【作者】 吴鑫；

【导师】 金泰廙；

【作者基本信息】 复旦大学 ， 卫生毒理学， 2003， 博士

【摘要】 丙烯腈(Acrylonitrile, ACN)是一种重要的化工生产原料，单体用作合成各种有机产品。到目前为止，有关于丙烯腈潜在的生殖危害，尤其是男性生殖危害，可提供的资料很少。而丙烯腈是对接触工人健康造成潜在危害的一种重要毒物，在我国，从事化工作业(包括丙烯腈)的工人多为男性，有必要对丙烯腈男(雄)性生殖系统毒性作用和可能机制作出较系统研究。 本课题结合人群流行病学调查、整体动物研究和体外实验，对丙烯腈接触男工配偶生殖结局以及男工生殖内分泌的情况进行了研究，对染毒动物生殖力、性腺形态改变、生化改变和基因表达、生殖细胞和性激素改变做出评价，并利用Sertoli细胞和蝾螈精原细胞探讨可能的毒性机制。 第一部分 职业流行病学研究 1)丙烯腈工人接触评价 结果显示该厂丙烯腈生产车间污染较严重，测定浓度最高达42.0mg／m~3，TWA最高达19.12mg／m~3，合格率仅仅为26.9％(现行作业场所有害物质卫生标准TWA1 mg／m~3)。其中不同车间浓度顺序依次为北纺车间≥南纺车间＞聚合车间＞＞溶剂车间。个人平均累计接触剂量为69.09g。 2)丙烯腈接触男工问卷调查 504份生殖问卷调查结果表明接触组工人妻子妊娠合并症发生率升高。空气中浓度最低的溶剂车间工人妻子不孕症和自然流产发生率显著的低于其他车间(P＜0.05)，夫妻双方都接触丙烯腈自然流产率明显高于仅男工一方接触组，接触丙烯腈男工性功能障碍显著高于对照组。 3)丙烯腈接触工人性激素水平研究 随年龄增加FSH略升高，40岁以上年龄段接触工人睾酮下降(P＜0.05)，随工龄增加工人血清FSH也略增高，而接触组睾酮随工龄增加逐渐下降，伴雌激素升高。空气中丙烯腈浓度相对较低的溶剂车间，男工血清睾酮水平高于浓度高的其他车间。接触工人血清FSH和T的水平随着接触剂量的增加呈下降趋势，累积接触剂量大于50g，睾酮下降有显著性，累及接触剂量大于100g，FSH和T下降均有显著性。复旦大学博士研究生毕业论文丙烯睛雄(男)性生殖毒性研究摘要第二部分动物实验 雄性Wistar大鼠分别以。，10，20，40mg瓜g灌胃染毒，连续13周，测定附争精子参数，相关酶活力和分析交配实验结局;雄性SD大鼠分别以O，7.5，15，30mg瓜g腹腔染毒，连续13周，恢复2周，观察辜丸病理损伤、抗氧化酶活力、脂质过氧化水平、雄激素结合蛋白和抑制素基因表达;雄性ICR小鼠以O，6，12，24m叭g灌胃染毒，连续5天，分析附肇尾精子畸形率和早期精细胞微核;分析大、小鼠血清性激素，并通过大鼠子宫增重实验判断丙烯睛是否具有雌激素活性。 结果表明:丙烯睛染毒各剂量组之间雌鼠受孕率，雄鼠生育率，交配指数无差异，剂量为4omg/kg时，孕鼠平均每窝活胎数为5.88个，明显低于对照组(P<0 .05)，胎吸收率为19.66%，明显高于对照组(P<0 .05)。各种酶活性与对照组比较，无显著性差异。 大鼠辛丸受损程度随染毒剂量的增大而加重，光镜下可见部分曲细精管出现退行性病变，管腔中生精细胞和精子数量减少，电镜下可看到正在凋亡变的，核畸形的以及坏死初级精母细胞和崎形的精子. 染毒4周，大鼠辛丸中GSH含量和GPX活力明显升高(P<0 .01);8周后，3omg/kg组oSH明显降低(P<0.01)，剂量大于15mg/kg组sOD活力明显升高(p<0.01):13周后，剂量大于15mg/kg组时，GSH水平明显降低(p<0.01)，GST活力下降，各染毒组GPX活力均明显低于对照组，而3Om留kg剂量组MDA含量显著高于对照组。 染毒4周结束，各剂量组的雄激素结合蛋白表达呈增强趋势，高剂量组出现显著升高，8周末各剂量组差异不明显，13周末，ABP表达显著下降。抑制素随时间延长和剂量增加，表达明显下降. 染毒剂量》12.om叭g，早期精细胞微核明显增加;各染毒组附条尾精子畸形率随染毒时间延长而增加。精子崎形种类主要以头部崎形为主，主要包括无钩、不定型、香蕉型。 丙烯睛染毒8周后小鼠血清T水平随染毒剂量增加显著下降;大鼠血清中T和EZ均表现为先升后降趋势，二者变化水平一致;丙烯睛染毒对未成年大鼠子宫重量无明显改变。复旦大学博士研究生毕业论文丙烯睛雄(男)性生殖毒性研究摘要第三部分体外研究 支持细胞是辛丸毒物的主要靶部位之一。建立了支持细胞体外无血清原代单室和双室培养。采用18天龄左右的大鼠作为细胞供体可获得纯度较高的培养细胞。跨细胞上皮电阻和转铁蛋白反应体外支持细胞紧密单层构建成功，培养基中加入辛酮和卵泡刺激激素可明显提高紧密连接形成效率。 50林g/m1浓度丙烯睛对体外培养的支持细胞具有明显的毒性，影响到支持细胞的活力。丙烯睛对体外双室培养的支持细胞跨上皮电阻形成能力有明显的抑制作用，存在剂量反应关系，同时对已经形成的跨上皮电阻，丙烯睛也能够有一定的破坏。在各个染毒浓度组中，活化前后跨上皮电阻的变化表现出同步的改变，活化前后没有明显的差异. 丙烯睛对支持细胞分泌转铁蛋白的影响不明显。在不影响到支持细胞活力下，丙烯睛浓度‘25林留ml时，培养双室中内外室转铁蛋白浓度更多还原

