石家庄灌溉用污水潜在致癌能力的研究
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摘要
为缓解水资源短缺的状况，世界上许多国家利用工业废水和生活污水进行农田灌溉。美国、德国和日本等发达国家，很早就意识到污水回用的重大意义，将城市污水再生处理后，用于农业灌溉、地下水回灌、景观生态用水、工业用水、绿化灌溉和其他用水。美国针对不同的回用对象，制定了严格的回用标准。美国城市污水处理等级基本都在二级以上，处理率可以达到100%。目前，发达国家污水灌溉主要用于园林、牧草以及饲料作物，也用于某些经济作物，而很少应用于粮食作物以及蔬菜。
在我国一方面水资源紧缺，另一方面废水的排放量逐年增加。为缓解水资源紧缺的现状，我国大规模地利用污水进行农田灌溉。城市污水集中处理后进行农田灌溉，可以节约大量的水资源，提高水的再利用率，缓解供水压力；可以充分利用其中的氮、磷资源，减少化肥的使用量。然而我国城镇污水处理率不高，处理水平低。国内外研究证实，污水中含有多种外来化学物，如铅（Pb）、铬（Cr）、镉（Cd）等金属以及多氯联苯、邻苯二甲酸酯类、多环芳烃类、杂环类化合物等有机污染物。利用含有金属和有机污染物的污水进行农田灌溉，可能导致一些地区土壤中金属和有机污染物蓄积，且有逐年加重的趋势，致使土壤生产力下降，农作物生长不良而造成减产，甚至颗粒无收。同时，由于这些化学污染物大多具有生物半减期长、脂溶性、难以降解、易于生物富集等特性，可以通过食物链逐级放大后进入到人体内，从而对人体健康产生潜在的危害。人体摄入被金属污染的饮用水或食物后，体内必需微量元素就会被消耗，从而带来一系列的健康问题，如免疫功能缺陷、宫内发育迟缓、营养缺乏相关的残疾以及肿瘤的高发等。
河北省石家庄市是我国主要污灌区之一，主要为市区以南的几个县的部分地区，总面积约438.8km2。我教研室曾先后两次对石家庄污灌区进行人口死因回顾性调查研究。20世纪80年代调查结果发现全死因标化死亡率污灌区为623.71/10万，显著高于净灌区的557.22/10万；恶性肿瘤标化死亡率，污灌区为155.45/10万，显著高于净灌区144.57/10万，并且有逐年上升的趋势，由1977年的145.79/10万上升到1982年的200.59/10万。本教研室2008年再一次对污灌区进行了死因回顾性调查，结果发现污灌区的全死因标化死亡率为525.78/10万，显著高于净灌区的453.14/10万；污灌区男性、女性恶性肿瘤标化死亡率均显著高于净灌区。两次调查结果表明污水灌溉可能是污灌区居民恶性肿瘤死亡率高于净灌区和全国平均水平的一个重要影响因素，提示灌溉用污水可能有潜在致癌能力。基于人群流行病学调查结果，很有必要对灌溉用污水的潜在致癌能力进行进一步研究，从而规范污水灌溉的管理，达到保护环境、预防疾病、促进健康的目的。
第一部分石家庄灌溉用污水中金属和有机污染物的分析
目的：了解石家庄灌溉用污水中的主要成分。
方法：用电感耦合等离子体质谱方法（ICP-MS）测定石家庄市灌溉用污水中金属的含量；采用固相萃取的方法，提取水样中的有机污染物，用气相色谱质谱联机（GC-MS）定性分析其中的有机污染物。
结果： ICP-MS测定农田灌溉用污水中金属元素铬（Cr）、锰（Mn）、镍（Ni）、铍（Be）、砷（As）、镉（Cd）、铅（Pb）的含量，应用在线加入内标的方法来校正干扰效应，加标回收率为96%~102.3%，相对标准偏差(RSD)小于3%，表明该方法的准确性和精密度良好。
所测水样中各金属Mn、Ni、Be、As、Cd含量均符合城市污水再生利用农田灌溉用水水质标准（GB20922-2007）；上游和中游的样本Cr浓度超过国家标准，分别超过标准2.3和0.1倍；上游和下游的样本Pb的浓度超过国家标准，分别超过标准的0.83和0.55倍。不同地点的混合样本中Cr和Pb的浓度分别超过国家标准0.78和0.43倍。GC-MS共检出86个峰，选择可信度大于70%的峰，共检出30种有机化学物，主要有邻苯二甲酸酯类、杂环类化合物、多环芳烃类化合物、苯胺类化合物等。
结论：石家庄灌溉用污水中Cr和Pb浓度超过国家标准；GC-MS共检出30种有机污染物，其中很多有机污染物是确认的人类致癌物或可疑致癌物。
第二部分石家庄灌溉用污水短期致癌作用研究
目的：应用配套短期致癌试验对石家庄市灌溉用污水的短期致癌效应进行评价。
方法：选择反映不同遗传学终点的短期致癌试验。
鼠伤寒沙门氏菌回复突变试验（Ames）：选用平板掺入法。37℃预培养后，将含有0.5mM组氨酸-生物素的顶层琼脂培养基分装于试管中，然后每管依次加入增菌液0.1ml，不同浓度的受试物0.1ml（需代谢活化时，还需要另外加入S9混合液0.5ml），混合均匀，迅速铺板。顶层琼脂凝固后，翻转平皿，37℃恒温箱内培养48h。受试物设四个剂量组，分别为25μl，50μl，75μl、100μl浓度的灌溉用污水样品。同时设溶剂对照（蒸馏水）、阳性对照以及未处理对照。每个剂量设3个平行皿，重复2次。受试物平皿的实际回变菌落数是自发回变的菌落数2倍及以上，并且有剂量-反应关系就可以认为该受试物诱变试验阳性。
小鼠骨髓嗜多染红细胞微核试验：昆明小鼠按体重随机分成5组，每组10只，雌雄各半。三个不同浓度的剂量组分别含25%、50%、100%的灌溉用污水，自由饮用。蒸馏水为阴性对照，阳性对照组采用环磷酰胺（40mg/kg腹腔注射）。选择两个点进行取材，一个时间点是连续两天暴露于受试物后取材；第二个时间点是亚急性连续15天暴露于受试物后取材。颈椎脱臼法处死小鼠后，取胸骨，涂片，自然干燥，甲醇固定后10%Giemsa染液染色10min，油镜镜检。采用盲法进行计数分析。试验组和阴性对照组相比，微核率差别有统计学意义并有剂量反应关系，即可判定为阳性结果。
单细胞凝胶电泳试验：昆明小鼠按体重随机分成5组，每组8只，雌雄各半。三个不同浓度的剂量组分别含25%、50%、100%灌溉用污水，自由饮用。蒸馏水为阴性对照，选用已知的能引起DNA损伤的苯巴比妥钠为阳性对照（140mg/kg腹腔注射）。处死小鼠，制备肝细胞悬液，细胞密度为104～105个/ml。按常规彗星试验三明治法制片。将载玻片置于新配置的预冷裂解液中，低温避光裂解1h，于电泳液避光解旋30min，室温避光电泳30min （电压25V，电流300mA）；电泳结束后取出载玻片用甲醇固定10min。用Milli-Q纯水漂洗3次，每次3min；3.2%的AgNO3染色30min，4℃预冷的显色液显色5min，甲醇脱色5min。每只动物计数100个细胞。普通光学显微镜下观察，CASP彗星分析软件分析Olive尾矩。
结果：
鼠伤寒沙门氏菌回复突变试验：不加S9体外代谢活化系统，不同浓度的灌溉用污水引起的TA98回变菌落数均大于未处理对照组的两倍，并有剂量反应（y=34.833+113.133x，rs=0.976，P=0.000）；加S9体外代谢活化系统，不同浓度的灌溉用污水引起的TA98回变菌落数均大于未处理对照组的两倍，并有剂量反应（y=42.833+124.133x，rs=0.954，P=0.000）。说明灌溉用污水具有致突变作用，内含有直接和间接性移码型的致突变物。
小鼠骨髓嗜多染红细胞微核试验：连续两天暴露于灌溉用污水，各组小鼠骨髓细胞微核率有显著性差异（F=171.746，P=0.000），阳性对照组小鼠骨髓细胞微核率与阴性对照组相比差异有统计学意义（P<0.01）；但不同剂量的污水处理组和阴性对照组相比，小鼠骨髓细胞微核率差别无统计学意义（P>0.05）。连续15天染毒后，随着染毒时间的延长，各组小鼠骨髓细胞微核率差别有统计学意义（F=71.706，P=0.000）；其中25%污水处理组小鼠骨髓细胞微核率和阴性对照组相比差别无统计学意义（P>0.05），50%和100%污水处理组小鼠骨髓细胞微核率和阴性对照组相比显著增加（P<0.01）；随着污水暴露浓度的增加，微核率显著增加（y=0.003+0.007x，rs=0.814，P=0.000）。
单细胞凝胶电泳试验：小鼠连续15天暴露于灌溉用污水，各组Olive尾矩数值显著升高(=35.815，P=0.000)；25%污水处理组Olive尾矩和阴性对照组相比差别无统计学意义（P>0.05），50%和100%污水处理组Olive尾矩和阴性对照组相比差别有统计学意义（P<0.01）；随着污水暴露剂量的增加，Olive尾矩显著增加（y=0.298+4.055x，rs=0.905，P=0.000）。
结论：
1Ames试验结果表明，石家庄灌溉用污水里面含有直接或间接地可以引起移码型突变的污染物。
2小鼠骨髓细胞微核试验的结果表明，石家庄灌溉用污水可能在小鼠体内发生了物质蓄积，并能引起染色体损伤。
3小鼠体内肝细胞彗星试验的结果表明，石家庄灌溉用污水可以引起小鼠肝细胞DNA链断裂，从而造成DNA损伤。
第三部分灌溉用污水对实验性大鼠肝癌的促进作用
目的：在短期致癌试验阳性结果的基础上，采用大鼠肝转变灶诱发试验对石家庄灌溉用污水的致癌作用进行进一步的研究。
方法：健康清洁级5周龄雄性SD大鼠，按体重随机分为5组，饲养于河北医科大学第四附属医院实验动物中心。大鼠适应一周后，按Ito模式构建大鼠两阶段肝癌促进模型， A组用生理盐水作为启动剂，B、C、D、E组根据动物体重给大鼠一次性腹腔注射二乙基亚硝胺（DEN）200mg/kg作为启动剂。经过为期两周的恢复期饲养后，B、C、D、E组大鼠给予不同浓度的污水样品（0%，25%，50%，100%）作为促进剂，暴露方式为自由饮用，A组大鼠自由饮用蒸馏水。第3周末按经典方法进行2/3肝大部分切除术，恢复两天后再暴露于不同浓度的污水，每周记录一次动物体重、饮水及饲料消耗情况。第8周末实验结束时，称重后将其断头处死，同时采集血样进行生化分析。小心分离肝脏并称重。一部分肝脏标本直接用多聚甲醛固定，一部分用液氮冷冻后保存于-80℃冰箱中待用。根据南京建成试剂盒说明书测定大鼠血清中谷丙转氨酶（ALT）、谷草转氨酶（AST）、碱性磷酸酶（AKP）和γ-谷氨酰转肽酶（γ-GT）的浓度；HE染色病理切片观察染毒大鼠肝组织病变；RT-PCR检测大鼠肝组织GST-Pi mRNA的表达；免疫组化及Western blot检测大鼠肝组织GST-Pi蛋白的表达。
