摘要
目的
砷是环境中广泛存在的有毒类金属元素之一,已被美国疾病预防控制中心和国际防癌研究机构确定为人类致癌物。饮水中含有高浓度的砷而导致的慢性砷中毒已经成为全世界广泛关注的卫生学问题。无机砷进入人体后,经代谢主要分布于皮肤、肝、肾、脾、肺和胃肠道,经尿排出,毛发、指甲也可排出少量砷。由于肝是重量最大的内脏器官,并且机体摄入iAs的首过效应,所以肝是iAs甲基化的主要场所,肝损伤有可能导致尿砷代谢模式的变化。近年来的动物实验和人群流行病学调查资料均表明,砷可以引起不同程度的肝损伤、肝纤维化、肝硬化和肝癌,这些都与肝脏对砷的代谢有关。因此,研究砷中毒与肝细胞及肝细胞线粒体受损相关的酶显得甚为重要。其中ALT(血清丙氨酸氨基转移酶)和AST(天冬氨酸氨基转移酶)为最重要的两种,ALT能反映肝细胞的完整性,是急性肝炎的早期敏感指标。血清mAST增高可反映亚细胞结构损害的严重性,是诊断肝细胞损伤的另外敏感指标之一。乙肝表面抗体是患有乙型肝炎的标志之一,乙肝的发病与否能够直接反映出砷对肝脏损伤指标,也是免疫系统损伤的一个间接指标。
本研究我们在大规模的横断面调查基础上,探讨相关肝指标异常和损伤的人群尿砷代谢模式与正常人群相比是否发生改变；并将尿总砷作为机体的内暴露指标对人群进行分组,观察砷暴露水平与肝指标异常发生之间的关系。
方法
一、调查对象
1、调查地区的选择
根据山西省地病中心以往水质调查及水砷测定的结果,选择饮水型砷中毒流行区,山西省大同市天镇县6个村和朔州市应县为调查区；调查地区的自然状况、经济条件、人口构成、农作物种类、生活习惯等条件相似,两地区均无工业砷污染。
2、调查对象的选择
调查对象为该病区常住人口,采取自愿和入户调查相结合的方法,收集在当地居住5年以上18岁成人为调查对象。排除慢性病患者,所有调查对象近3天没有食用海产品。
二、调查内容
1、一般状况
采用统一印制的调查表、直接询问并填写问卷。内容包括：(1)年龄、性别、职业、文化程度、婚姻状况、子女数等。(2)生活、环境因素：吸烟史和吸烟量、饮酒史和饮酒量、饮食习惯、家族史、病史等。
2、体格检查：
身高、体重、血压、空腹血糖、尿糖、肝胆B超、心电图等,所有测量均由专业医护人员按统一的规范进行。砷中毒症状识别依据《中国砷中毒诊断标准》(WS／T211-2001)进行慢性砷中毒皮肤症状的检查。
3、采集生物样品：
(1)尿液样品
采集人群即时尿,立刻放置于0-4℃冰盒中,当日尿样收集后统一保存于-20℃。待调查结束时所有尿样于冰盒中运送回实验室,保存于-80℃待测。
(2)血液样品
采集清晨空腹静脉血5 ml,置于肝素抗凝管中并颠倒混匀,于液氮中保存运送回实验室,-80℃保存待测。
4、实验室检查：
(1)尿砷测定；
融化冷冻尿样,经过消化处理,使用冷阱捕集氢化物发生原子分光光度计测定待测样品中的砷含量。该方法测定3种形态砷化物检测限均为1 ng,变异系数<5%。标准物质为日本环境研究所提供。
(2)肝功能和乙肝表面抗原的测定：
应用ACTO1半自动生化分析仪测定ALT(血清丙氨酸氨基转移酶)、AST(天冬氨酸氨基转移酶)和HBs-Ag(乙肝病毒表面抗原)。凡上述指标异常者纳入肝指标异常组。
三、统计学处理
采用SPSS 16.0统计软件对数据进行分析。对尿砷数据进行对数转化,使数据服从或近似服从正态分布后进行相应统计,数据结果以几何均数(geometric mean,GM)表示。采用x2检验比较病例组和对照组在性别、吸烟和饮酒分布。采用两独立样本t检验对两组间人群尿砷水平和砷甲基化能力进行比较。应用二分类多元的logistic回归对尿TAs≤50μg/g Cr组和尿TAs>50μg/g Cr组中肝指标异常各组进行比较。统计学上的显著性水平规定为α=0.05。
结果
本次共调查1006人,剔除资料不全者,肝指标异常(AST异常或ALT异常或乙肝患者)共有166人(AST异常为84人、ALT异常为13人、乙肝患者为28人、AST+ALT异常为28人、乙肝携带+AST异常为7人和乙肝携带+AST+ALT异常为6人)。剔除患有肝脏、高血压等其他慢性疾病,纳入423位身体健康的人作为对照组。肝指标异常各组与对照组进行在性别、年龄、吸烟和饮酒方面的分布无显著性差异。
1、尿砷代谢模式和性别、吸烟及饮酒间关系的比较
在对照组发现,女性尿TAs、DMA和SMR显著高于男性；不吸烟组尿TAs、DMA和SMR显著高于吸烟的人群。
2、尿砷代谢模式各指标在肝指标异常各组与对照组的比较
肝指标异常各组分别与对照组比较,尿砷代谢模式各项指标均无显著性差异。
3、肝脏指标异常在不同尿TAs水平人群分布
通过尿TAs分组发现,AST异常在尿TAs>50μg/g Cr组发生的危险度显著高于尿TAs≤50μg/g Cr组,调整后的RR值是1.94(95%CI=1.00-3.76),而其他肝指标异异常组的RR值均无统计学意义。
采用病例对照的研究方法分析,肝指标异常各组中,也只有AST的发生在尿TAs>50 gg／g Cr组的危险度显著升高,调整后的OR值是1.97(95%CI=1.00-3.51)。
结论
1、与正常人群比较,AST、ALT异常和乙肝表面抗体阳性的人群尿砷代谢模式无显著性变化。
2、在本研究人群砷暴露水平下,随着尿TAs水平的升高,人体对无机砷的甲基化能力增强。
3、当人群的尿砷水平高于50μg/g Cr时, AST异常率增高,提示肝损伤危险度增加。
Objective
Arsenic is a sort of venomous metalloid elements extensively exist in the nature-confirmed carcinogen-authenticated by both CDC and IARC (International Agency for Research on Cancer). Chronic arsenic poisoning from drinking water has become a widespread concern throughout the world health issues. Inorganic arsenic in human body is metabolized mainly in the liver, distributed in kidney, spleen, lung and gastrointestinal tract, through the urine, hair, nails can also emit a small amount of arsenic. Because the liver is the largest weight of internal organs, and the first-pass effect of inorganic arsenic, the liver is the principal place