Effect Of Heroin On Genital Endocrine System In Female Rats

The abuse of opioid has caused serious medical and social problems. However,the generation and development of tolerance, dependence and addiction ofopioid has long been an interesting subject. Yet the mechanism of tolerance andphysical dependence has not been clarified. It was said that heroin was the maindrug which was used. The number of Female heroin abusers raised quickly inthe last ten years. Menstrual disorder, amenia, abortion and sterility are theharmful results caused by heroin use. The paper tends to investigate the effectsof heroin on genital endocrine system in female rats by the following aspects:effects of heroin on five sexual hormones in plasma;effects of heroin on thegene expression of prolactin (PRL), luteinizing hormone (LH) andfollicle-stimulating hormone (FSH) in pituitary;effects of heroin on themorphologic changes of pituitary, uterus and ovary;effects of heroin on themetabolism of purine nucleotide. According to these four aspects , the researchtry to probe the substantial basis of heroin dependence and tolerance in femalerats and provide a new way for the mechanisms research in females.1. Establishment of heroin addiction and withdrawal model: Adult femaleWistar rats were randomly divided into randomly divided into six groupsrandomly (10 rats each group): control, heroin-treated-3d, heroin-treated-9d,heroin-withdrawal-3d, heroin-withdrawal-9d and heroin-treated-withdrawalgroups. Whether the model was established successfully or not was assessed bythe conditioned place preference experiment reflects the psychologic or mentaldependence and by the naloxone precipitated withdrawal syndromes whichreflect the physical dependence. The weight and tail flick test were alsoobserved during the test.In this experiment it was found that rats developed obvious place preference onday 4th in heroin-treated-withdrawal group as compared with control (salinetreated) group, which preferring recording times was less than 500 second, andthe rats also preferred to stay in the heroin-related-side although they werestopped being administered heroin for 3days. The place preference vanished onthe day when the rats haven't been treated by heroin for 9days. Naloxoneprecipitated withdrawal syndromes show that the scores of rearing, jumping,wet dog shakes, diarrhea or defecation and the stretch of body weresignificantly higher than that in group control. The results of conditioned placepreference experiment and the observation of naloxone precipitated withdrawalsyndromes showed that the rats had developed dependence and tolerance toheroin.Weight of rats didn't declined significantly in the heroin-treated-withdrawalgroup compared with group control, and the tail flick test didn't show theantinociception of heroin. These results might be related to the time and way ofheroin treated.2. Effects of heroin on the sexual hormone in female rats.2.1 Effects of heroin on the level of five sexual hormones in plasmaRadioimmunoassay (RIA) was used to test prolactin (PRL), luteinizinghormone (LH), follicle-stimulating hormone (FSH), estradiol (E2) andprogesterone (P).The level of PRL and LH were declined significantly inheroin-treated-3d, 9d groups compared with control group, and these twohormones decreased continuously in heroin-withdrawal-3d group. PRL and LHwere increased in heroin-withdrawal-9d group compared with control group.The level of FSH in these five groups was almost the same. E2 and P, twosteroid hormones, were increased significantly in heroin-treated-3d, 9d groupscompared with control group, and these two hormones increased continuouslyin heroin-withdrawal-3d group. E2 and P were decreased inheroin-withdrawal-9d group compared with control group.2.2 Effects of heroin on the level of m RNA of PRL, LH and FSH inpituitaryReverse -transcription-polymerase-chain-reaction (RT-PCR) method wasemployed to research on the effects of heroin on the level of mRNA of PRL,LH and FSH which were secreted by pituitary cells. The expression levels ofPRL mRNA of pituitary were declined significantly in heroin-treated-3d, 9dgroups compared with control group(p<0.01), while the expression levels ofPRL mRNA of pituitary showed increase trends in heroin-withdrawal-3d, 9dgroups compared with heroin-treated-9d group with no significant differences.The expression levels of LH mRNA of pituitary showed increase