结果：
1大体观察和脏器系数变化：在实验结束的第8周，各组动物体重差别有统计学意义（F=27.925，P=0.000），阴性对照组和25%污水处理组与DEN对照组相比动物体重无显著差异（P＞0.05），50%和100%污水处理组动物体重和DEN对照组相比显著降低（P<0.01）；随着污水暴露剂量的增加，大鼠体重显著下降（y=414.146-53.136x，rs=-0.822，P=0.000）。不同浓度污水处理组大鼠肝脏脏器系数有显著性差异（F=6.961，P=0.000），其中阴性对照组和25%污水处理组与DEN对照组相比大鼠肝脏脏器系数无显著差异（P＞0.05），50%和100%污水暴露组大鼠肝脏脏器系数显著高于DEN对照组（P<0.05），随着污水暴露剂量的增加，大鼠肝脏脏器系数显著增加（y=2.935+0.359x，rs=0.459，P=0.004），表明灌溉用污水具有促进大鼠肝细胞增殖的作用。各组大鼠摄食量和饮水量差别均无统计学意义（P>0.05）。
2肝脏大体及病理形态学观察：HE染色高倍镜下25%灌溉用污水暴露组大鼠肝脏汇管区可见少量增生的卵圆细胞；50%剂量组的大鼠肝小叶组织结构破坏，可见肝细胞再生性结节和小细胞增生灶；在100%剂量组，大鼠肝小叶组织结构破坏更为严重，有纤维组织增生和假小叶形成，汇管区可见大量卵圆细胞增生。
3灌溉用污水对肝功能的影响：各组大鼠的血清中AST浓度有显著性差异（=35.735，P=0.000）；阴性对照组和25%污水处理组的大鼠血清AST浓度与DEN对照组相比差别无统计学意义（P>0.05）；50%和100%污水处理组大鼠血清中AST浓度也显著高于DEN对照组（P<0.01）；随着污水暴露剂量的增加，AST浓度也显著增加（y=57.620+248.277x，rs=0.928，P=0.000）。各组大鼠血清ALT浓度差别有统计学意义（=38.684，P=0.000），阴性对照组与DEN对照组相比大鼠血清ALT浓度无显著性差异（P>0.05），而各污水处理组的大鼠血清ALT浓度显著高于DEN对照组（P<0.01）；随着染毒剂量的增加，ALT浓度也显著增加（y=14.540+246.921x，rs=0.960，P=0.000）。各组大鼠血清γ-GT浓度差别有统计学意义（F=12.987，P=0.000），其中阴性对照组、25%和50%污水处理组的大鼠血清γ-GT浓度与DEN对照组比较，差别无统计学意义（P>0.05），100%污水处理组的大鼠血清中γ-GT浓度与DEN对照组比较，差别有统计学意义（P<0.01）。各剂量组的大鼠血清AKP浓度未观察到显著差异（F=1.824，P=0.144）。
4肝组织中GST-Pi mRNA的表达：在DEN启动作用下，给予大鼠暴露不同浓度的污水后，各组大鼠肝脏GST-Pi mRNA表达量差别有统计学意义（F=37.904，P=0.000）。阴性对照组和25%污水处理组，大鼠肝脏GST-Pi mRNA表达量与DEN对照组相比差别无统计学意义（P>0.05）；大鼠暴露50%和100%的灌溉用污水后，诱导大鼠肝脏GST-PimRNA表达量与DEN对照组相比均显著增加（P<0.01）；随着污水暴露剂量的升高，诱导GST-Pi mRNA表达量显著增加，在100%污水处理组表达量最高（y=1.029+1.060x，rs=0.950，P=0.000）。
5肝组织中GST-Pi蛋白的表达：GST-Pi蛋白在阴性对照组和DEN对照组的大鼠肝组织中仅有少量表达。在DEN启动作用下，给予大鼠暴露不同浓度的污水后，诱导GST-Pi蛋白表达量各组之间差别有统计学意义（F=268.176，P=0.000），其中阴性对照组和DEN对照组相比大鼠肝脏GST-Pi蛋白表达量无显著性差异（P>0.05），而不同浓度的污水处理组与DEN对照组相比大鼠肝脏GST-Pi蛋白表达量显著增加（P<0.01），随着污水暴露浓度的增加，GST-Pi蛋白表达量显著增加（y=0.470+0.823x，rs=0.972，P=0.000）。
免疫组织化学的方法在阴性对照组未观察到GST-Pi蛋白的表达。在DEN启动作用下，DEN对照组的大鼠肝组织中仅有少量表达。在DEN启动作用下，给予大鼠暴露不同浓度的污水后，诱导GST-Pi蛋白表达量与DEN对照组相比均明显增加，在100%污水处理组表达量最高，污水暴露浓度与GST-Pi蛋白表达量之间呈明显的剂量效应关系。
结论：石家庄灌溉用污水有促癌作用，能引起Ito模型大鼠体重显著下降，肝脏脏器系数显著升高，并能引起肝功能异常，诱导肝组织中GST-Pi在mRNA水平和蛋白表达升高。第四部分CYP1A1和CYP1B1在灌溉用污水促癌实验中的作用
目的：研究细胞色素CYP1A1和CYP1B1在灌溉用污水促癌试验中的作用
方法：动物模型同第三部分。用RT-PCR和Western blot方法观察CYP1A1和CYP1B1在mRNA及蛋白水平的表达情况。
结果：
1灌溉用污水对大鼠肝脏CYP1A1mRNA及蛋白表达的影响：各组大鼠肝脏CYP1A1mRNA表达有显著性差异（F=73.054，P=0.000）；阴性对照组的大鼠肝脏CYP1A1mRNA表达和DEN对照组相比差别无统计学意义（P>0.05），但不同浓度的污水暴露组的大鼠肝脏CYP1A1mRNA表达和DEN对照组相比显著增加（P<0.01）；随着污水暴露剂量的增加，其表达量显著增加（y=0.346+0.621x，rs=0.941，P=0.000）。各组CYP1A1蛋白的表达量差别有统计学意义（F=6.788，P=0.007）；阴性对照组和25%污水暴露组的大鼠肝脏CYP1A1蛋白表达量和DEN对照组相比差别无统计学意义（P>0.05），50%和100%污水暴露组的大鼠肝脏CYP1A1蛋白表达量和DEN对照组相比显著增高（P<0.05），随着污水暴露剂量的增加，其表达量显著增加（y=0.557+0.316x，rs=0.799，P=0.001）。
2灌溉用污水对大鼠CYP1B1mRNA及蛋白表达的影响：各组大鼠肝脏CYP1B1mRNA表达差别有统计学意义（F=11.539，P=0.001）；其中阴性对照组的大鼠肝脏CYP1B1mRNA表达和DEN对照组相比差别无统计学意义（P>0.05），但不同浓度的污水暴露组大鼠肝脏CYP1B1mRNA表达量和DEN对照组相比显著增加（P<0.05）；随着污水暴露剂量的增加，CYP1B1mRNA表达量显著增加（y=0.527+0.313x，rs=0.907，P=0.000）。各组大鼠CYP1B1蛋白表达有显著性差异（F=8.247，P=0.003）；其中阴性对照组的大鼠肝脏CYP1B1蛋白表达水平和DEN对照组相比差别无统计学意义（P>0.05），但不同浓度的污水暴露组大鼠肝脏CYP1B1蛋白表达水平和DEN对照组相比显著增加（P<0.05）；随着污水暴露剂量的增加， CYP1B1蛋白表达量显著增加（y=0.530+0.528x，rs=0.789，P=0.002）。
结论：石家庄市灌溉用污水可以诱导细胞色素P4501A1和P4501B1的异常表达，发挥促癌剂的作用，最终导致大鼠肝脏发生癌前病变。
To alleviate the shortage of water resources, many countries in theworld utilize industrial and domestic wastewater for irrigation. Thedeveloped countries, such as the United States, Japan and Germany, realizedthe significance of wastewater reuse many years ago, regenerating andtreating for agricultural irrigation, groundwater recharge, landscape ecologywater, industrial water, green irrigation and so on. According to differentreused objects, different strict standards were developed by the United States,and the urban wastewater treatment level is basically above secondarytreatment, with the processing rates up to100%. At present, in the developedcountries, wastewater irrigation is mainly used for gardens, pasture, foragecrops, and for certain cash crops, while less for food crops and vegetablesapplications.
