172中國(guó)工業(yè)醫(yī)學(xué)雜志2005年6月第18卷第3期 Chinese J Ind Med June2005,Vol.18No.3乙醇的雄性生殖毒性解麗君!,趙松2,胡文媛2(1.河北省疾病預(yù)防控制中心藥物研究室,河北石家莊050021;2.河北醫(yī)科大學(xué),河北石家莊050017)摘要:40只健康雄性成年SD大鼠隨機(jī)分為4組,各組毎日分別灌胃給予乙醇θ2.7、4.5、7.5gkg,連續(xù)13測(cè)定各組大鼠血清睪酮(T)促黃體生成素(LH)促卵泡刺激素(FSH)含量,光、電鏡觀察睪丸組織的形態(tài)學(xué)改變,同時(shí)測(cè)定睪丸線粒體中丙二醛(M〕A)的含量。結(jié)果顯示各乙醇組動(dòng)物血清T水平較對(duì)照組明顯降低(P<0.01),血清IH、FSH含量亦明顯降低〔P<0.05)睪丸的組織病理學(xué)觀察顯示染毒動(dòng)物睪丸生精細(xì)胞核固縮、變性,曲細(xì)精管腔中脫落細(xì)胞增多;超微結(jié)構(gòu)顯示睪丸生精上皮結(jié)構(gòu)破壞,攴持細(xì)胞和各級(jí)生精細(xì)胞均有退化變性,且損傷程度與乙醇劑量明顯相關(guān)。乙醇4.5、7.5g/kg組動(dòng)物睪丸線粒體MDA含量明顯高于對(duì)照組(P<0.05)說(shuō)明乙醇既可以直接作用于睪丸,引起生精細(xì)胞損傷和睪丸類固醇合成抑制,還可使下丘腦垂體軸生殖內(nèi)分泌功能受損。脂質(zhì)過(guò)氧化可能是乙醇致睪丸損傷的機(jī)制之關(guān)鍵詞:乙醇;大鼠;睪丸;生殖毒性中圖分類號(hào):R994.6文獻(xiàn)標(biāo)識(shí)碼:A文章編號(hào):1002-221X(2005)3-0172-02Effects of alcohol on reproductive system of male ratsXIE Li-jun', ZHAO Song, HU W(1. Hebei Provincial Center for Disease Prerention and Control, Shijiazhuang 050021, China 2. Hebei Medical UniversityShijiazhuang 050017, ChinaAbstract: Forty healthy Sprague-Dawley adult male rats were randomly divided into four groups with varied doses of alcohol (0g/kg, 2.7 g/kg, 4.5 g/kg and 7.5 g/kg)administrated for 13 weeks by gastric tubes. Serum sexual hormones testosterone(T)luteinizing hormones( LH and follicle stimulating hormones( FSH) were determined. Patholgical changes of tesits tissue of the ratswere simultaneously observed by light microscopy and electron microscopy. Malondialdehyde( MDA content of testis mitochondria wasdetermined as well. Result showed that Serum T level in all alcohol-treated groups wes significantly lower than that in control group( p<0.01). Serum levels of LH and FSH in alcohol-treated rats were also significantly lower than those in controls( P<0.05). Lightmicroscopic evaluation of the testis of the rats treated with alcohol revealed their germ cell nuclear pyknosis and degeneration and increased exfoliated cells in tubular lumina. Electron microscopic analysis showed that seminiferous epithelium of alcohol-treated ratdisorganized and Sertoli cells and germ cells at all stages were degenerated in a dose-dependent pattern. Content of MDA in the testismitochondria of the rats treated with 45g/kg