trends inheroin-treated-3d, 9d groups compared with control group with no significantdifferences. It showed decrease trend in heroin-withdrawal-3d, 9d groupscompared with heroin-treated-9d group. The expression levels of FSH mRNAof pituitary increased significantly in heroin-treated-9d group compared withcontrol group. Although the level also higher in heroin-withdrawal-3d, 9dgroups than that in control group, compared with heroin-treated-9d group, itwas lowered gradually.As far as the effects of heroin on the level of PRL, LH and FSH, sexualhormones in plasma and gene expression in pituitary were concerned, the levelsof concentration and mRNA of each hormone were not paralleled. It showedthat heroin affected both gene expression on the level of transcription and otherprocedures which related to the secretion and decomposition of PRL, LH andFSH proteins. Apart from the reasons of gene expression, the co-relationshipamong sexual hormones might be other reasons of why the changes ofconcentration and mRNA were different with heroin use. Dopamine (DA)inhibits the secretion of PRL and promotes decomposition of PRL. Opioiddeclined the PRL in both concentration and mRNA level might be by the way ofincrease the DA in nucleus accumbens. The mechanisms of the effects of heroinon the LH and FSH were different, although they were synthesized and secretedby the same cell in pituitary. Protein hormone and the steroid hormone wereaffected by each others. Higher PRL might inhibit the secretion of E2 and P, sothe higher E2 and P after using heroin for 3days and 9days might be theconsequences of the lower PRL in plasma. In a word, heroin inhibitor theprotein hormone, while steroid hormones were promoted by heroin.3. Effects of heroin on the metabolism of purine nucleotide in pituitary,ovary and uterus.3.1 Effects of heroin on the catabolism of purine nucleotideAdenosine deaminase (ADA) and xanthine oxidase (XOD) in pituitary, ovaryand uterus were assessed in order to investigate the effects of heroin on thecatabolism of purine nucleotide. ADA and XOD in uterus showed increasetrends no matter the rats treated with heroin or had been stopping injectionheroin. To be specific, ADA and XOD increased significantly inheroin-treated-9d group compared with control group, and they were still higherin heroin-withdrawal-9d group than that in control group. ADA in ovaryshowed no remarkable changes heroin-treated-3d, 9d groups, while it wasincreased significantly in heroin-withdrawal-3d group compared with controlgroup and decreased to the normal level in heroin-withdrawal-9d group. ADAin ovary showed no remarkable changes in these five groups.In short, heroin increased the concentration of key enzymes of the catabolism ofpurine nucleotide in both uterus and ovary. This effect still can be observed inheroin-withdrawal groups and different organizes showed different reactions.3.2 Effects of heroin on the gene expression of key enzymes of purinenucleotide catabolism and anabolism in pituitary and uterusThe expression of ADA,XOD mRNA in pituitary in heroin-treated-9d groupincreased compared with control group. Although in the heroin-withdrawal-3dgroup, it still higher than that in control group. Compared withheroin-treated-9d group, the level of ADA,XOD mRNA in pituitary inheroin-withdrawal-3d ,9d group showed declined trends. The expression ofADA,XOD mRNA in uterus in heroin-treated-3d ,9d groups increasedsignificantly compared with control group. Although in theheroin-withdrawal-3d group, it still higher than that in control group. Comparedwith heroin-treated-9d group, the level of ADA mRNA in uterus inheroin-withdrawal-3d, 9d group showed declined trends, while the level ofXOD mRNA still showed increased trends. In a word, heroin increased the levelof mRNA of key enzymes of the catabolism of purine nucleotide in bothpituitary and uterus. This effect still can be observed in heroin-withdrawalgroups and different organizes showed different reactions.The expression of adenosine kinase (AK) mRNA was higher inheroin-treated-3d group compared with control group, however, it was loweredslightly in heroin-treated-9d group. Compared with heroin-treated-9d group, thelevel of AK mRNA in pituitary in heroin-withdrawal-3d, 9d group showeddeclined trends. The expression of hypoxanthine-guanine phosphate ribosetransferase (HGPRT) mRNA was increased gradually in