China has a shortage of water resources; on the other hand, theemissions of China's wastewater are increasing year by year. To alleviate thewater shortage situation, China initiatively utilizes wastewater for irrigationon a large scale. Urban wastewater irrigation, after being collectively treatedby the sewage plants, can save massive water resources, improve waterre-utilization rate, alleviate water pressure, fully take advantage of thenitrogen, phosphorus resources and reduce the usage of fertilizers. However,low treatment rate and low level of sewage treatment exsit in China.Domestic and international studies have confirmed that the sewage containsa variety of xenobioties such as metals (lead, chromium, cadmium) andorganic pollutants (polychlorinated biphenyls, phthalates, polycyclicaromatic hydrocarbons, heterocyclic compounds). These pollutants can notcompletely be removed by conventional sewage treatment. Utilizingwastewater containing metals and organic pollutants in agriculture irrigation, may lead to the accumulation of metals and organic pollutants in the soil, andthere is increasing trend year by year, resulting in the decline of soilproductivity, crop production or even no yield. Also, these chemicalcontaminants with the characteristic of lipid solubility, hardly degradationand easily bioconcentration, can enter into the human body through the foodchain and then pose the potential hazards to human health. Once drinkingwater or food contaminated by metals are ingested by human, the body ofnecessary trace elements will be consumed, resulting in a series of healthproblems, such as immune dysfunction, intrauterine growth retardation, thehigh incidence of nutritional deficiency-related disability and malignanttumor and etc.
Shijiazhuang City of Hebei Province is also one of the main wastewaterirrigation areas in China, Mainly including several areas of the countieslocated in the south of the city with a total area of about438.8km2. Tworetrospective studies of the population cause of death in wastewater irrigationareas were done by our department. One study in1980s found that all-causestandardized mortality of sewage irrigation area was623.71/105,significantly higher than557.22/105of the control irrigation area; and cancerstandardized mortality of sewage irrigation area, significantly higher than144.57/105of the control irrigation area, was155.45/105, with an increasingtrend from145.79/105in1977to200.59/105in1982. Another retrospectivestudy was conducted by our department in2008. It found that all-causestandardized mortality of sewage irrigation area was525.78/105,significantly higher than453.14/105of the control irrigation area; cancerstandardized mortality of sewage irrigation area was significantly higher thanthat of the control irrigation area. The results of the studies showed thatwastewater irrigation might be a very important factor responsible for thesignificantly higher cancer mortality of the people lived in sewage irrigationarea than both that in control irrigation area and the national average,suggesting that irrigative wastewater may be potentially carcinogenic. Basedon epidemiological survey results, it is necessary to further research on the potential carcinogenicity of irrigative wastewater in order to standardize themanagement of wastewater irrigation, and to achieve the purposes ofenvironment protection, disease prevention and health promotion.
Part one: Analysis of metals and organic pollutants in irrigativewastewate from Shijiazhuang City
Objective：Analysis the main components in irrigative wastewater fromShijiazhuang City
Methods: The concentrations of metals in irrigative wastewater inShijiazhuang City were determination by Inductively Coupled Plasma MassSpectrometry (ICP-MS). Organic pollutants were qualitative analysis by GasChromatography Mass Spectrometry (GC-MS) after water sample wasextracted by solid-phase extraction method.
Results: The concentrations of metal elements (Cr, Mn, Ni, Be, As, Cdand Pb) in wastewater were detected by ICP-MS by adding the internalstandard method to correct the interference effect. The recovery rates werebetween96%and102.3%, and relative standard deviations (RSD) were lessthan3%, indicating good accuracy and precision of the method. Theconcentrations of Mn、Ni、Be、As、Cd in water samples were in line with thereuse of urban recycling water-Quality of farmland irrigation water(GB20922-2007). The chromium concentrations of the upstream andmidstream specimens exceeded the national standard by2.3and0.1fold,respectively; the lead concentrations of upstream and downstream specimensexceeded the national standard by0.83and0.55fold, respectively. Thechromium and lead concentrations of the composite sample exceeded thenational standard by0.78and0.43fold.86peaks were detected by GC/MS.In light of the chosen peaks of credibility above70%, a total of30kinds oforganic compounds were detected, including phthalates, heterocycliccompounds, polycyclic aromatic hydrocarbon compounds and anilinecompounds.
Conclusions: The concentrations of chromium and lead in irrigativewastewater of Shijiazhuang exceeded the national standard. A total of30 kinds of organic compounds were detected, including many organiccontaminants which are confirmed human carcinogens or suspectedcarcinogens.
Part two: Short-term carcinogenic effects of irrigative wastewaterfrom Shijiazhuang City
Objective：Short-term carcinogenic effects of irrigative wastewaterfrom Shijiazhuang City were evaluted by applying a battery of short-termcarcinogenicity bioassays.
Methods: The short-term carcinogenicity bioassays of different geneticendpoints were chosed.
Salmonella typhimurium reverse mutation assay (Ames): The Amesassay was performed using the standard plate incorporation method and alsoby the pre-incubation procedure. A mixture containing0.1ml wastewatersample of different concentrations and0.1ml test strain was initiallyincubated at37℃for20min in culture tubes (If metabolic activation wasrequired, the S9mixture of0.5ml was added too). Subsequently the mixturewas added to a tube containing2ml of top agar with0.5mM biotin-histidinewas then gently vortexed and poured onto a minimal glucose plate. Aftersolidification of the top agar, the plates were turned over. After incubation at37℃for48h, the numbers of revertant colonies were counted. Four differentconcentrations of wastewater sample were examined:25μl/plate,50μl/plate,75μl/plate,100μl/plate. The samples were tested with and without S9mix.The solvent control, positive control and untreated control were alsoconducted at the same time. Triplicate plates were done for each dose andrepeated twice. A compound was considered a mutagen if there was atwofold or greater increase in the number of revertants compared with thenumber of spontaneous revertants and a dose-related increase in the numberof revertants for one or more strains.
Micronucleus test of mice bone marrow polychromatic erythrocytes（PCE-MN）：Kunming mice were randomly divided into five groups, eachof which has10, five males and five females. The negative and positive control groups received distilled water and intraperitoneal injection ofcyclophosphamide (40mg/kg), respectively. The other three groups receivedirrigative wastewater ad libitum by drinking water, with the concentrations of25%,50%, and100%wastewater, respectively. Two sampling points werechosen. One was harvested after two consecutive days exposed towastewater; the other was derived afte15consecutive days of subacuteexposure to wastewater. Mice were sacrificed by cervical dislocation method,subsequently sternums were taken. After smeared, all slides were air-dried,fixed in methanol and stained with10%Giemsa stain for10min. All slideswere observed under oil microscopic lens. Micronucleus was counted in ablind manner. A compound can be considered as positive result ifmicronucleus were statistically significant compared with negative controlgroup and a dose-related increase in the number of micronucleus wasobserved.
Single cell gel electrophoresis assay (SCGE): Kunming mice wererandomly divided into five groups, each of which has8, four males and fourfemales. The negative and positive control groups received distilled waterand intraperitoneal injection of phenobarbital sodium (140mg/kg) which isknown chemical with DNA primary lesions, respectively. Three groupsreceived irrigative wastewater ad libitum by drinking water, with theconcentrations of25%,50%, and100%wastewater, respectively. Mice weresacrificed by cervical dislocation method and then liver cell suspension withcell density of104~105/ml was prepared.
The slides were prepared by the conventional sandwich method ofcomet assay. The coverslips were removed gently and the slides were thenimmersed in cold, freshly prepared lysing solution. Protected from light, theslides were left to stand at4℃for1h and then placed in electrophoresisbuffer at4℃for30min to allow the DNA to unwind before electrophoresis.Electrophoresis was performed for20min at25V and300mA in dark atroom temperature. After electrophoresis, the slides were fixed in methanolfor10min. The slides were then submerged in Milli-Q water for3min, repeated three times. The slides were stained with3.2%of AgNO3for30min,and then developed by chromogenic reagent at4℃for5min. The slideswere decolorization with methanol for5min. For each animal,100cells wereevaluated. The olive tail moments were observed under ordinary microscopeusing comet analysis software CASP.
Results:
Ames: Without S9metabolic activation system in vitro, the number ofrevertant colonies of TA98induced by different concentrations of irrigativewastewater was more than twice that of the untreated controls, and thedose-response relationship was observed(y=34.833+113.133x, rs=0.976,P=0.000). With S9metabolic activation system in vitro, the number ofrevertant colonies of TA98induced by different concentrations of irrigativewastewater was more than twice that of the untreated controls, and thedose-response relationship was observed (y=42.833+124.133x, rs=0.954,P=0.000). Irrigative wastewater in Shijiazhuang City had mutagenicitywhich contained direct and indirect frameshift-type mutagens.
PCE-MN： Micronucleus rates of mice PCE exposed to irrigativewastewater for two consecutive days were significant different among groups(F=171.746, P=0.000). Micronucleus rates of positive control group weresignificantly increased compared with that of negative control group(P<0.01). Yet, micronucleus rates induced by different concentations ofirrigative wastewater were not significantly different compared with thatinduced by negative control (P>0.05). After the mice were exposed toirrigative wastewater for consecutive15days, PCE-MN rates of mice weresignificant difference (F=71.706，P=0.000). PCE-MN rates of mice given25%irrigative wastewater were not significantly increased compared withthat induced by negative control (P>0.05); PCE-MN rates of mice given50%and100%irrigative wastewater were significantly increased (P<0.01), andthere was the dose-response relationship (y=0.003+0.007x, rs=0.814,P=0.000).
SCGE: After the mice were exposed to irrigative wastewater for consecutive15days, Olive tail moment values were significantly differentamong groups (=35.815, P=0.000). Olive tail moment values of the micegiven25%irrigative wastewater were no significantly increased comparedwith that of negative control (P>0.05). Olive tail moment values of the micegiven50%and100%irrigative wastewater were significantly increasedcompared with that of negative control (P<0.01), and dose-responserelationship was observed (y=0.298+4.055x，rs=0.905，P=0.000).