for arsenic methylation, the liver may affect inorganic arsenic metabolism. Epidemiology studies and animal experiments have clearly indicated an association between chronic arsenic exposure and abnormal liver function, hepatomegaly, hepatoportal sclerosis, liver fibrosis and cirrhosis. It is important that studies between arsenic poisoning and the damage of liver cells and mitochondria. ALT (serum almandine aminotransferase) and AST (aspirate aminotransferase) was the most important in liver cell enzymes. ALT can reflect the integrity of liver cells and an early sensitive indicator of acute hepatitis. Elevated serum mAST subcellular structures can reflect the severity of the damage, the diagnosis of liver cell injury, one of the other sensitive indexes. HBsAg is the mark of hepatitis B, hepatitis B not only can directly reflect arsenic on liver damage indicator, but also an indirect indicator on damage of the immune system.
In this study, residents with damage of ALT, AST and HBsAg have whether the change in urinary arsenic patterns, compared to the normal population; By total arsenic in urine as indicators of body exposure, we study the relationship on the level of body exposure and liver damage.
Methods
1. Study Subjects
(1) Study areas:According to water quality surveys and the results of arsenic determination in Shanxi CDC in the past, we select areas of arsenic poisoning from drinking water (six villages of Tianzhen County in Datong and ying County of Shuozhou City). It is similar that their natural conditions, economic conditions, demographic composition, crop type, living habits, and so on. there was no industrial arsenic contamination in the two regions.
(2) Study subjects:We select the long-term residents. We use two methods of take-home and voluntary. The adults have health examination, according to examination results, we select population with nearly 3 days without eating seafood.
2. Investigation of Study
(1) General situation:We use uniform printed questionnaire, uniform medical standards for the survey, direct questions and complete the questionnaires. Include: including age, sex, occupation, education level, marital status, number of children and so on. Life, and environmental factors:smoking history and smoking, drinking history and alcohol consumption, eating habits, family history, medical history and so on.
(2) Physicians use a professional medical examination, including:height, weight, blood pressure, fasting blood sugar, urine sugar, and so on. Trained doctors conducted detailed physical examinations and arsenicosis identification according to the Diagnosis Standards on Arsenicosis of China (WS/T211-2001).
(3) Collection of biological samples
Urine samples:We collect urine, immediately placed in ice box at 0-4℃. Saved the day and collected urine samples at-20℃. In the end, all urine samples tested until the investigation in ice box to return the laboratory, stored at-80℃.