heroin-treated-9d,heroin-withdrawal-3d and 9d groups. In conclusion, heroin might inhibit thegene expression of the key enzyme AK of the anabolism of purine nucleotidesat the level of transcription, while increase the gene expression of another keyenzyme HGPRT at the level of transcription.The expression of AK and HGPRT mRNA in uterus in heroin-treated-3d, 9dgroups and heroin-withdrawal-3d, 9d groups were lower than that in controlgroup. Heroin might inhibit the gene expression of the key enzyme AK andHGPRT in uterus at the level of transcription.In conclusion, heroin might promote the catabolism of purine nucleotides byincreasing the gene expression of the key enzymes: ADA and XOD in thisprocess, and inhibit the anabolism of purine nucleotides by decreasing the geneexpression of the key enzymes: AK and HGPRT in this process. Differentorgans showed different reactions to heroin.4. Effects of heroin on the morphologic changes of pituitary, uterus andovaryAngiectasia was observed in pituitary in heroin-treated-9d group with opticalmicroscope, while necrosis were seen in part of the pituitary inheroin-treated-21 group except angiectaia. Apoptosis of gonadotroph cells andlactotrophic cells in heroin-treated-9d and 21d groups were seen withtransmission electron microscope (TEM). Secretory granules (SG) in these twotypes of cells were decreased compared with control group, and roughendoplasmic reticulum (RER) and the mitochondria were distend with themitochondrial crista fragmentation.No differences of the uterus and ovary structure could be observed amongcontrol group and heroin-treated-9d and 21d groups under the opticalmicroscope. While agglutination of chromatin to the edge, distention ofmitochondria and fragmentation of mitochondrial crista were the changes ofultra-microstructure in both tunica intimae cells and matrix cells in uterus inheroin-treated-9d and 21d groups. Eosinophile granulocytes and neutrophilicgranulocytes were more in heroin-treated-9d and 21d groups than that in controlgroup. Agglutination of chromatin to the edge, distention of RER, increasing ofthe number of secondary lysosome, distention of mitochondria andfragmentation of mitochondrial crista were the changes of ultra-microstructurein both granulosa cells and granulosa lutein cells in ovary in heroin-treated-9dand 21d groups.These results showed that heroin had effects not only on the function of a cellbut also on the structure of a cell in pituitary, uterus and ovary. The changes ofthe ultra-microstructure indicated that heroin might cause apoptosis. Changes inmitochondria might cause energy shortage which might be a reason of apoptosis,and metabolism of the cell might be also inhibited due to short of energyproduced by mitochondria. On the whole, heroin might affect cell metabolismthrough the way of inhibition of energy production in a cell, and the apotoisismight be a consequence of ATP shortage.5. Effects of heroin on proliferating cell nuclear antigen (PCNA)Immunohistochemistry technique was used to test the changes of PCNA inpituitary, ovary and uterus, and the results demonstrated that heroin inhibitedthe expression of PCNA at protein level in heroin-treated-9d and 21d groupscompared with control group. RT-PCR was employed to assess the expressionof PCNA mRNA in pituitary, and the results indicated that the level ofexpression of PCNA mRNA in pituitary in heroin-treated-9d group decreasedcompared with control group, and the level in heroin-withdrawal-3d was stilllower than that in control group. The level of PCNA mRNA increasedsignificantly in heroin-withdrawal-9d group. Heroin might depress the geneexpression of PCNA at the level of both transcription and translation. No newcells proliferating after administration of heroin due to PCNA being inhibited,and the parental cells would be senescent gruadually. In this circumstance, thefunction of pituitary, ovary and uters were depressed at the end.To sum up, heroin was harmful to both the function and the structure of genitalendocrine system in female rats. The harmful effects included hormonesecretion lost normal control, catabolism of purine nucleotide was enhanced,anabolism of purine nucleotide was inhibited, and proliferation of cells wasdecreased. These results hinted that the adverse effects of heroin on the genitalendocrine system in female rats might be resulted from the influence of heroinon the metabolism of nucleotide.