Conclusions:
1The results of Ames showed that direct and indirect frameshift-typemutagens existed in irrigative wastewater from Shijiazhuang City.
2The results of micronucleus test showed that substances accumulationof irrigative wastewater might occur in mice and irrigative wastewater couldcause chromosomal damage.
3The results of comet assay showed that irrigative wastewater fromShijiazhuang City could cause DNA strand breaks of mouse hepatocytes,resulting in DNA primary lesion.
Part three: Enhancement of preneoplastic lesion yield by irrigativewastewater in Shijiazhuang in a rat liver medium-term carcinogenesisbioassay
Objective: On the basis of the positive results of the short-termcarcinogenicity test, rat liver medium-term bioassay was applied for furtherresearch on the carcinogenic effects of the irrigative wastewater fromShijiazhuang City.
Methods: Healthy male SD rats of6-week-old were randomly dividedinto five groups and maintained in the animal facility of experimental animalcenter of Hebei Medical University Affiliated4th Hospital. After one weekacclimation period, the experiment was started. The two stages of rat livermedium-term carcinogenesis model were chosen. At the commencement, rats(group A) were intraperitoneally injected with0.9%physiological saline.Rats (groups B-E) were intraperitoneally injected with200mg/kg bodyweight of diethylnitrosamine (DEN) which is well-knowed initiator of liver carcinogenicity. After two weeks, irrigative wastewater was given by freedrinking ad libitum at doses of0,25%,50%and100%wastewater in groupsB-E and rats in group A were given distilled water. At the end of week3, allrats were underwent two-thirds partial hepatectomy. Rats were continuouslyexposed to different concentrations of irrigative wastewater after restorationof two days. During the experimental period, body weights, foodconsumption and water intake were recorded weekly. The experiment wasterminated at week8. Rats were decapitated after being weighed and bloodsamples were collected for biochemical analysis. The liver of rats wascarefully separated and weighed. Parts of the liver specimens were directlyfixed in paraformaldehyde, and the other part frozen in liquid nitrogen andstored at-80℃refrigerator. Serum concentrations of alanineaminotransferase, aspartate aminotransferase, alkaline phosphatase andgamma-glutamyl transfer peptide enzyme were determinated according toNanjing jiancheng kit. Liver tissue lesions were observed by HE stained.GST-Pi mRNA expression of rat liver was detected by RT-PCR; GST-Piprotein expression of rat liver was detected by immunohistochemistry andwestern blot.
Results:
1General observation and changes of organ coefficients: At week8when the experiment was terminated, weights of the rats were significantlydifferent among groups (F=27.925，P=0.000). Weights of the rats in negativecontrol and given25%irrigative wastewater and were no significantlyreduced compared with that of DEN control group (P>0.05). And weights ofthe rats given50%and100%irrigative wastewater were significantlyreduced compared with that of DEN control group (P<0.01). With theincreasing doses of wastewater, weights of the rats were significantlyreduced (y=414.146-53.136x, rs=-0.822, P=0.000). Organ coefficients ofthe liver among groups were significantly different (F=6.961，P=0.000).Liver Organ coefficients of the rats in negative control and given25%irrigative wastewater were no significantly increased compared with that of the DEN control group (P>0.05). Liver organ coefficients of the rats given50%and100%irrigative wastewater were significantly increased comparedwith that of the DEN control group (P<0.05). With the increasing doses ofwastewater, liver organ coefficients of the rats were significantly increased(y=2.935+0.359x, rs=0.459, P=0.004). No significant difference wasobserved in food consumption and water intake (P>0.05).
2Liver general observation and pathological morphological alteration:Liver tissue of HE stained tissue sections was observed at high magnificationunder general microscope. The liver of the rats given25%irrigativewastewater showed a small amount of proliferation of oval cells in portalarea. Of the rats given50%irrigative wastewater, organizational structures ofhepatic lobule were destroyed, showing regeneration nodules and small cellfoci; of the rats given100%irrigative wastewater, organizational structuresof hepatic lobule were destroyed seriously, forming fibrous tissues andpseudolobule, and a large number of oval cell proliferation in portal area.
3Effect of irrigative wastewater on liver function: AST serumconcentrations of the rats were significantly different among groups（=35.735, P=0.000）. Compared to the DEN control group, AST serum levelsof the rats in negative control and given25%irrigative wastewater were nosignificantly increased (P>0.05); AST serum levels of the rats given50%and100%irrigative wastewater were significantly increased (P<0.01) and thedose-response relationship was observed (y=57.620+248.277x, rs=0.928，P=0.000). ALT serum concentrations of the rats were significantly diffentent(=38.684, P=0.000); ALT serum concentrations of the rats in negativecontrol group were no significantly higher than that of the DEN controlgroup (P>0.05). ALT serum concentrations of the rats given differentconcentrations irrigative wastewater were significantly higher than that ofthe DEN control group (P<0.01) and dose-response relationship wasobserved (y=14.540+246.921x, rs=0.960, P=0.000). γ-GT serum levels ofthe rats were significantly different (F=12.987, P=0.000). γ-GT serum levelsof the rats in negative control group and given25%,50%irrigative wastewater were no significantly increased (P>0.05); γ-GT serum levels ofthe rats given100%irrigative wastewater were significantly increased(P<0.01). No significant difference was observed of AKP serum levels of therats (F=1.824, P=0.144).
4GST-Pi mRNA expression of rat liver: Rats initiated by DEN, mRNAexpression of GST-Pi in the rat liver tissue were significantly differentamong groups (F=37.904, P=0.000). Compared with that of the DEN-treatedgroup, GST-Pi mRNA expression of the rats in negative control group andgiven25%irrigative wastewater were not significantly upregulated (P>0.05);GST-Pi mRNA expression of the rats given50%and100%irrigativewastewater were significantly upregulated (P<0.01). GST-Pi mRNAexpression of the rat liver were increased in a dose-dependent manner(y=1.029+1.060x, rs=0.950, P=0.000).
5GST-Pi expression of rat liver: Results of western blot showed lowGST-Pi expressions of the rat liver were observed in negative control andDEN-treated control. Rats initiated by DEN, GST-Pi expression of the ratliver were significantly different (F=268.176, P=0.000). Compared with thatof the DEN-treated group, GST-Pi protein expression of the rats in negativecontrol group were not significantly upregulated (P>0.05); GST-Piexpression of the rats in different concentration wastewater groups weresignificantly upregulated (P<0.01). With the increasing concentrations ofirrigative wastewater, GST-Pi expression levels were upregulatedsignificantly (y=0.470+0.823x, rs=0.972, P=0.000).
GST-Pi protein expression was not observed in the negative controlgroup by immunohistochemical method. Rats initiated by DEN, GST-Piexpressions of the rat liver in DEN control group were low. Rat givendifferent concentrations wastewater afer initiated by DEN, GST-Pi proteinexpression were significantly increased compared with that of the DENcontrol in a dose-response manner and it was highest in100%wastewatergroup.
Conclusions: Irrigative wastewater in Shijiazhuang City has the function of tumor promotion. By the effect of irrigative wastewater inShijiazhuang City, significantly weight losses of rats were observed, liverorgan coefficients of the rats were significantly increased and abnormal liverfunctions were observed. GST-Pi mRNA and GST-Pi expression levels of therat liver induced by irrigative wastewater of different concentrations weresignificantly upregulated in the dose-response manner.
Part four: Effect of CYP1A1and CYP1B1on rat livermedium-term carcinogenesis bioassay of irrigative wastewater
Objective: To study the effect of CYP1A1and CYP1B1on rat liver inmedium-term carcinogenesis bioassay of irrigative wastewater
Methods: Animal model was same as the third part. CYP1A1andCYP1B1expression at the mRNA and protein level were examined byRT-PCR and western blot.
Results:
1CYP1A1mRNA and protein expressions of rat liver induced byirrigative wastewater: CYP1A1mRNA expression of the rat liver wassignificantly different (F=73.054, P=0.000). Compared to that of theDEN-treated group, CYP1A1mRNA expressions in the liver tissue of therats in negative control group were not significantly upregulated (P>0.05);CYP1A1mRNA expressions in the liver tissue of the rats given irrigativewastewater of different concentrations were significantly upregulated(P<0.01) in a dose-dependent manner (y=0.346+0.621x, rs=0.941, P=0.000).CYP1A1expressions of the rat liver were significantly different (F=6.788,P=0.007). Compared to that of the DEN-treated group, CYP1A1expressionsin the liver tissue of the rats in negative control group and given25%irrigative wastewater were not significantly upregulated (P>0.05); CYP1A1expressions in the liver tissue of the rats given50%and100%irrigativewastewater were significantly upregulated (P<0.05) and a dose-responserelationship was observed (y=0.557+0.316x, rs=0.799，P=0.001).
2CYP1B1mRNA and protein expressions of rat liver induced byirrigative wastewater: CYP1B1mRNA expressions of the rat liver were significant different (F=11.539, P=0.001). Compared to that of theDEN-treated group, CYP1B1mRNA expressions in the liver tissue of therats in negative control were not significantly upregulated (P>0.05);CYP1B1mRNA expressions in the liver tissue of the rats given irrigativewastewater of different concentrations were significantly upregulated(P<0.05) in a dose-dependent manner (y=0.527+0.313x, rs=0.907, P=0.000).CYP1B1expressions of the rat liver were significant different (F=8.247,P=0.003). Compared to that of the DEN-treated group, CYP1B1expressionsin the liver tissue of the rats in negative control group were not significantlydifferent (P>0.05); CYP1B1expressions in the liver tissue of the rats givenirrigative wastewater of different concentrations were significantlyupregulated (P<0.05). CYP1B1expressions were significantly upregulatedwith the increasing doses of irrigative wastewater (y=0.530+0.528x,rs=0.789, P=0.002).
