Establishment Of A Rat Model For Developmental Immunotoxicity Assessment And Its Application In Safety Evaluation Of Transgenic Cryab/Ac Rice

ObjectivesA rat model was developed for developmental immunotoxicity study. After validating its feasibility through a extended-one generation study with MeHg, it was utilized to assess the safety of transgenic CryAb/Ac rice, including the systemic immune toxicity and gut mucosal immune toxicity.Methods1Development of a rat model for developmental immunotoxity(DIT) study1.128-day oral toxicity study in female rats30Wistar rats were administrated MeHg by garage at doses of1.5mg/(kg·bw)/d、0.75mg/(kg·bw)/d for28days.During the experiment, food intake were measured once a week, and body weight twice a week. The immunologic endpoints included: flow cytometric analysis of lymophocyte subsets of blood and spleen, evaluation of the lymphoproliferation activity of splenocytes in response to ConA and sheep red blood cell(SRBC) immune response test(PFC).1.2Development of a rat model for DIT study60female Wistar rats and30male Wistar rats were randomized and divided into2experimental groups:MeHg group and the control group. The study began with mating. Day0of pregnancy is defined as the day a vaginal plug was found. And each group should contain no less than20pregnant females at or near parturition. The FO female rates were dosed daily by oral gavage with MeHg from gestational day(GD)6to lactation day(LD)21at a dose of1.5mg/(kg·bw)/d. Body weight was measured twice a week while food intake once a week. And the FO female rats were sacrificed at LD21for immunotoxicity tests.The immunotoxicity assessment of offsprings were examined on postnatal day(PND)0、 PND21、PND42. For immunological endpoints,10female pups and10male pups, originating from different litters, were included at any timepoint. And the immunological endpoints included:hematology, the absolute weight and relative weight of organs, flow cytometric analysis of lymphocyte subsets of blood, spleen, and Peyer’s patch, natural killer(NK) cell activity of splenocytes in response to ConA, PFC assay, flow cytometric analysis of TNF-a in Peyer’s patch and the numbers of goblet cell in the small intestine.2DIT study of exposure to transgenic CryAb/Ac rice2.1Nutrients’ analysisSamples of the study included three kinds of rice, TT51, parent-control rice and control rice.Protein, fat, fiber, vatamin, minerals and amino acid in TT51were determined. And the nutrient, heavy metal, pesticide and aflatoxin in the feed(both maintenance and growth diet), were determined as well.2.2Qualitative and quantitative detection of CryAb/Ac protein in TT51The qualitative detection was performed by Quickstix strip. And the quantitative detection was performed by ELIS A, using CryAb/Ac standard protein.2.3DIT study of exposure to transgenic CryAb/Ac rice90female Wistar rats and45male Wistar rats were randomized and divided into three groups:TT51group, parent-control group and control group. The study began with the10-week period. The female and male rats were fed with the diets mixed with rice for10weeks before mating. And food intake and body weight were measured once a week. When mating, each female was placed with a single male from the same group, until copulation occurred. And then change to another female.Day0of pregnancy is defined as the day a vaginal plug was found. And each group should contain no less than20pregnant females at or near parturition. The F0female rats were sacrificed at LD21for immunotoxicity study.The immunotoxicity assessment of F1offsprings were examined on postnatal day(PND)0、PND21、PND42. For immunological endpoints,10female pups and10male pups, originating from different litters, were included at any timepoint. And the immunological endpoints included:hematology, the absolute weight and relative weight of organs, flow cytometric analysis of lymphocyte subsets of blood, spleen, and Peyer’s patch, natural killer(NK) cell activity of splenocytes in response to ConA, PFC assay, flow cytometric analysis of TNF-a in Peyer’s patch and the numbers of goblet cell in the small intestine.For mating the F1offspring, at least one male and one female should be selected at weaning from each litter for mating with other pups of the same group but different litter, to produce the F2generation. And the rats for F1mating included30female and15male rats each group.And before mating, there was a10-week period. And food intake and body weight were measured once a week. When mating, each female was placed with a single male from the same group, until copulation occurred. And then change to another female.Day0of pregnancy is defined as the day a vaginal plug was found. And each group should contain no less than20pregnant females at or near parturition. The F1female rats were sacrificed at LD21for immunotoxicity study.The immunotoxicity assessment of F2offsprings were examined on postnatal day(PND)0、PND21、PND42. For immunological endpoints,10female pups and10male pups, originating from different litters, were included at any timepoint. And the immunological endpoints included:hematology, the absolute weight and relative weight of organs, flow cytometric analysis of lymphocyte subsets of blood, spleen, and Peyer’s patch, natural killer(NK) cell activity of splenocytes in response to ConA, PFC assay, flow cytometric analysis of TNF-a in Peyer’s patch and the numbers of goblet cell in the small intestine.Results1Development of a rat model for developmental immunotoxity(DIT) study1.128-day oral toxicity study in female ratsCompared with control group, in both low-dose group and high-dose group, absolute and relative weight of liver were reduced while those of kidney were increased, and the number of PFC, reflecting humoral immune function, was reduced as well.And CD4/CD8of splenocytes and peripheral-blood lymphocytes was reduced in high-dose group.1.2Development of a rat model for DIT studyAs regard of the reduction of CD4/CD8in high-dose but not in low-dose group, we chose1.5mg/(kg-bw)/d as the dose for DIT study.Of both parental female rats and offsprings on PND42, abnormal changes were observed in systemic immune function and gut mucosal immune function.The changes of rat pups on PNDO revealed that the immune function was involved by indirect exposure in utero.On PND21, changes were observed in systemic immune function except the NK cell activity, and no adverse effect was observed towards gut mucosal immune function.2DIT study of exposure to transgenic CryAb/Ac rice2.1Nutrients’ analysisFeed formulation was based on results of the nutrients’ analysis of rice.And the component analysis of both maintenance and growth purified diet revealed that feed mixed with60%rice was able to satisfy the rats.2.2Qualitative and quantitative detection of CryAb/Ac protein in TT51The result of qualitative and quantitative detection of CryAb/Ac protein in TT51revealed that TT51did contain CryAb/Ac protein, and it’s about0.03187ug/g in it.2.3DIT study of exposure to transgenic CryAb/Ac riceThe condition of F0female rats during10-week period was good, and the Fl offsprings growed well. And the same was Fl parental female rats and the F2offsprings.In respect of systemic immune function, there was no significant differences found among three groups in F0and F1parental female rats,and in F1and F2 offsprings.And no adverse effects were observed towards gut mucosal immune function.Conclusions1The rat model for DIT study developed in this study is able to find the immunotoxicity of MeHg, so it can be utilized in DIT assessment.2No effect of developmental immunotoxicity was found in Wistar rats after exposure to transgenic CryAb/Ac rice TT51.3No adverse effect on gut mucosal immune function was found in F0, F1parental female rats and F1, F2offsprings after exposure to transgenic CryAb/Ac rice TT51.