Study On Toxic Mechanisms Of Chloropropanol In Reproduction

Objective:In this study, the effects of3-monochloropropane-1,2-diol （3-MCPD） and(1,3-dichloro-2-propanol （1,3-DCP） on reproduction were investigated using R2Crat Leydig tumor cells, fertilized eggs and early embryonic.Methods:R2C cells were exposed to0.5,1,2,4,6mmol/L CP for48h. IC₂₅, ICC₅₀and IC₇₅of3-MCPD or1,3-DCP were elected by MTT assay. R2C cells were stimulated byabove three concentration for4h or24h. Cellular surface information was collected byatomic force microscopy （AFM）; The damage of DNAs in R2C cells was investigatedby single cell gel electrophoresis （SCGE）; Progesterone level in supernatant of cellculture medium was measured by radioimmunoassav （RIA）. The level of mRNAexpression of steroidogenic acute regulatory protein （StAR）, cholesterol side-chaincleavage enzyme (P4C₅₀scc) and3β-hydroxysteroid dehydrogenase （3β-HSD） in R2Ccells was determined by real-time quantitative PCR; The protein of StAR and3β-HSDin R2C cells was determined by Western blot; Flow cytometry （FCM） was applied toestimate the changes in mitochondrial membrane potential （MMP）. Fluorescencemeasurement was performed to determine intracellular levels of reactive oxygenspecies （ROS） and cyclic adenosine monophosphate （cAMP）. Effects of3-MCPD onfertilized eggs and early embryonic development in mouse was confirmed by in-vitrofertilization （IVF）.Results:3-MCPD or1,3-DCP caused inhibition of cell viability at the IC₂₅, ICC₅₀, and IC₇₅levels of1.027、1.802、3.160mM or1.161,1.897,3.100mM. AFM showed that3-MCPD-or1,3-DCP-induced R2C cells turned from the spindle into triangle. While3-MCPD induced R2C cells with4h, Ra of control, IC₂₅, ICC₅₀and IC₇₅were14.70±2.44nm,36.10±4.46nm,40.80±10.04nm,82.92±12.73nm, respectively; Rq ofcontrol, IC₂₅, ICC₅₀and IC₇₅was18.48±3.06nm,45.72±4.84nm, C₅₀.70±12.70nm,101.80±15.04nm, respectively; Rpm of control, IC₂₅, ICC₅₀and IC₇₅were18.88± 3.867nm,33.00±11.19nm,36.42±11.21nm,92.36±17.18nm, respectively.While1,3-DCP induced R2C cells with4h, Ra of IC₂₅, ICC₅₀and IC₇₅were38.10±5.90nm,48.80±14.29nm,84.92±15.47nm, respectively; Rq of IC₂₅, ICC₅₀and IC₇₅were51.98±6.35nm,60.08±13.25nm,108.5±7.47nm, respectively; Rpm of IC₂₅,ICC₅₀and IC₇₅was35.00±11.52nm,46.42±13.72nm,100.4±18.54nm,respectively. The Ra, Rq and Rpm differences between stimulation group and controlgroup were statistically significant （p<0.05）. SCGE showed that after3-MCPDinduced R2C cells with4h, tail DNA%or tail length or OTM values of control, IC₂₅,ICC₅₀and IC₇₅were4.288±0.660%,10.243±1.073%,11.427±1.865%,18.984±1.640%; or12.286±0.808μm,17.429±1.913μm,19.333±1.647μm,29.167±2.332μm; or1.759±0.144μm,3.247±0.471μm,4.844±0.807μm,8.965±0.800μm. When1,3-DCP induced R2C cells with4h, tail DNA%or tail length or OTMvalues of control, IC₂₅, ICC₅₀and IC₇₅were4.29±0.66%,23.80±2.48%,34.33±3.18%,38.03±3.63%;or12.29±0.81μm,58.67±6.38μm,79.67±5.86μm,84.86±6.07μm; or1.76±0.14μm,7.80±1.13μm,18.C₅₀±1.22μm,21.05±2.74μm. Thetail DNA%, tail length and OTM values differences between stimulation group andcontrol group were statistically significant （p<0.05）. RIA showed that after3-MCPDinduced R2C cells with4h, progesterone production of3-MCPD groups and thecontrol group were no significant differences by statistical analysis （p>0.05）. When3-MCPD induced R2C cells with24h, progesterone production of control, IC₂₅, ICC₅₀and IC₇₅were140.24±6.89ng,126.83±6.53ng,111.56±3.04ng and54.28±4.67ng. Progesterone production of3-MCPD groups and the control group weresignificant differences by statistical analysis （p<0.05）. When1,3-DCP induced R2Ccells with4h or24h, progesterone production of control, IC₂₅, ICC₅₀and IC₇₅were12.90±0.58ng,11.37±0.42ng,11.43±0.37ng,10.98±0.37ng or138.92±3.59ng,131.41±2.37ng,115.32±1.87ng,23.85±3.63ng. Progesterone production of1,3-DCP groups and the control group were significant differences by statisticalanalysis （p<0.05）. QRT-PCR showed that after3-MCPD induced R2C cells with4h,the expressions of StAR and P4C₅₀scc mRNA were no significant differences bystatistical analysis （p>0.05）, but the expressions of3β-HSD mRNA decreased46.8± 6.90%in the concentration of IC₇₅（p<0.05）, the expressions of3β-HSD mRNAbetween IC₂₅and ICC₅₀concentration of3-MCPD groups and the control group wereno significant differences by statistical analysis （p>0.05）. While3-MCPD inducedR2C cells with24h, the expressions of StAR mRNA in ICC₅₀and IC₇₅decreased25.1±3.0%and51.7±3.0%; the expressions of P4C₅₀scc mRNA in IC₂₅, ICC₅₀and IC₇₅decreased32.6±2.3%,32.5±2.7%and31.9±1.9%; the expressions of3β-HSD mRNAin IC₇₅decreased21.8±4.4%. There was significant differences above comparison（p<0.05）. When1,3-DCP induced R2C cells with4h, the expressions of StAR mRNAwere no significant differences by statistical analysis（p>0.05）; the expressions ofP4C₅₀scc mRNA in IC₇₅decreased20.8±3.5%; the expressions of3β-HSD mRNA inIC₂₅, ICC₅₀and IC₇₅decreased44.6±2.9%,45.6±2.3%and C₅₀.3±4.6%. There wassignificant differences above comparison （p<0.05）. As1,3-DCP induced R2C cellswith24h, the expressions of StAR mRNA in IC₂₅, ICC₅₀and IC₇₅decreased44.6±2.9%,45.6±2.3%and C₅₀.3±4.6%; the expressions of P4C₅₀scc mRNA in ICC₅₀andIC₇₅decreased24.3±3.8%and45.5±4.4%; the expressions of3β-HSD mRNA inICC₅₀and IC₇₅decreased27.6±2.6%and28.0±5.1%. There was significantdifferences above comparison （p<0.05）. Western blot showed that after3-MCPDinduced R2C cells with4h, the expressions of3β-HSD protein in IC₇₅decreased60.5±5.1%compared to that of control group （p<0.05）. As3-MCPD induced R2C cellswith24h, the expressions of StAR protein in IC₇₅decreased44.0±4.1%; theexpressions of3β-HSD protein in ICC₅₀and IC₇₅decreased38.2±6.3%and63.0±7.1%.There was significant differences above comparison （p<0.05）;1,3-DCP induced R2Ccells with4h, the expressions of StAR and3β-HSD protein in IC₇₅decreased25.8±5.8%and C₅₀.2±12.0%compared to that of control group （p<0.05）.1,3-DCPinduced R2C cells with24h, the expressions of StAR protein in IC₇₅decreased28.6±2.9%; the expressions of3β-HSD protein in IC₂₅, ICC₅₀and IC₇₅decreased42.0±2.5%,34.6±5.2%and43.3±5.1%. There was significant differences abovecomparison （p<0.05）. FCM showed that after3-MCPD induced R2C cells with4h or24h, MMP in IC₂₅, ICC₅₀and IC₇₅decreased35.0±4.5%,35.5±3.9%,33.1±5.5%or23.7±5.9%,49.0±7.8%,53.9±9.4%. There was significant differences above comparison （p<0.05）; and1,3-DCP induced R2C cells with4h or24h, MMP in threegroups of ICC₅₀4h, IC₇₅4h, and IC₇₅24h decreased24.3%,30.7%, and53.7%,respectively. There was significant differences above comparison （p<0.05）. ROS assayshowed that after3-MCPD induced R2C cells with4h or12h, ROS in IC₂₅, ICC₅₀andIC₇₅decreased60.9±17.1%,58.7±28.8%,93.5±20.1%;or C₅₀.5±23.9%,62.9±15.2%and71.4±20.3%, respectively. There was significant differences above comparison（p<0.05）. After1,3-DCP induced R2C cells with4h or12h, ROS in IC₂₅, ICC₅₀andIC₇₅decreased110.9±19.1%,138.7±20.8%,113.5±25.1%; C₅₀.5±13.98%,92.9±17.2%and111.4±24.3%, respectively. Results showed significant differences by statisticalanalysis except the group of IC₂₅for4h.3-MCPD or1,3-DCP acted on R2C cells1hor24h, there was no significant differences between3-MCPD-or1,3-DCP-inducedgroup and control group （p>0.05）. cAMP assay showed that3-MCPD induced R2Ccells with4h, cAMP in ICC₅₀and IC₇₅decreased30.0±7.2%and33.6±12.2%;1,3-DCPinduced R2C cells with4h, cAMP in IC₇₅decreased34.4±11.9%. There wassignificant differences above comparison （p<0.05）. IVF showed that3-MCPD (0, IC₂₅,ICC₅₀and IC₇₅) acted on oocytes for6h, fertilization rates of oocytes were90.52±15.41%,77.00±12.79%,17.86±10.C₅₀%,13.24±6.38%; fracture rates were0,10.14±3.82%,37.96±8.63,46.15±6.94%. There was significant differences abovecomparison （p<0.05）. After3-MCPD (0, IC₂₅, ICC₅₀and IC₇₅) acted on early embryosfor72h, the rate of2-cell were90.01±8.52%,72.96±12.27%,60.76±12.68%and61.27±14.68, respectively; the rate of4-cell were24.41±8.33%,6.73±3.95%,5.63±3.74%and0.10±0.10%, respectively; the rate of8-cell were7.04±3.10%,0%,0%and0%, respectively. There was significant differences above comparison（p<0.05）Conclusion:CP induced the morphological changes and DNA damage in R2C cells. In addition,CP exposure leads to increased levels of ROS in R2C cells.The findings suggest thatthe CP-induced progesterone production in R2C cells is mediated by StAR, P4C₅₀sccand3β-HSD. Take the ROS, MMP and cAMP results, the present findings indicatethat steroid hormone biosynthesis is inhibited by CP, which we tried to explain, at least in part, by ROS-and cAMP-induced StAR activity. CP could decline thefertilization rate and inhabit the early embryos from normal development in mice.