Conclusions: Irrigative wastewater in Shijiazhuang City had thefunction of the induction of abnormal expression of cytochrome P4501A1and P4501B1and played the role of tumor promoters, eventually resulting inthe precancerous lesions of the rat liver
在我国一方面水资源紧缺，另一方面废水的排放量逐年增加。为缓解水资源紧缺的现状，我国大规模地利用污水进行农田灌溉。城市污水集中处理后进行农田灌溉，可以节约大量的水资源，提高水的再利用率，缓解供水压力；可以充分利用其中的氮、磷资源，减少化肥的使用量。然而我国城镇污水处理率不高，处理水平低。国内外研究证实，污水中含有多种外来化学物，如铅（Pb）、铬（Cr）、镉（Cd）等金属以及多氯联苯、邻苯二甲酸酯类、多环芳烃类、杂环类化合物等有机污染物。利用含有金属和有机污染物的污水进行农田灌溉，可能导致一些地区土壤中金属和有机污染物蓄积，且有逐年加重的趋势，致使土壤生产力下降，农作物生长不良而造成减产，甚至颗粒无收。同时，由于这些化学污染物大多具有生物半减期长、脂溶性、难以降解、易于生物富集等特性，可以通过食物链逐级放大后进入到人体内，从而对人体健康产生潜在的危害。人体摄入被金属污染的饮用水或食物后，体内必需微量元素就会被消耗，从而带来一系列的健康问题，如免疫功能缺陷、宫内发育迟缓、营养缺乏相关的残疾以及肿瘤的高发等。
河北省石家庄市是我国主要污灌区之一，主要为市区以南的几个县的部分地区，总面积约438.8km2。我教研室曾先后两次对石家庄污灌区进行人口死因回顾性调查研究。20世纪80年代调查结果发现全死因标化死亡率污灌区为623.71/10万，显著高于净灌区的557.22/10万；恶性肿瘤标化死亡率，污灌区为155.45/10万，显著高于净灌区144.57/10万，并且有逐年上升的趋势，由1977年的145.79/10万上升到1982年的200.59/10万。本教研室2008年再一次对污灌区进行了死因回顾性调查，结果发现污灌区的全死因标化死亡率为525.78/10万，显著高于净灌区的453.14/10万；污灌区男性、女性恶性肿瘤标化死亡率均显著高于净灌区。两次调查结果表明污水灌溉可能是污灌区居民恶性肿瘤死亡率高于净灌区和全国平均水平的一个重要影响因素，提示灌溉用污水可能有潜在致癌能力。基于人群流行病学调查结果，很有必要对灌溉用污水的潜在致癌能力进行进一步研究，从而规范污水灌溉的管理，达到保护环境、预防疾病、促进健康的目的。
第一部分石家庄灌溉用污水中金属和有机污染物的分析
目的：了解石家庄灌溉用污水中的主要成分。
方法：用电感耦合等离子体质谱方法（ICP-MS）测定石家庄市灌溉用污水中金属的含量；采用固相萃取的方法，提取水样中的有机污染物，用气相色谱质谱联机（GC-MS）定性分析其中的有机污染物。
结果： ICP-MS测定农田灌溉用污水中金属元素铬（Cr）、锰（Mn）、镍（Ni）、铍（Be）、砷（As）、镉（Cd）、铅（Pb）的含量，应用在线加入内标的方法来校正干扰效应，加标回收率为96%~102.3%，相对标准偏差(RSD)小于3%，表明该方法的准确性和精密度良好。
所测水样中各金属Mn、Ni、Be、As、Cd含量均符合城市污水再生利用农田灌溉用水水质标准（GB20922-2007）；上游和中游的样本Cr浓度超过国家标准，分别超过标准2.3和0.1倍；上游和下游的样本Pb的浓度超过国家标准，分别超过标准的0.83和0.55倍。不同地点的混合样本中Cr和Pb的浓度分别超过国家标准0.78和0.43倍。GC-MS共检出86个峰，选择可信度大于70%的峰，共检出30种有机化学物，主要有邻苯二甲酸酯类、杂环类化合物、多环芳烃类化合物、苯胺类化合物等。
结论：石家庄灌溉用污水中Cr和Pb浓度超过国家标准；GC-MS共检出30种有机污染物，其中很多有机污染物是确认的人类致癌物或可疑致癌物。
第二部分石家庄灌溉用污水短期致癌作用研究
目的：应用配套短期致癌试验对石家庄市灌溉用污水的短期致癌效应进行评价。
方法：选择反映不同遗传学终点的短期致癌试验。
鼠伤寒沙门氏菌回复突变试验（Ames）：选用平板掺入法。37℃预培养后，将含有0.5mM组氨酸-生物素的顶层琼脂培养基分装于试管中，然后每管依次加入增菌液0.1ml，不同浓度的受试物0.1ml（需代谢活化时，还需要另外加入S9混合液0.5ml），混合均匀，迅速铺板。顶层琼脂凝固后，翻转平皿，37℃恒温箱内培养48h。受试物设四个剂量组，分别为25μl，50μl，75μl、100μl浓度的灌溉用污水样品。同时设溶剂对照（蒸馏水）、阳性对照以及未处理对照。每个剂量设3个平行皿，重复2次。受试物平皿的实际回变菌落数是自发回变的菌落数2倍及以上，并且有剂量-反应关系就可以认为该受试物诱变试验阳性。
小鼠骨髓嗜多染红细胞微核试验：昆明小鼠按体重随机分成5组，每组10只，雌雄各半。三个不同浓度的剂量组分别含25%、50%、100%的灌溉用污水，自由饮用。蒸馏水为阴性对照，阳性对照组采用环磷酰胺（40mg/kg腹腔注射）。选择两个点进行取材，一个时间点是连续两天暴露于受试物后取材；第二个时间点是亚急性连续15天暴露于受试物后取材。颈椎脱臼法处死小鼠后，取胸骨，涂片，自然干燥，甲醇固定后10%Giemsa染液染色10min，油镜镜检。采用盲法进行计数分析。试验组和阴性对照组相比，微核率差别有统计学意义并有剂量反应关系，即可判定为阳性结果。
单细胞凝胶电泳试验：昆明小鼠按体重随机分成5组，每组8只，雌雄各半。三个不同浓度的剂量组分别含25%、50%、100%灌溉用污水，自由饮用。蒸馏水为阴性对照，选用已知的能引起DNA损伤的苯巴比妥钠为阳性对照（140mg/kg腹腔注射）。处死小鼠，制备肝细胞悬液，细胞密度为104～105个/ml。按常规彗星试验三明治法制片。将载玻片置于新配置的预冷裂解液中，低温避光裂解1h，于电泳液避光解旋30min，室温避光电泳30min （电压25V，电流300mA）；电泳结束后取出载玻片用甲醇固定10min。用Milli-Q纯水漂洗3次，每次3min；3.2%的AgNO3染色30min，4℃预冷的显色液显色5min，甲醇脱色5min。每只动物计数100个细胞。普通光学显微镜下观察，CASP彗星分析软件分析Olive尾矩。
结果：
鼠伤寒沙门氏菌回复突变试验：不加S9体外代谢活化系统，不同浓度的灌溉用污水引起的TA98回变菌落数均大于未处理对照组的两倍，并有剂量反应（y=34.833+113.133x，rs=0.976，P=0.000）；加S9体外代谢活化系统，不同浓度的灌溉用污水引起的TA98回变菌落数均大于未处理对照组的两倍，并有剂量反应（y=42.833+124.133x，rs=0.954，P=0.000）。说明灌溉用污水具有致突变作用，内含有直接和间接性移码型的致突变物。
小鼠骨髓嗜多染红细胞微核试验：连续两天暴露于灌溉用污水，各组小鼠骨髓细胞微核率有显著性差异（F=171.746，P=0.000），阳性对照组小鼠骨髓细胞微核率与阴性对照组相比差异有统计学意义（P<0.01）；但不同剂量的污水处理组和阴性对照组相比，小鼠骨髓细胞微核率差别无统计学意义（P>0.05）。连续15天染毒后，随着染毒时间的延长，各组小鼠骨髓细胞微核率差别有统计学意义（F=71.706，P=0.000）；其中25%污水处理组小鼠骨髓细胞微核率和阴性对照组相比差别无统计学意义（P>0.05），50%和100%污水处理组小鼠骨髓细胞微核率和阴性对照组相比显著增加（P<0.01）；随着污水暴露浓度的增加，微核率显著增加（y=0.003+0.007x，rs=0.814，P=0.000）。
单细胞凝胶电泳试验：小鼠连续15天暴露于灌溉用污水，各组Olive尾矩数值显著升高(=35.815，P=0.000)；25%污水处理组Olive尾矩和阴性对照组相比差别无统计学意义（P>0.05），50%和100%污水处理组Olive尾矩和阴性对照组相比差别有统计学意义（P<0.01）；随着污水暴露剂量的增加，Olive尾矩显著增加（y=0.298+4.055x，rs=0.905，P=0.000）。
结论：
1Ames试验结果表明，石家庄灌溉用污水里面含有直接或间接地可以引起移码型突变的污染物。
2小鼠骨髓细胞微核试验的结果表明，石家庄灌溉用污水可能在小鼠体内发生了物质蓄积，并能引起染色体损伤。
3小鼠体内肝细胞彗星试验的结果表明，石家庄灌溉用污水可以引起小鼠肝细胞DNA链断裂，从而造成DNA损伤。
第三部分灌溉用污水对实验性大鼠肝癌的促进作用
目的：在短期致癌试验阳性结果的基础上，采用大鼠肝转变灶诱发试验对石家庄灌溉用污水的致癌作用进行进一步的研究。
方法：健康清洁级5周龄雄性SD大鼠，按体重随机分为5组，饲养于河北医科大学第四附属医院实验动物中心。大鼠适应一周后，按Ito模式构建大鼠两阶段肝癌促进模型， A组用生理盐水作为启动剂，B、C、D、E组根据动物体重给大鼠一次性腹腔注射二乙基亚硝胺（DEN）200mg/kg作为启动剂。经过为期两周的恢复期饲养后，B、C、D、E组大鼠给予不同浓度的污水样品（0%，25%，50%，100%）作为促进剂，暴露方式为自由饮用，A组大鼠自由饮用蒸馏水。第3周末按经典方法进行2/3肝大部分切除术，恢复两天后再暴露于不同浓度的污水，每周记录一次动物体重、饮水及饲料消耗情况。第8周末实验结束时，称重后将其断头处死，同时采集血样进行生化分析。小心分离肝脏并称重。一部分肝脏标本直接用多聚甲醛固定，一部分用液氮冷冻后保存于-80℃冰箱中待用。根据南京建成试剂盒说明书测定大鼠血清中谷丙转氨酶（ALT）、谷草转氨酶（AST）、碱性磷酸酶（AKP）和γ-谷氨酰转肽酶（γ-GT）的浓度；HE染色病理切片观察染毒大鼠肝组织病变；RT-PCR检测大鼠肝组织GST-Pi mRNA的表达；免疫组化及Western blot检测大鼠肝组织GST-Pi蛋白的表达。
