Developmental Neurotoxicity Of Rats Exposed To Yttrium Nitrate And The Mechanism Exploration

ObjectiveExploring the mechanism of yttrium nitrate [Y(NO3)3] on neurodevelopment by using ITSFn neural developmental toxicity evaluation in vitro model, to provide cytological evidences for animal experimental endpoints. Assessing the effects of Y(NO3)3 on neurobehavioral development in Sprague-Dawley rats according to OECD guideline 426, and the cell signaling mechanisms were explored. The results can provide important scientific basis for risk assessment of yttrium.Methods1 Effects of Y(NO3)3 on developmental neurontoxicity and the mechanisms involved① Using MTT method to calculate cell viability and IC50 ②The concentration of Y(NO3)3 was 0.55,2.75,13.7,27.5 u.g/mL separately, the control group was provided medium only. Neurons was induced by using ITSFn culture, and tested in the following experiments:The rate of cell apoptosis was detected by the comet assay and flow cytometry. The mRNA expression including Bcl-2, Bax and Caspase-3 were checked by real time reverse transcription-polymerase chain reaction(RT-PCR). The Bcl-2 and Bax protein were measured by western blotting; the Caspase-3 activity was determined by spectrophotometric method2 Developmental neurotoxicity assessment of rats exposed to Y(NO3)3 and the mechanisms involved2.1 Pre developmental neurotoxicity experiments①Using Horn method to determine LD50. The animals were given 0,215,464,1000,2150 mg/kgBW Y(NO3)3, then observed for 14 days, and the dead numbers of rats were noted.②Repeated dose 28-day oral toxicity study for females:three experimental groups were fed with Y(NO3)3 at doses of 1/10,1/20,1/40 LD50(80,40,20 mg/kg/day) by gavage, and one control group was given water. Body weight gain and food consumption were measured and recorded weekly. At the end of the experiment, the rats were sacrificed; the organs were removed, weighted and examined grossly. Hematology, serum chemistry parameters were measured.2.2 Developmental neurotoxicity assessment of rats exposed to Y(NO3)3Each sexually mature virgin female Sprague-Dawley (SD) rats mated with a resident male rat of the same strain. Evidence of a vaginal plug was used to indicate successful mating and Day 0 of pregnancy. Mated female rats were randomly divided into four groups of 20 animals each. From gestation day (GD) 6 to postnatal day (PND) 21, the pregnant rats were housed singly and received one of four doses (0,5,15, or 45 mg/kg) of the Y(NO3)3 solution by gavage each day. On PND 4, the number of pups in each litter was reduced to 8:(4 males and 4 females). At the weaning day, the dams were sacrificed, some litters were fed to PND70, the others were given the same dose Y(NO3)3 until the end of the experiments. The pattern of pup assignment was based on OECD 426 guidelines, with slight modifications.①Observation indexes for dams:maternal weight and food consumption were noted, food utilization rates were calculated for each group. At the weaning day, the dams were sacrificed; the organs were removed, weighted and examined with HE staining. Hematology, serum chemistry parameters were measured.②Observation indexes for litters (feeding group):litters weight and post-weaning food consumption were noted, food utilization rates were calculated for each group. Litter parameters including survival rate at PND 0,4,21, pups per litter, sex ratio of male pups, average pup weight were recorded and calculated. Physical developmental parameters such as testes descent and vaginal opening were observed and evaluated. At the schedule day, the rats were sacrificed; the organs were removed, weighted and examined with HE staining, The numbers of nissl-positive cell in hippocampal dentate gyrus (DG) were counted. Hematology, serum chemistry parameters were measured. Early neural development parameters of pups (including surface righting reflex, cliff avoidance, fore-limb grip strength, startle response, homing reflex, air righting reflex) were observed and evaluated. Sensory-motor function, motor activity and learning-memory behavior were tested at adolescent and early stage of maturity.③Observation indexes for litters (gavage group):post-weaning pups weight and food consumption were noted, food utilization rates were calculated. Parameters such as hematology, serum chemistry, organs examination and nissl-positive cells count were measured as described above. Sensory-motor function, motor activity and learning-memory behavior were also tested at the early stage of maturity.2.3 Study on the mechanism of Y(NO3)3 on developmental neurotoxicityThe hippocampus of PND21 pups and PND70 pups (gavage group) were detached, and used for following parameters:the mRNA expression including Bcl-2, Bax, Caspase-3, CaM, CaMKⅡ, CREB and BDNF were checked by RT-PCR; the Bcl-2, Bax, CaMKⅡ, CREB and BDNF protein were measured by western blotting; the Caspase-3 activity was determined by spectrophotometric method; the content of amino acid neurotransmitters was determined by LC-MS/MS.Results1 Effects of Y(NO3)3 on developmental neurontoxicity and the mechanisms involved① With the increase of concentration, the inhibition rate of cell growth went up. The IC50were calculated to 224.16μg/mL. ②13.7 and 27.5 μg/mL groups showed a significant increased rates of apoptosis in comet assay (P<0.05), flow cytometry results demonstrated rates of apoptosis decreased in 0.55μg/mL group, while increased in 27.5 μg/mL group, the differences were significant comparing with the control group (P<0.05). RT-PCR and western blotting results showed that Y(NO3)3 could up-regulate the expression of Bcl-2 and down-regulate the expression of Bax and Caspase-3 in 0.55μg/mL group, but down-regulate the expression of Bcl-2 and up-regulate the expression of Bax and Caspase-3 in 27.5μg/mL group. The ratio of Bcl-2/Bax ascended first then decreased, displaying an inverted U-shape.2 Developmental neurotoxicity assessment of rats exposed to Y(NO3)3 and the mechanisms involved2.1 Pre developmental neurotoxicity experiments① LD50 for females was 794mg/kg BW.