and 7.5 g/kg was significantly higher than that in the controls( P<0.01 ). It issuggested that alcohol is a known testis toxin which can directly act on the testis causing damage to germ cells and inhibition of synthesiof steroids as well as dysfuncation of hypothalamic-pituitary axis in gonadotropin release. Lipid peroxidation may be atative mecnism that contributes at least in part to the testis damage associated with chronic alcohol abuseKey words: Alcohol Rat Testis Reproductive toxicity研究表明,乙醇是一種確認(rèn)致畸物,母親孕期大量飲酒1.1實(shí)驗(yàn)動(dòng)物及分組可致”胎兒酒精綜合征;男性酗酒可影響身體健康并引起選體重180~20g健康性成熟雄性SD大鼠40只(河北后代發(fā)育和行為異常2為提高人類生命質(zhì)量和優(yōu)生優(yōu)育,實(shí)驗(yàn)動(dòng)物中心提供),隨機(jī)分為4組:0(對(duì)照組)2.7、4研究乙醇的生殖遺傳毒性具有深遠(yuǎn)意義,而囯內(nèi)在乙醇對(duì)雄7.5g/kg乙醇組(按等效劑量計(jì)算,染毒乙醇劑量分別相當(dāng)于性性腺的影響方面尚少見系統(tǒng)報(bào)道。本研究旨在觀察乙醇對(duì)體重60睪丸形態(tài)及生殖內(nèi)分泌激素的影響,并初步探討了乙醇引起每組10中國(guó)煤化工135m、165m、250m)睪丸損傷的作用機(jī)制。胃,灌昌YHaCNMHG醇加入不同量蒸餾水灌wg,需=給予等量蒸餾水,每日11材料與方法次,連續(xù)13周。于末次染毒24h后用乙醚麻醉動(dòng)物,眼眶采血后處死,取雙側(cè)睪丸,稱重,一側(cè)待做睪丸線粒體MDA含收稿日期:2004-09-20;修回日期:2005-02-18作者筒(12-),女,碩士,主管醫(yī)師,主要從事藥量測(cè)定,另一側(cè)用于形態(tài)學(xué)觀察。物毒理學(xué)研究1.2觀察指標(biāo)及方法中國(guó)工業(yè)醫(yī)學(xué)005年6月第18卷第3期 Chinese J Ind Med June2005,Vdl.18N.31731.2.1睪丸光鏡病理切片制作取一側(cè)睪丸部分組織 Bouine各乙醇處理組血清T水平均較對(duì)照組明顯降低(P<0.01)氏液固定,常規(guī)石蠟包埋切片,巸E染色,光鏡下觀察生精細(xì)血清IH水平與對(duì)照組相比均眀顯下降(P<0.05),高劑量ε胞形態(tài)結(jié)構(gòu)。血清FH含量明顯低于對(duì)照組(P<0.051.2.2睪丸超薄切片制作及觀察取一側(cè)睪丸部分組織,雙乙醇4.5、7.5g/kg組睪丸線粒體MDA含量明顯高于對(duì)照蒸水沖洗,在冰面上分別切割成2mm×2mmx4m小塊,組(P<0.01),2.7g/kg組與對(duì)照組差異無(wú)顯著性(P>4%戊二醛固定,環(huán)氧樹脂包埋,超薄切片,醋酸雙氧鈾和枸0.05)櫞酸鉛雙重染色法染色,于日本產(chǎn)H-7500型透射電鏡下觀察表1乙醇對(duì)血清T、IH、FSH及睪丸線粒體睪丸超微結(jié)構(gòu)。MDA含量的影響(x±s,n=10)1.2.3血清睪酮(T)促黃體生成素(IH)促卵泡刺激素(FH)測(cè)定采用放射免疫法。由北京核儀器廠產(chǎn)F646A(nmol/L) ( mlU/L) (mlU/)( nmol/ mg prot微機(jī)單頭放免儀進(jìn)行測(cè)定,試劑盒由中國(guó)原子能科學(xué)院同位對(duì)照組13.25±3.481.42±0.141.74±0.5412.7±4.5素研究所提供。乙醇2.7gkg組884±2,48*0.74±0.18*1.40±0.7314.3±5.4124睪丸線粒體MDA含量測(cè)定取一側(cè)睪丸,摘除被膜乙醇4.5g/kg組6.52±2.98·061±0.4·1.11+0.311,7±3.2后,加入6倍體積的含0.25mmL蔗糖、1mmol/L氯化鎂、乙醇7.5g/kg組3.18±2.16*·0.54±0.550.92±0.3420.2±3.710mmol/ L Tris-HCl緩沖液(pl=74),在冰浴下制成勻漿與對(duì)照組相比,P<0.05,P<0.01使用日本產(chǎn)RP20型高速冷凍離心機(jī)在4℃條件下600g/mn3討論離心得上清液,再以9700g/min離心30min,得沉淀氯化鉀本研究表明各乙醇組尤其是7.5g/kg組大鼠睪丸生精細(xì)磷酸鉀(0.15moL)緩沖液(p=7.4)沖洗,再以同樣速胞核固縮變性,曲細(xì)精管腔中脫落細(xì)胞增多,超微結(jié)構(gòu)檢査度離心30min,最后得睪丸線粒體。