.05)Conclusion1All rats and their offsprings were in good health condition, behavior noramally; no animal found deformities o rother adverse effects, nor death and poisoning symptoms observred. In this two-generation reproduction study, TT51has no significant impacts on reproductive and developmental parameters of rats Compared with parental MingHui63rice and the control (p>0.05)2Effects of exposure to TT51on testis enzyme activity and relative mRNA expression in hypothalamus-pituitary gonad axis of male rats were firstly evaluated in this stydy. No significant differences were observed on testis enzyme activity and relative mRNA expression in hypothalamus-pituitary gonad axis of F1male rats (p>0.05).3Effects of exposure to TT51on early neural development parameters in offsring rats and learning-memory ability of rats were firstly evaluated through this stydy. No significant differences were observed on early neural development parameters in F1and F2offsring rats and learning-memory ability of F2rats (p>0.05). |
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