The Toxicity Of Copper Nanoparticles On Pregnant Rats And Its Effects On CYP450 Enzymes Related To Ovarian Sex Hormone Metabolism

Copper nanoparticles（Cu NPs）have been widely used in industry,agriculture,bio-medicine,environmental protection,etc.In animal husbandry,Cu NPs is an effective substitute for the current dietary copper sources because of its antibacterial,antiviral and immune-enhancing activity.In the toxicological study of Cu NPs to pregnant animals and its effect on sex hormone metabolism in ovary have not been reported.There is a safety risk about the use of Cu NPs during pregnancy.So,this study was carried out to research the toxicity of Cu NPs to pregnant rats and the influence of Cu NPs on regulation of cytochrome P450 enzyme（Cytochrome P450,CYP450）to metabolism endogenous hormones in pregnant rats’ovary.1.Characterization of Cu NPs and its acute toxicity to female rats:the average particle sizes of Cu NPs（50 nm）and micron copper（Cu MPs,1μm）were respectively 54.77±15.86 nm and 990.96±163.18 nm,by scanning electron microscopy,transmission electron microscopy and laser particle size analyzer.According to the results of up and down method,the acute peroral Lethal dose（LD50）of Cu ions,Cu NPs and Cu MPs were respectively 121.7 mg/kg,1573 mg/kg and>2000 mg/kg.2.The effect of Cu NPs to pregnant rats and fetal:Cu NPs can decrease the pregnant rats’body weight in a dose-dependent manner.High doses（180 mg/kg）of Cu NPs lowered the reproductive performance of pregnant rats.Blood physiological and biochemical results in high doses of Cu NPs showed that white blood cells（WBC）,glutamic-pyruvic transaminase（ALT）,alkaline phosphate kinase（ALP）,triglyceride（TG）,total cholesterol（TC）,UREA nitrogen（UREA）,low density lipoprotein（LDL-C）and lactate dehydrogenase（LDH）were significantly increased.Besides,the serum levels of interleukin-1β（IL-1β）,interleukin-18（IL-18）and tumor necrosis factor-α（TNF-α）in pregnant rats were also significantly increased in high doses of Cu NPs.The growth performance,total protein and iron contents of fetal were decreased in a dose-dependent manner.The content of malondialdehyde（MDA）,cyclooxygenase-2（COX-2）,inducible nitric oxide synthase（i NOS）,interleukin-2（IL-2）and interleukin-18（IL-18）were significantly increased in the fetal liver.3.The damage of Cu NPs to pregnant rats’ovary:Cu NPs as feed addictive in pregnancy caused the accumulation of copper element in the ovary.With the increasing dose of Cu NPs,it could cause ovarian tissue atresia,follicular interstitium edema.High dose of Cu NPs and copper ions lead to mitochondrial damage.Medium and high dose of Cu NPs and micron copper（180 mg/kg）caused vacuolization of endoplasmic reticulum in the ovary.At the same time,the level of MDA and i NOS were increased in high dose of Cu NPs,and the secretion of inflammatory factors of IL-2,TNF-αand MIP-αin medium and high dose of Cu NPs were also increased.The result of ovary metabonomics showed that the inflammatory and oxidative metabolic pathways were activated with the lipid metabolism disorders in pregnant rats’ovary.4.The effect of Cu NPs on sex hormone-related CYP450 enzymes in pregnant rats’ovary:medium and high dose of Cu NPs can significantly reduce the m RNA and protein expression of CYP450 1A2,1B1 and 3A4 in rats’ovary,and significantly increase the m RNA and protein expressions of CYP450 11A1,17A1 and 19A1.In addition,medium and high dose of Cu NPs could activate the phosphorylation levels of Nf-κb,Nrf2,ERK and the protein expression of aromatization receptor（AHR）,and inhibite the expression of famesoid X receptor（FXR）,pregnane X receptor（PXR）,constitutive androstane receptor（CAR）and Peroxisome proliferators-activated receptors（PPAR-α）.5.The mechanism research of Cu NPs to regulate CYP450 enzymes related to sex hormone metabolism in rat ovarian granulosa cells:the effect of Cu NPs to cell viability was determined by chemiluminescence method.The results showed the cell vitality was significantly reduced by Cu NPs from 6.25μg/m L～62.5μg/m L.The results of ELISA showed that SOD and GPx were significantly reduced,and the production of MDA and inflammatory cytokines IL-1β,IL-18 and TNF-αwere promoted by Cu NPs.6.25μg/m L Cu NPs can reduce the protein expression of CYP450 1A2,1B1 and 3A4,and activate the protein expressions of CYP450 11A1,17A1 and 19A1.In addition,chemical inhibitors were used to study the mechanism of oxidative stress and inflammatory response pathway regulating CYP450 enzyme to metabolism the ovarian sex hormones.The results showed that Cu NPs（6.25 mg/kg）can activate the expression of p-Nrf2 and NQO1 in the oxidative pathway and p-Nf-κB,p-ERK and p-p38 in the inflammatory pathway to induce the increase of expression of estrogen（E₂）restrictive synthase CYP19 protein,causing the increase of E₂secretion in rat ovarian granulosa cells.Above studies indicated that pregnant rats exposured to Cu NPs can reduce the reproductive performance of pregnant rats,inhibite the growth of the fetus,lead to ovarian injury,activate oxidative stress and inflammation and cause disorders of lipid metabolism in rats’ovary.Our vitro and vivo experiments elucidated that the mechanism of sex hormone disorder caused by Cu NPs poisoning is the regulation of CYP450 enzyme activity by Cu NPs activating the oxidative stress and inflammatory signaling pathways.