结果：
1大体观察和脏器系数变化：在实验结束的第8周，各组动物体重差别有统计学意义（F=27.925，P=0.000），阴性对照组和25%污水处理组与DEN对照组相比动物体重无显著差异（P＞0.05），50%和100%污水处理组动物体重和DEN对照组相比显著降低（P<0.01）；随着污水暴露剂量的增加，大鼠体重显著下降（y=414.146-53.136x，rs=-0.822，P=0.000）。不同浓度污水处理组大鼠肝脏脏器系数有显著性差异（F=6.961，P=0.000），其中阴性对照组和25%污水处理组与DEN对照组相比大鼠肝脏脏器系数无显著差异（P＞0.05），50%和100%污水暴露组大鼠肝脏脏器系数显著高于DEN对照组（P<0.05），随着污水暴露剂量的增加，大鼠肝脏脏器系数显著增加（y=2.935+0.359x，rs=0.459，P=0.004），表明灌溉用污水具有促进大鼠肝细胞增殖的作用。各组大鼠摄食量和饮水量差别均无统计学意义（P>0.05）。
2肝脏大体及病理形态学观察：HE染色高倍镜下25%灌溉用污水暴露组大鼠肝脏汇管区可见少量增生的卵圆细胞；50%剂量组的大鼠肝小叶组织结构破坏，可见肝细胞再生性结节和小细胞增生灶；在100%剂量组，大鼠肝小叶组织结构破坏更为严重，有纤维组织增生和假小叶形成，汇管区可见大量卵圆细胞增生。
3灌溉用污水对肝功能的影响：各组大鼠的血清中AST浓度有显著性差异（=35.735，P=0.000）；阴性对照组和25%污水处理组的大鼠血清AST浓度与DEN对照组相比差别无统计学意义（P>0.05）；50%和100%污水处理组大鼠血清中AST浓度也显著高于DEN对照组（P<0.01）；随着污水暴露剂量的增加，AST浓度也显著增加（y=57.620+248.277x，rs=0.928，P=0.000）。各组大鼠血清ALT浓度差别有统计学意义（=38.684，P=0.000），阴性对照组与DEN对照组相比大鼠血清ALT浓度无显著性差异（P>0.05），而各污水处理组的大鼠血清ALT浓度显著高于DEN对照组（P<0.01）；随着染毒剂量的增加，ALT浓度也显著增加（y=14.540+246.921x，rs=0.960，P=0.000）。各组大鼠血清γ-GT浓度差别有统计学意义（F=12.987，P=0.000），其中阴性对照组、25%和50%污水处理组的大鼠血清γ-GT浓度与DEN对照组比较，差别无统计学意义（P>0.05），100%污水处理组的大鼠血清中γ-GT浓度与DEN对照组比较，差别有统计学意义（P<0.01）。各剂量组的大鼠血清AKP浓度未观察到显著差异（F=1.824，P=0.144）。
4肝组织中GST-Pi mRNA的表达：在DEN启动作用下，给予大鼠暴露不同浓度的污水后，各组大鼠肝脏GST-Pi mRNA表达量差别有统计学意义（F=37.904，P=0.000）。阴性对照组和25%污水处理组，大鼠肝脏GST-Pi mRNA表达量与DEN对照组相比差别无统计学意义（P>0.05）；大鼠暴露50%和100%的灌溉用污水后，诱导大鼠肝脏GST-PimRNA表达量与DEN对照组相比均显著增加（P<0.01）；随着污水暴露剂量的升高，诱导GST-Pi mRNA表达量显著增加，在100%污水处理组表达量最高（y=1.029+1.060x，rs=0.950，P=0.000）。
5肝组织中GST-Pi蛋白的表达：GST-Pi蛋白在阴性对照组和DEN对照组的大鼠肝组织中仅有少量表达。在DEN启动作用下，给予大鼠暴露不同浓度的污水后，诱导GST-Pi蛋白表达量各组之间差别有统计学意义（F=268.176，P=0.000），其中阴性对照组和DEN对照组相比大鼠肝脏GST-Pi蛋白表达量无显著性差异（P>0.05），而不同浓度的污水处理组与DEN对照组相比大鼠肝脏GST-Pi蛋白表达量显著增加（P<0.01），随着污水暴露浓度的增加，GST-Pi蛋白表达量显著增加（y=0.470+0.823x，rs=0.972，P=0.000）。
免疫组织化学的方法在阴性对照组未观察到GST-Pi蛋白的表达。在DEN启动作用下，DEN对照组的大鼠肝组织中仅有少量表达。在DEN启动作用下，给予大鼠暴露不同浓度的污水后，诱导GST-Pi蛋白表达量与DEN对照组相比均明显增加，在100%污水处理组表达量最高，污水暴露浓度与GST-Pi蛋白表达量之间呈明显的剂量效应关系。
结论：石家庄灌溉用污水有促癌作用，能引起Ito模型大鼠体重显著下降，肝脏脏器系数显著升高，并能引起肝功能异常，诱导肝组织中GST-Pi在mRNA水平和蛋白表达升高。第四部分CYP1A1和CYP1B1在灌溉用污水促癌实验中的作用
目的：研究细胞色素CYP1A1和CYP1B1在灌溉用污水促癌试验中的作用
方法：动物模型同第三部分。用RT-PCR和Western blot方法观察CYP1A1和CYP1B1在mRNA及蛋白水平的表达情况。
结果：
1灌溉用污水对大鼠肝脏CYP1A1mRNA及蛋白表达的影响：各组大鼠肝脏CYP1A1mRNA表达有显著性差异（F=73.054，P=0.000）；阴性对照组的大鼠肝脏CYP1A1mRNA表达和DEN对照组相比差别无统计学意义（P>0.05），但不同浓度的污水暴露组的大鼠肝脏CYP1A1mRNA表达和DEN对照组相比显著增加（P<0.01）；随着污水暴露剂量的增加，其表达量显著增加（y=0.346+0.621x，rs=0.941，P=0.000）。各组CYP1A1蛋白的表达量差别有统计学意义（F=6.788，P=0.007）；阴性对照组和25%污水暴露组的大鼠肝脏CYP1A1蛋白表达量和DEN对照组相比差别无统计学意义（P>0.05），50%和100%污水暴露组的大鼠肝脏CYP1A1蛋白表达量和DEN对照组相比显著增高（P<0.05），随着污水暴露剂量的增加，其表达量显著增加（y=0.557+0.316x，rs=0.799，P=0.001）。
2灌溉用污水对大鼠CYP1B1mRNA及蛋白表达的影响：各组大鼠肝脏CYP1B1mRNA表达差别有统计学意义（F=11.539，P=0.001）；其中阴性对照组的大鼠肝脏CYP1B1mRNA表达和DEN对照组相比差别无统计学意义（P>0.05），但不同浓度的污水暴露组大鼠肝脏CYP1B1mRNA表达量和DEN对照组相比显著增加（P<0.05）；随着污水暴露剂量的增加，CYP1B1mRNA表达量显著增加（y=0.527+0.313x，rs=0.907，P=0.000）。各组大鼠CYP1B1蛋白表达有显著性差异（F=8.247，P=0.003）；其中阴性对照组的大鼠肝脏CYP1B1蛋白表达水平和DEN对照组相比差别无统计学意义（P>0.05），但不同浓度的污水暴露组大鼠肝脏CYP1B1蛋白表达水平和DEN对照组相比显著增加（P<0.05）；随着污水暴露剂量的增加， CYP1B1蛋白表达量显著增加（y=0.530+0.528x，rs=0.789，P=0.002）。
结论：石家庄市灌溉用污水可以诱导细胞色素P4501A1和P4501B1的异常表达，发挥促癌剂的作用，最终导致大鼠肝脏发生癌前病变。
To alleviate the shortage of water resources, many countries in theworld utilize industrial and domestic wastewater for irrigation. Thedeveloped countries, such as the United States, Japan and Germany, realizedthe significance of wastewater reuse many years ago, regenerating andtreating for agricultural irrigation, groundwater recharge, landscape ecologywater, industrial water, green irrigation and so on. According to differentreused objects, different strict standards were developed by the United States,and the urban wastewater treatment level is basically above secondarytreatment, with the processing rates up to100%. At present, in the developedcountries, wastewater irrigation is mainly used for gardens, pasture, foragecrops, and for certain cash crops, while less for food crops and vegetablesapplications.