Blood samples:Blood collected 5 ml, placed in heparin tubes and reverses the mix, tested after transport back to the lab in liquid nitrogen.
(4) Examination in lab
Determination of arsenic metabolites:Urine samples were mix with 2 mol/L NaOH (1/1, v/v) and digested at 100℃for 3 h. Then cold trap hydride generation-atomic absorption spectrometry were applied to determine the content of iAs, MMA, dimethylated arsenic (DMA) and trimethylated arsenic (TMA). Detection limits were 1 ng with this method the three kinds of forms of arsenic, the coefficient of variation<5%. Standard material was provided for the Japanese Environmental Institute.
Determination of liver indexes:ALT (alanine aminotransferase), AST (aspartate aminotransferase), and HBS-Ag (Hepatitis B Virus Surface Antigen) is determined by ACTO1 Photometer Application.
4. Statistical analysis
Data analysis was used by SPSS software (version 16.0). for. Logarithmic transformation of data made the data normal distribution. The results express as the geometric mean. We used x2 test in gender, lifestyle distribution. We used two independent samples t test between two groups of urinary arsenic levels and arsenic methylation capacity. Statistically significant level:a=0.05.
Results
We selected 1006 people, according to urine and blood samples taken, as well as gender, age, smoking and drinking and other related factors, the principle of excluding incomplete data, there are 166 people of abnormal liver function (AST or ALT abnormal or unusual hepatitis B patients), abnormal AST and ALT abnormalities, hepatitis B patients were 84 people,13 and 28, respectively. Excluding suffering from liver disease, high blood pressure and other chronic diseases, there are 423 healthy people. There is no significant difference in the gender, age, smoking and drinking compared all groups of abnormal liver function with the control group.
1. Urinary arsenic patterns in gender, age, smoking and alcohol
With regard to gender, urinary TAs、DMA and SMR in female were significantly higher than male; urinary TAs、DMA and SMR in non-smoking group were significantly higher than smoking.
2. Urinary arsenic patterns in groups of liver function abnormalities
There is no significant difference compared all groups of abnormal liver function with the control group.
3. The effects of arsenic exposure in body to the liver
According to urinary arsenic values, After excluding confounding factors, urinary TAs> 50μg/g Cr was an independent risk factor for abnormal AST, the adjusted RR value of 1.94 (95% CI= 1.00-3.76), There is no significant difference compared arsenic exposure group with the control group.
The study population divided into control group and arsenic exposure group. After excluding confounding factors, urinary TAs> 50μg/g Cr was an independent risk factor for abnormal AST, the adjusted OR value of 1.97 (95% CI= 1.00-3.51), showed that when the urinary TAs> 50μg/g Cr is 1.97 fold of total urinary arsenic≤50μg/g Cr. There is no significant difference compared arsenic exposure group with the control group.
Conclusion
1. Compared with the normal population, There is no significant difference in all groups of abnormal liver function.
2. In this study, in an certain arsenic exposure, the capacity of inorganic arsenic methylation is increased when the levels of urinary TAs is increased.
3. The rate of AST abnormal is increased, it suggests the risk of liver injury is increased, when urinary arsenic levels above 50μg/g Cr, there is high risk in AST abnormal.
引文
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1 Yoshida T, Yamauchi H, Sun GF. Chronic health effects in people exposed to arsenic via the drinking water:dose-response relationships in review. Toxicol Appl Pharmacol.2004, 198(3):243-252.
2 Sun G Arsenic contamination and arsenicosis in China. Toxicol Appl Pharmacol,2004,198: 268-271.
3 Vahter ME. Interactions between arsenic-induced toxicity and nutrition in early life. J Nutr. 2007 Dec;137(12):2798-804.
4 Andreae MO. Determination of arsenic species in natural waters. Anal. Chem. 1977;49:820-823.
5 Shraim A, Sekaran NC, Anuradha CD, Hirano S. Speciation of arsenic in tubewell water samples collected from West Bengal, India, by high-performance liquid chromatography-inductively coupled plasma mass spectrometry. Appl. Organomet.Chem. 2002; 16:1-8.
6 Tseng CH.Arsenic methylation, urinary arsenic metabolites and human diseases:current perspective. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev.2007 Jan-Mar;25(1):1-22.
7 Aposhian HV, Zakharyan RA, Avram MD, et al. A review of the enzymology of arsenic metabolism and a new potential role of hydrogen peroxide in the detoxication of the trivalent arsenic species. Toxicol Appl Pharmacol.2004; 198(3):327-335.
8 Hayakawa T, Kobayashi Y, Cui X, et al. A new metabolic pathway of arsenite: arsenic-glutathione complexes are substrates for human arsenic methyltransferase Cyt 19. Arch Toxicol.2005; 79:183-191.
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