【Abstract】 Acrylonitrile ( ACN ) is an important intermediary for the synthesis of a variety of industrial organic products. Little attention was paid to the toxicity effects of ACN on the reproductive systems, especially on males. In China, males are the major part of workers in those operations. The reproductive toxicity of ACN has to be considered as an important hazardous effect on exposed workers, particularly on males.In order to study male reproductive toxicity of ACN, epidemiological research and animal experiment in vivo and in vitro were conducted. In human study, the reproductive outcome of exposed male’s wife and the alteration of male sexual hormone were investigated; In animals experiments, reproductive performance, morphology alteration, biochemical changes, expression of gene, and toxic effects on germ cells and on sex hormone were assayed; In vitro study, sertoli cell and spermatogonia of newt were applied to explore the potential toxic mechanism.Parti. Epidemiological investigationExposure assessment was performed in male workers. The results showed that 73.1 percent of the air ACN concentration data in the workplaces exceeded the national hygienic standard. The maximum concentration was 42.00mg/m3 and the maximum PEC-TWA was 19.12mg/m3. The concentrations in the polymer and spinning workshop were much higher than in the solvent workshop. The average estimated cumulative uptake of exposed workers was 69.09 g.The incidence of pregnancy complication in males’ wives was higher than that in control group and the incidence of infertility and spontaneous abortion in the solvent workshop with the lowest concentration was significantly lower than those in other workshops (P<0.05). Spontaneous abortion incidence in couples both exposed to ACN was significantly higher than that in male only exposed to ACN.FSH was increased with aging and T was declined in the ACN exposed workersafter 40 years old (P<0.05). Following working age increased, it also tended to increase in FSH while T declined and estrogens increased. T in the workers working in the solvent workshop with the lowest air ACN concentration was higher than those in other workshops. Both serum FSH and T in exposed workers were declined with the increase of cumulative dose. When ACN uptake was over 50g, T was significantly decreased, and when dose over lOOg, both FSH and T were decreased significantly.Parti. Experimental Study in vivoMale Wistar rats were daily administrated ACN by gavage 5 times a week for 13 weeks at the dose of 0, 10, 20 and 40mg/kg body weight, respectively. Mating experiment was conducted, and then the sperms parameters and the activities of enzymes were detected. Male Sprague-Dawley rats were daily administrated ACN by intraperitoneal 5 times a week for 13 weeks at the dose of 0, 7.5, 15 and 30 mg/kg body weight, respectively. The activities of antioxidant enzyme, the levels of glutathione and malonaldehyde, the mRNA expressions of androgen binding protein (ABP) and inhibin gene, and histopathology in testes were observed. ACN at dose of 6, 12 and 24mg/kg body weight were administrated by gavage to ICR mice for 5 consecutive days, the rate of abnormal sperms and micronucleus in early spermatogenic cells was analyzed. The effects on pituitary-gonad axis were investigated through the sex hormones detection from mice and rats and uterotrophic activity test.The results showed that there was no significant difference in those detected enzymes of testis and epididymis. Mating experiment indicated that ACN has no effect on sexual function of rats, but a decrease in the number of live fetuses per litter (5.58) and an increase in the rate of embryonic absorption (19.66%) at dose of 40mg/kg b.w.Testicular impairment in rats was observed with dose increasing. ACN caused degeneration of seminiferous tubule, decrease in the number of spermatogenic cells and spermatozoa in the tubule. Furthermore, apoptosis, necrosis and abnormal nucleus were observed on primary spermatocytes.After 4 weeks of ACN treatment, the levels更多还原