China has a shortage of water resources; on the other hand, theemissions of China's wastewater are increasing year by year. To alleviate thewater shortage situation, China initiatively utilizes wastewater for irrigationon a large scale. Urban wastewater irrigation, after being collectively treatedby the sewage plants, can save massive water resources, improve waterre-utilization rate, alleviate water pressure, fully take advantage of thenitrogen, phosphorus resources and reduce the usage of fertilizers. However,low treatment rate and low level of sewage treatment exsit in China.Domestic and international studies have confirmed that the sewage containsa variety of xenobioties such as metals (lead, chromium, cadmium) andorganic pollutants (polychlorinated biphenyls, phthalates, polycyclicaromatic hydrocarbons, heterocyclic compounds). These pollutants can notcompletely be removed by conventional sewage treatment. Utilizingwastewater containing metals and organic pollutants in agriculture irrigation, may lead to the accumulation of metals and organic pollutants in the soil, andthere is increasing trend year by year, resulting in the decline of soilproductivity, crop production or even no yield. Also, these chemicalcontaminants with the characteristic of lipid solubility, hardly degradationand easily bioconcentration, can enter into the human body through the foodchain and then pose the potential hazards to human health. Once drinkingwater or food contaminated by metals are ingested by human, the body ofnecessary trace elements will be consumed, resulting in a series of healthproblems, such as immune dysfunction, intrauterine growth retardation, thehigh incidence of nutritional deficiency-related disability and malignanttumor and etc.
Shijiazhuang City of Hebei Province is also one of the main wastewaterirrigation areas in China, Mainly including several areas of the countieslocated in the south of the city with a total area of about438.8km2. Tworetrospective studies of the population cause of death in wastewater irrigationareas were done by our department. One study in1980s found that all-causestandardized mortality of sewage irrigation area was623.71/105,significantly higher than557.22/105of the control irrigation area; and cancerstandardized mortality of sewage irrigation area, significantly higher than144.57/105of the control irrigation area, was155.45/105, with an increasingtrend from145.79/105in1977to200.59/105in1982. Another retrospectivestudy was conducted by our department in2008. It found that all-causestandardized mortality of sewage irrigation area was525.78/105,significantly higher than453.14/105of the control irrigation area; cancerstandardized mortality of sewage irrigation area was significantly higher thanthat of the control irrigation area. The results of the studies showed thatwastewater irrigation might be a very important factor responsible for thesignificantly higher cancer mortality of the people lived in sewage irrigationarea than both that in control irrigation area and the national average,suggesting that irrigative wastewater may be potentially carcinogenic. Basedon epidemiological survey results, it is necessary to further research on the potential carcinogenicity of irrigative wastewater in order to standardize themanagement of wastewater irrigation, and to achieve the purposes ofenvironment protection, disease prevention and health promotion.
Part one: Analysis of metals and organic pollutants in irrigativewastewate from Shijiazhuang City
Objective：Analysis the main components in irrigative wastewater fromShijiazhuang City
Methods: The concentrations of metals in irrigative wastewater inShijiazhuang City were determination by Inductively Coupled Plasma MassSpectrometry (ICP-MS). Organic pollutants were qualitative analysis by GasChromatography Mass Spectrometry (GC-MS) after water sample wasextracted by solid-phase extraction method.
Results: The concentrations of metal elements (Cr, Mn, Ni, Be, As, Cdand Pb) in wastewater were detected by ICP-MS by adding the internalstandard method to correct the interference effect. The recovery rates werebetween96%and102.3%, and relative standard deviations (RSD) were lessthan3%, indicating good accuracy and precision of the method. Theconcentrations of Mn、Ni、Be、As、Cd in water samples were in line with thereuse of urban recycling water-Quality of farmland irrigation water(GB20922-2007). The chromium concentrations of the upstream andmidstream specimens exceeded the national standard by2.3and0.1fold,respectively; the lead concentrations of upstream and downstream specimensexceeded the national standard by0.83and0.55fold, respectively. Thechromium and lead concentrations of the composite sample exceeded thenational standard by0.78and0.43fold.86peaks were detected by GC/MS.In light of the chosen peaks of credibility above70%, a total of30kinds oforganic compounds were detected, including phthalates, heterocycliccompounds, polycyclic aromatic hydrocarbon compounds and anilinecompounds.
Conclusions: The concentrations of chromium and lead in irrigativewastewater of Shijiazhuang exceeded the national standard. A total of30 kinds of organic compounds were detected, including many organiccontaminants which are confirmed human carcinogens or suspectedcarcinogens.
Part two: Short-term carcinogenic effects of irrigative wastewaterfrom Shijiazhuang City
Objective：Short-term carcinogenic effects of irrigative wastewaterfrom Shijiazhuang City were evaluted by applying a battery of short-termcarcinogenicity bioassays.
Methods: The short-term carcinogenicity bioassays of different geneticendpoints were chosed.
Salmonella typhimurium reverse mutation assay (Ames): The Amesassay was performed using the standard plate incorporation method and alsoby the pre-incubation procedure. A mixture containing0.1ml wastewatersample of different concentrations and0.1ml test strain was initiallyincubated at37℃for20min in culture tubes (If metabolic activation wasrequired, the S9mixture of0.5ml was added too). Subsequently the mixturewas added to a tube containing2ml of top agar with0.5mM biotin-histidinewas then gently vortexed and poured onto a minimal glucose plate. Aftersolidification of the top agar, the plates were turned over. After incubation at37℃for48h, the numbers of revertant colonies were counted. Four differentconcentrations of wastewater sample were examined:25μl/plate,50μl/plate,75μl/plate,100μl/plate. The samples were tested with and without S9mix.The solvent control, positive control and untreated control were alsoconducted at the same time. Triplicate plates were done for each dose andrepeated twice. A compound was considered a mutagen if there was atwofold or greater increase in the number of revertants compared with thenumber of spontaneous revertants and a dose-related increase in the numberof revertants for one or more strains.
Micronucleus test of mice bone marrow polychromatic erythrocytes（PCE-MN）：Kunming mice were randomly divided into five groups, eachof which has10, five males and five females. The negative and positive control groups received distilled water and intraperitoneal injection ofcyclophosphamide (40mg/kg), respectively. The other three groups receivedirrigative wastewater ad libitum by drinking water, with the concentrations of25%,50%, and100%wastewater, respectively. Two sampling points werechosen. One was harvested after two consecutive days exposed towastewater; the other was derived afte15consecutive days of subacuteexposure to wastewater. Mice were sacrificed by cervical dislocation method,subsequently sternums were taken. After smeared, all slides were air-dried,fixed in methanol and stained with10%Giemsa stain for10min. All slideswere observed under oil microscopic lens. Micronucleus was counted in ablind manner. A compound can be considered as positive result ifmicronucleus were statistically significant compared with negative controlgroup and a dose-related increase in the number of micronucleus wasobserved.
Single cell gel electrophoresis assay (SCGE): Kunming mice wererandomly divided into five groups, each of which has8, four males and fourfemales. The negative and positive control groups received distilled waterand intraperitoneal injection of phenobarbital sodium (140mg/kg) which isknown chemical with DNA primary lesions, respectively. Three groupsreceived irrigative wastewater ad libitum by drinking water, with theconcentrations of25%,50%, and100%wastewater, respectively. Mice weresacrificed by cervical dislocation method and then liver cell suspension withcell density of104~105/ml was prepared.
The slides were prepared by the conventional sandwich method ofcomet assay. The coverslips were removed gently and the slides were thenimmersed in cold, freshly prepared lysing solution. Protected from light, theslides were left to stand at4℃for1h and then placed in electrophoresisbuffer at4℃for30min to allow the DNA to unwind before electrophoresis.Electrophoresis was performed for20min at25V and300mA in dark atroom temperature. After electrophoresis, the slides were fixed in methanolfor10min. The slides were then submerged in Milli-Q water for3min, repeated three times. The slides were stained with3.2%of AgNO3for30min,and then developed by chromogenic reagent at4℃for5min. The slideswere decolorization with methanol for5min. For each animal,100cells wereevaluated. The olive tail moments were observed under ordinary microscopeusing comet analysis software CASP.
Results:
Ames: Without S9metabolic activation system in vitro, the number ofrevertant colonies of TA98induced by different concentrations of irrigativewastewater was more than twice that of the untreated controls, and thedose-response relationship was observed(y=34.833+113.133x, rs=0.976,P=0.000). With S9metabolic activation system in vitro, the number ofrevertant colonies of TA98induced by different concentrations of irrigativewastewater was more than twice that of the untreated controls, and thedose-response relationship was observed (y=42.833+124.133x, rs=0.954,P=0.000). Irrigative wastewater in Shijiazhuang City had mutagenicitywhich contained direct and indirect frameshift-type mutagens.
PCE-MN： Micronucleus rates of mice PCE exposed to irrigativewastewater for two consecutive days were significant different among groups(F=171.746, P=0.000). Micronucleus rates of positive control group weresignificantly increased compared with that of negative control group(P<0.01). Yet, micronucleus rates induced by different concentations ofirrigative wastewater were not significantly different compared with thatinduced by negative control (P>0.05). After the mice were exposed toirrigative wastewater for consecutive15days, PCE-MN rates of mice weresignificant difference (F=71.706，P=0.000). PCE-MN rates of mice given25%irrigative wastewater were not significantly increased compared withthat induced by negative control (P>0.05); PCE-MN rates of mice given50%and100%irrigative wastewater were significantly increased (P<0.01), andthere was the dose-response relationship (y=0.003+0.007x, rs=0.814,P=0.000).
SCGE: After the mice were exposed to irrigative wastewater for consecutive15days, Olive tail moment values were significantly differentamong groups (=35.815, P=0.000). Olive tail moment values of the micegiven25%irrigative wastewater were no significantly increased comparedwith that of negative control (P>0.05). Olive tail moment values of the micegiven50%and100%irrigative wastewater were significantly increasedcompared with that of negative control (P<0.01), and dose-responserelationship was observed (y=0.298+4.055x，rs=0.905，P=0.000).
Conclusions:
1The results of Ames showed that direct and indirect frameshift-typemutagens existed in irrigative wastewater from Shijiazhuang City.