MDA的測(cè)定用南京建成顯示乙醇組動(dòng)物睪丸生精上皮結(jié)構(gòu)破壞,支持細(xì)胞和各級(jí)生生物研究所試劑盒,嚴(yán)格按操作說(shuō)明書進(jìn)行;考馬斯亮藍(lán)法精細(xì)胞、精子均有退化變性。這與 El-Sokkary gh的研究報(bào)道測(cè)定蛋白含量類似3。13統(tǒng)計(jì)學(xué)處理結(jié)果還表明,各乙醇組血清T水平均明顯低于對(duì)照組應(yīng)用SPSs10.0統(tǒng)計(jì)軟件進(jìn)行 Dunnett檢驗(yàn)說(shuō)明乙醇可以抑制睪丸間質(zhì)細(xì)胞合成睪酮。從而提示乙醇所2結(jié)果致的睪丸損傷的另一特征是抑制睪丸類固醇合成45]。乙醇抑2.1睪丸組織病理切片觀察制睪酮合成的機(jī)制可能為乙醇或其代謝產(chǎn)物影響了睪酮合成光鏡下對(duì)照組睪丸生精上皮呈規(guī)則圓形,排列緊密,結(jié)的酶。本研究顯示各乙醇處理組大鼠血清IH均明顯低于對(duì)照構(gòu)清晰,層次分明,精原細(xì)胞大而圓,規(guī)則地緊貼曲細(xì)精管組,高劑量組血清FSH水平也明顯低于對(duì)照組。這說(shuō)明乙醇的基底膜,向內(nèi)依次為初級(jí)精母細(xì)胞、精子細(xì)胞、精子。乙可以直接或間接作用于下丘腦或垂體來(lái)干擾下丘腦垂體性腺醇組睪丸曲細(xì)精管有不同程度的損傷,表現(xiàn)為生精細(xì)胞核固軸的功能,以進(jìn)一步影響睪丸的結(jié)構(gòu)和功能縮、變性。損傷程度與乙醇劑量密切相關(guān),低劑量組只有少睪丸線粒體膜、精子均富含多不飽和脂肪酸,易受過(guò)氧量曲細(xì)精管受損,變性的生精細(xì)胞少;高劑量組幾乎毎個(gè)曲化攻擊,乙醇及其代謝物乙醛可以在體內(nèi)產(chǎn)生自由基,自由細(xì)精管均有核固縮、變性的玍精細(xì)胞,曲細(xì)精管腔中脫落細(xì)基攻擊膜上的多不飽和脂肪酸即可誘發(fā)脂質(zhì)過(guò)氧化。通常以胞增多。丙二醛的生成判斷脂質(zhì)過(guò)氧化程度。本研究表明乙醇4.5、2.2睪丸超薄切片觀察7.5g/kg組睪丸線粒體MDA含量明顯高于對(duì)照組,說(shuō)明乙醇電鏡下觀察睪丸組織的超微結(jié)構(gòu),對(duì)照組基膜平整,支可以引起睪丸的過(guò)氧化損傷。持細(xì)胞腔凹窩內(nèi)嵌著各級(jí)處于不同時(shí)期的生精細(xì)胞,有精原參考文獻(xiàn)細(xì)胞、初級(jí)精母細(xì)胞和變態(tài)期精子細(xì)胞。各乙醇組大鼠生精[1]Abel, Sokol. Incidence of fetal alcohol syndrome and economic impact of上皮超微結(jié)構(gòu)顯示,各級(jí)生精細(xì)胞和支持細(xì)胞均有損傷,包fetal alcohol syndrome related anomalies [JAlcohol括:(1)支持細(xì)胞內(nèi)洛酶體增多,支持細(xì)胞滑面內(nèi)質(zhì)網(wǎng)變性【2]1dgM,MmnR,Veg4E,etd. Lateral alcohol exposure: develop退化;(2)精原細(xì)胞核空泡化;(3)初級(jí)精母細(xì)胞核變性溶mental and behavioral effects on the offspring of rats J ]. Neuropharna-解;(4)精子細(xì)胞變態(tài)期核溶解,局部核內(nèi)陷,核周隙擴(kuò)大(5)變態(tài)期精子細(xì)胞移至基膜處,示生精上皮紊亂。且隨劑[3]EsYH中國(guó)煤化工effects of chronic ethanol admin-量加大,損傷加重。CNMHGI Neuroendocrinol Lett, 20012.3乙醇對(duì)血清睪酮、促黃體生成素、促卵泡刺激素及睪丸[4] Kashko m, Khokha am, Antsulevich sn,etal. Influence of ethanol線粒體MDA含量的影響(見表1)and ethanol-induced lipid peroxidation on the steroidogenic activity of testies[j]. Ukr Biokhim Zh, 1993, 65(2):89-94睪丸間質(zhì)細(xì)胞的主要功能是分泌睪酮,促黃體生成素、[5] Salonen I. Effects of chronic ethanol diet on pituitary- testicular function of促卵泡刺激蘩爛鮭咊分泌的兩種促性腺激素。如表1所示,[J]. Biol Reprod,1990,42:5562.