The Female Reproductive Toxicity Of Phthalic Acid Esters On C. Elegans

Research Background and Research PurposesPhthalic acid esters (PAEs) which used as PVC plastic plasticizer that released into the environment in the process of production and life using can be checked out in various of environmental media. As a kind of environmental endocrine,they have caused different influence on the reproduction and growth of algae and apuatic organisms.This study used c. elegans to explore the female reproductive toxicity of PAEs, look for a sensitive index to evaluate PAEs reproductive toxicity and explore the damage effect of PAEs on oogenesis process and discusse its mechanisms of reproductive toxicity.Methods and ResultsReproductive toxicity effect of PAEs in c. elegans1. Effect on c. elegans fertility of PAEsTo measure the acute toxicity of PAEs on nematodes, each test exposure group is synchronization L4 period after 24 h of N2 nematodes that was set up in five infected dose groups (100.10,1,0.1,0.01 mg/L), solvent control group and blank control group. After exposing to PAEs for 24 h, the mortality of nematodes in each group was observed and the LC50 of PAEs on nematodes was calculated.Experimental results showed that the LC50 of PAEs was much higher than 100 mg/L, each dose group of nematode mortality had no statistical difference compared with the solvent control group; To evaluate the offspring number and generation time effect on the nematode fertility of PAEs, synchronization L4 period after 24h of N2 nematodes for each test exposure group was set up in three infected dose groups (1,1,0.1 mg/L), solvent control group and blank control group. After exposing to PAEs for 24 h, the number of offspring and generation time of nematodes was measured.Only high doses of DEHP infected group has a statistically significant decline, other dose groups have no significant change; There was no statistically significant extend or shorten of the generation time after exposing to different dose of PAEs.2. PAEs in c. elegans eggsUsing germ cell number to evaluate the effect of PAEs in nematodes eggs, synchronization L4 after 24h of N2 nematodes was used in the exposure group, dose and exposure was the same with the above case, DAPI staining after the exposure.Results showed that the number of germ cells in middle and high dose groups of DEHP. DBP and DIBP have a statistically significant decrease compared with the solvent control group, each low dose group did not shown any statistically significant change. The DTC cell fluorescence intensity of JK2868 mutant strains (Plag-2::GFP) reflects the gonads structure is normal or not, synchronization L4 period after 24h of JK2868 mutant strains nematodes was used as the exposure group, dose and exposed was the same with the above case, the DTC cell fluorescence intensity was detected after the exposure.Fluorescence analysis results showed that DTC fluorescence intensity was unchanged after PAEs exposure, which suggest that PAEs unaffected the gonads structure in reproductive toxicity.The mechanism of oogenesis of PAEs in c. elegans1. The effect of PAEs on c. elegans germ cell apoptosisCounting number of germ cell apoptosis reflected the influence of PAEs in nematodes apoptosis, the dose and exposure of each test exposure group for MD701 mutant strains (Plim-7::ced-1::GFP) nematodes was the same with the above case, the apoptosis of c. elegans germ cells were detected under the fluorescence microscope after the exposure. The experimental results showed that nematodes apoptosis levels raised up after the PAEs exposure, the apoptotic cells in middle and high dose groups were statistically significant by comparing with the solvent control group. WS1433 mutant strains (HUS-1::GFP) indicated the apoptosis induced by germ cell DNA damage. WS1433 mutant strains nematodes were used in each test exposure group, dose and exposure was the same with the above case. Results showed that DNA repair instructions fluorescent of c. elegans germ cell was increased significantly in high dose infected group of PAEs under the fluorescence microscope after the exposure, which suggest that elegans germ cell apoptosis was induced by DNA damage. The mechanism research of germ cell apoptosis for PAEs in c. elegansDetection of apoptosis related gene expression level reflected its role in apoptosis pathway, each test exposure group of N2 nematodes was used with a volume of 80-100μL, dose and exposure was the same with the above case.The total RNA of nematodes was extracted and RT-qPCR was used to detecte the expression level of apoptosis related gene(cep-1、egl-1、ced-9、 ced-4 and ced-3) after the exposure. Results showed that the expression level of cep-1、egl-1、 ced-4 and ced-3 were increased significantly while ced-9 expression level decreased significantly. The effect of apoptosis related genes in the pathway was analyzed by using the apoptosis gene mutation. The dose and exposure of knockout gene mutant strain without cep-1、 egl-1、ced-9、ced-4 and ced-3 expressed was the same with the above case. The apoptosis number of germ cell was measured after the exposure. The test results showe that the apoptosis number was obviously decreased after the cep-1、egl-1、ced-4 and ced-3 gene mutant strain exposure while raised after the ced-9 gene mutant strains exposure. Effect of PAEs on oxidative stress in c. elegans PAEs affected ROS level in c. elegansDetermination of c. elegans ROS reflected the nematodes oxidative stress level, the CM-H2DCFDA probe reflected the ROS in c.elegan. Synchronization L4 period after 24 h of N2 nematodes were used in the test exposure group, dose and exposure was the same with the above case. After the exposure, the ROS level in nematode is measured by the CM-H2DCFDA probe.Results showed that the ROS levels in the middle and high dose groups were raised after exposing to PAEs.To detect the level of H2O2 that was the main components in ROS, each test exposure group of N2 nematodes was used with a volume of 80-100μL, dose and exposure was the same with the above case. The test results showed that H2O2 level has no difference change in every dose group. Oxidative stress mechanism research of PAEs in c. elegansDetection the expression level of oxidative stress related gene reflected its role in the oxidative stress pathway.Each test group was exposed to 80-100μL synchronization L4 period after 24 h of N2 nematodes, dose and exposure was the same with the above case. The total RNA of nematodes was extracted and RT-qPCR was used to detecte the expression level of apoptosis related gene(mev-1、gas-1、isp-1、clk-2) after the exposure. The results showed that the expression level of isp-1, clk-2 increased significantly while the expression level of mev-1、 gas-1 were significantly decreased. Germ cell DNA damage research of PAEs in c. elegansThe numbers of c. elegans DNA AP was applied to reflect the response to DNA damage, each test group was exposed to 80-100μL synchronization L4 period after 24h of N2 nematodes, dose and exposure was the same with the above case. The DNA of nematodes was extracted and the numbers of c. elegans DNA AP points were counted. The results showed that the number of AP points were increased with the increasing dose of PAEs, there was a statistically significant in middle and high dose group of DEHP and DBP, DIBP by comparing to the solvent control group.ConclusionPAEs is a kind of low toxic substances, the number of offspring and generation time are not the sensitive indicators to evaluate the reproductive toxicity of PAEs in c. elegans, while the change of germ cell number is more sensitive than the number of offspring and generation time. PAEs could increase the DNA damage by increasing the ROS levels and the AP points in the nematodes, which leads to a DNA damage induced apoptosis in germ cells and may play an important role in the reproductive toxicity.