2The results of micronucleus test showed that substances accumulationof irrigative wastewater might occur in mice and irrigative wastewater couldcause chromosomal damage.
3The results of comet assay showed that irrigative wastewater fromShijiazhuang City could cause DNA strand breaks of mouse hepatocytes,resulting in DNA primary lesion.
Part three: Enhancement of preneoplastic lesion yield by irrigativewastewater in Shijiazhuang in a rat liver medium-term carcinogenesisbioassay
Objective: On the basis of the positive results of the short-termcarcinogenicity test, rat liver medium-term bioassay was applied for furtherresearch on the carcinogenic effects of the irrigative wastewater fromShijiazhuang City.
Methods: Healthy male SD rats of6-week-old were randomly dividedinto five groups and maintained in the animal facility of experimental animalcenter of Hebei Medical University Affiliated4th Hospital. After one weekacclimation period, the experiment was started. The two stages of rat livermedium-term carcinogenesis model were chosen. At the commencement, rats(group A) were intraperitoneally injected with0.9%physiological saline.Rats (groups B-E) were intraperitoneally injected with200mg/kg bodyweight of diethylnitrosamine (DEN) which is well-knowed initiator of liver carcinogenicity. After two weeks, irrigative wastewater was given by freedrinking ad libitum at doses of0,25%,50%and100%wastewater in groupsB-E and rats in group A were given distilled water. At the end of week3, allrats were underwent two-thirds partial hepatectomy. Rats were continuouslyexposed to different concentrations of irrigative wastewater after restorationof two days. During the experimental period, body weights, foodconsumption and water intake were recorded weekly. The experiment wasterminated at week8. Rats were decapitated after being weighed and bloodsamples were collected for biochemical analysis. The liver of rats wascarefully separated and weighed. Parts of the liver specimens were directlyfixed in paraformaldehyde, and the other part frozen in liquid nitrogen andstored at-80℃refrigerator. Serum concentrations of alanineaminotransferase, aspartate aminotransferase, alkaline phosphatase andgamma-glutamyl transfer peptide enzyme were determinated according toNanjing jiancheng kit. Liver tissue lesions were observed by HE stained.GST-Pi mRNA expression of rat liver was detected by RT-PCR; GST-Piprotein expression of rat liver was detected by immunohistochemistry andwestern blot.
Results:
1General observation and changes of organ coefficients: At week8when the experiment was terminated, weights of the rats were significantlydifferent among groups (F=27.925，P=0.000). Weights of the rats in negativecontrol and given25%irrigative wastewater and were no significantlyreduced compared with that of DEN control group (P>0.05). And weights ofthe rats given50%and100%irrigative wastewater were significantlyreduced compared with that of DEN control group (P<0.01). With theincreasing doses of wastewater, weights of the rats were significantlyreduced (y=414.146-53.136x, rs=-0.822, P=0.000). Organ coefficients ofthe liver among groups were significantly different (F=6.961，P=0.000).Liver Organ coefficients of the rats in negative control and given25%irrigative wastewater were no significantly increased compared with that of the DEN control group (P>0.05). Liver organ coefficients of the rats given50%and100%irrigative wastewater were significantly increased comparedwith that of the DEN control group (P<0.05). With the increasing doses ofwastewater, liver organ coefficients of the rats were significantly increased(y=2.935+0.359x, rs=0.459, P=0.004). No significant difference wasobserved in food consumption and water intake (P>0.05).
2Liver general observation and pathological morphological alteration:Liver tissue of HE stained tissue sections was observed at high magnificationunder general microscope. The liver of the rats given25%irrigativewastewater showed a small amount of proliferation of oval cells in portalarea. Of the rats given50%irrigative wastewater, organizational structures ofhepatic lobule were destroyed, showing regeneration nodules and small cellfoci; of the rats given100%irrigative wastewater, organizational structuresof hepatic lobule were destroyed seriously, forming fibrous tissues andpseudolobule, and a large number of oval cell proliferation in portal area.
3Effect of irrigative wastewater on liver function: AST serumconcentrations of the rats were significantly different among groups（=35.735, P=0.000）. Compared to the DEN control group, AST serum levelsof the rats in negative control and given25%irrigative wastewater were nosignificantly increased (P>0.05); AST serum levels of the rats given50%and100%irrigative wastewater were significantly increased (P<0.01) and thedose-response relationship was observed (y=57.620+248.277x, rs=0.928，P=0.000). ALT serum concentrations of the rats were significantly diffentent(=38.684, P=0.000); ALT serum concentrations of the rats in negativecontrol group were no significantly higher than that of the DEN controlgroup (P>0.05). ALT serum concentrations of the rats given differentconcentrations irrigative wastewater were significantly higher than that ofthe DEN control group (P<0.01) and dose-response relationship wasobserved (y=14.540+246.921x, rs=0.960, P=0.000). γ-GT serum levels ofthe rats were significantly different (F=12.987, P=0.000). γ-GT serum levelsof the rats in negative control group and given25%,50%irrigative wastewater were no significantly increased (P>0.05); γ-GT serum levels ofthe rats given100%irrigative wastewater were significantly increased(P<0.01). No significant difference was observed of AKP serum levels of therats (F=1.824, P=0.144).
4GST-Pi mRNA expression of rat liver: Rats initiated by DEN, mRNAexpression of GST-Pi in the rat liver tissue were significantly differentamong groups (F=37.904, P=0.000). Compared with that of the DEN-treatedgroup, GST-Pi mRNA expression of the rats in negative control group andgiven25%irrigative wastewater were not significantly upregulated (P>0.05);GST-Pi mRNA expression of the rats given50%and100%irrigativewastewater were significantly upregulated (P<0.01). GST-Pi mRNAexpression of the rat liver were increased in a dose-dependent manner(y=1.029+1.060x, rs=0.950, P=0.000).
5GST-Pi expression of rat liver: Results of western blot showed lowGST-Pi expressions of the rat liver were observed in negative control andDEN-treated control. Rats initiated by DEN, GST-Pi expression of the ratliver were significantly different (F=268.176, P=0.000). Compared with thatof the DEN-treated group, GST-Pi protein expression of the rats in negativecontrol group were not significantly upregulated (P>0.05); GST-Piexpression of the rats in different concentration wastewater groups weresignificantly upregulated (P<0.01). With the increasing concentrations ofirrigative wastewater, GST-Pi expression levels were upregulatedsignificantly (y=0.470+0.823x, rs=0.972, P=0.000).
GST-Pi protein expression was not observed in the negative controlgroup by immunohistochemical method. Rats initiated by DEN, GST-Piexpressions of the rat liver in DEN control group were low. Rat givendifferent concentrations wastewater afer initiated by DEN, GST-Pi proteinexpression were significantly increased compared with that of the DENcontrol in a dose-response manner and it was highest in100%wastewatergroup.
Conclusions: Irrigative wastewater in Shijiazhuang City has the function of tumor promotion. By the effect of irrigative wastewater inShijiazhuang City, significantly weight losses of rats were observed, liverorgan coefficients of the rats were significantly increased and abnormal liverfunctions were observed. GST-Pi mRNA and GST-Pi expression levels of therat liver induced by irrigative wastewater of different concentrations weresignificantly upregulated in the dose-response manner.
Part four: Effect of CYP1A1and CYP1B1on rat livermedium-term carcinogenesis bioassay of irrigative wastewater
Objective: To study the effect of CYP1A1and CYP1B1on rat liver inmedium-term carcinogenesis bioassay of irrigative wastewater
Methods: Animal model was same as the third part. CYP1A1andCYP1B1expression at the mRNA and protein level were examined byRT-PCR and western blot.
Results:
1CYP1A1mRNA and protein expressions of rat liver induced byirrigative wastewater: CYP1A1mRNA expression of the rat liver wassignificantly different (F=73.054, P=0.000). Compared to that of theDEN-treated group, CYP1A1mRNA expressions in the liver tissue of therats in negative control group were not significantly upregulated (P>0.05);CYP1A1mRNA expressions in the liver tissue of the rats given irrigativewastewater of different concentrations were significantly upregulated(P<0.01) in a dose-dependent manner (y=0.346+0.621x, rs=0.941, P=0.000).CYP1A1expressions of the rat liver were significantly different (F=6.788,P=0.007). Compared to that of the DEN-treated group, CYP1A1expressionsin the liver tissue of the rats in negative control group and given25%irrigative wastewater were not significantly upregulated (P>0.05); CYP1A1expressions in the liver tissue of the rats given50%and100%irrigativewastewater were significantly upregulated (P<0.05) and a dose-responserelationship was observed (y=0.557+0.316x, rs=0.799，P=0.001).
2CYP1B1mRNA and protein expressions of rat liver induced byirrigative wastewater: CYP1B1mRNA expressions of the rat liver were significant different (F=11.539, P=0.001). Compared to that of theDEN-treated group, CYP1B1mRNA expressions in the liver tissue of therats in negative control were not significantly upregulated (P>0.05);CYP1B1mRNA expressions in the liver tissue of the rats given irrigativewastewater of different concentrations were significantly upregulated(P<0.05) in a dose-dependent manner (y=0.527+0.313x, rs=0.907, P=0.000).CYP1B1expressions of the rat liver were significant different (F=8.247,P=0.003). Compared to that of the DEN-treated group, CYP1B1expressionsin the liver tissue of the rats in negative control group were not significantlydifferent (P>0.05); CYP1B1expressions in the liver tissue of the rats givenirrigative wastewater of different concentrations were significantlyupregulated (P<0.05). CYP1B1expressions were significantly upregulatedwith the increasing doses of irrigative wastewater (y=0.530+0.528x,rs=0.789, P=0.002).
Conclusions: Irrigative wastewater in Shijiazhuang City had thefunction of the induction of abnormal expression of cytochrome P4501A1and P4501B1and played the role of tumor promoters, eventually resulting inthe precancerous lesions of the rat liver
引文
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