②Repeated dose 28-day oral toxicity study:the 40 mg/kg/day group showed a decreased body weight at the third and the fourth week, the 80 mg/kg/day group showed a decreased body weight at the second, the third and the fourth week. Both groups had a lower weight gain. All these differences were significant comparing with the control(p<0.05). There were some statistically significant differences in hematology and serum chemistry, but all these data were in our lab history control range.2.2 Developmental neurotoxicity assessment of rats exposed to Y(NO3)3① Results for dams:There was no significant difference in body weight, food consumption and food utilization rates among the animal groups during gestation and lactation (p>0.05). There were some statistically significant differences in hematology and serum chemistry, but all these data were in our lab history control range. No histological abnormalities were found.② Results for litters (feeding group):Both male and female groups exposed to Y(NO3)3 during gestation and lactation demonstrated a significant increase in mean body weight compared with controls on PND 21 (p<0.05). However, the differences disappeared two weeks or more after the yttrium solution was discontinued. The postnatal survival data, the mean number of pups, the percentage of males per litter, the average pup weight, and the PND of testes descent or vaginal opening were not affected by any dosage of the yttrium solution. No differences were found in food consumption or the food utilization rates among all the groups. No histological abnormalities were found in HE stain or Nissl stain. Some statistically significant changes were noticed in hematology, serum chemistry, early neural development tests and neurobehavioral tests, but the difference had no biological significances.③Results for litters (gavege group):45mg/kg/day male offspring body weight decreased significantly from PND 42 to the end of the experiment (p<0.05), parameters like food utilization rates, hematology or serum chemistry did not show biological significant changes. No histological abnormalities were found in HE stain or Nissl stain.45mg/kg/day females offspring showed a higher latency on the fifth day (p<0.05) in Morris water maze test, results of other neurobehavioral tests were similar among all the groups.2.3 Study on the mechanism of Y(NO3)3 on developmental neurotoxicityRT-PCR results showed that the relative level of CaM decreased in male 5 and 15 mg/kg/day groups on PND21, increased in female 5 and 45 mg/kg/day groups on PND21 and male 15 mg/kg/day group on PND70, all these difference were significant comparing with the controls (P<0.05). GABA content of brain in male 45 mg/kg/day group on PND21 and Gly content in female 45 mg/kg/day group on PND21 were significantly higher than control group (p<0.05) No significant differences were found in the expressions of Bcl-2,Bax, Caspase-3, CaMKⅡ、 CREB and BDNF protein.Conclusion1. The effects of Y(NO3)3 on developmental neurontoxicity has two sides. Low concentration of Y(NO3)3 could up-regulate the expression of Bcl-2, down-regulate the expression of Bax and Caspase-3,inhibit the rate of apoptosis; while high concentration of Y(NO3)3 down-regulate the expression of Bcl-2, up-regulate the expression of Bax and Caspase-3, and induce the rate of apoptosis. The results revealed that Y(NO3)3 has neural development toxicity.2.Both male and female groups exposed to Y(NO3)3 during gestation and lactation demonstrated a significant increase in mean body weight compared with controls on PND 21 (p<0.05). However, the differences would disappear two weeks or more after the yttrium solution was discontinued. No significant impacts were found on early neural development tests and neurobehavioral tests among all the groups (p>0.05). The NOAEL of Y(NO3)3 for developmental neurotoxicity is 45 mg/kg/day, equal 14.6 mg/kg/day for yttrium.3. The additional experiment(gavage group) showed that 45mg/kg/day male offspring body weight decreased significantly from PND 42 to the end of the experiment (p<0.05), the females showed a similar trend, but no significant differences were observed. The results hinted that with the increase of exposure time, Y(NO3)3 has a potential developmental neurotoxicity.4. Y(NO3)3 had an effect on the expression level of CaM mRNA, possibly due to the influx of Ca2+.No significant differences were observed in the expression of proteins related with apoptosis, CaMKⅡ, CREB and BDNF proteins (P>0.05).