黄曲霉毒素B_1和脱氧雪腐镰刀菌烯醇对锦鲤及原代培养肝细胞的毒性研究
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摘要
真菌毒素是由真菌产生的有毒次级代谢产物,广泛污染各种饲料及饲料原料。动物摄入被真菌毒素污染的饲料会造成动物的真菌毒素中毒。黄曲霉毒素是由黄曲霉和寄生曲霉等真菌产生的一大类结构相似的次级代谢产物,是目前已知的最稳定、毒性最强的毒素。脱氧雪腐镰刀菌烯醇是禾谷镰刀菌在生长过程中产生的一种主要代谢产物,是单端孢霉烯族毒素的一种,毒性和黄曲霉毒素相比较弱,但广泛存在于各种谷物和饲料。
本论文主要研究这两种毒素在常规剂量下对锦鲤的毒性作用,分别从体内试验和体外试验两个方面对两种毒素单独和联合作用对锦鲤的毒性作用进行了研究。
试验Ⅰ江苏省水产饲料及饲料原料中黄曲霉毒素B,和脱氧雪腐镰刀菌烯醇的污染情况调查
分别从江苏省各地(扬州、盐城、苏州和徐州等)收集的水产饲料及饲料原料,其中粉状料15份,颗粒料15份。毒素经提取净化后,HPLC检测含量。
测定结果表明,在粉状料和颗粒料中,AFB1的阳性检出率分别为40%和26.7%,DON的阳性检出率均为100%,从真菌毒素的绝对含量来看,AFB1的最高含量为16.4μg kg-1,低于国家规定的饲料及饲料原料最大允许量kg-1);而DON的最高含量为7046μg kg-1,高于国家配合饲料允许含量标准(1000μg kg-1).
试验Ⅱ黄曲霉毒素B1和脱氧雪腐镰刀茵烯醇对锦鲤生长性能、血液生化和组织病理学的影响
锦鲤270尾(均重为18±0.1g),分为9组,每组10尾,每组设三个平行,2个平行组进行试验观察和统计,1个平行组作为试验过程中解剖检查用(在第1、20、40天各取样3尾剖检)。分别饲喂基础日粮、基础日粮+AFB1 (0.05mg kg-1)基础日粮+AFB1 (0.1mg kg-1)、基础日粮+DON (lmg kg-1)、基础日粮+DON (5mg kg-1)、基础日粮+AFB1 (0.05mg kg-1)+DON (1mg kg-1)、基础日粮+AFB1(0.05mg kg-1)+DON (5 mg kg-1)、基础日粮+AFB1(0.1 mg kg-1)+DON (1 mg kg-1)、基础日粮+AFB1(0.1mg kg-1)+DON (5mg kg-1).试验为期40天。每20天进行体重、体长、丰满系数、肝胰脏重／体重比、血清生化指标检测(ALP、ALB、ALT、AST、TP)以及肝胰脏、脾脏和肠道的组织学检查。试验结果表明：不管是毒素单独作用还是联合作用,均能不同程度的引起锦鲤生长性能的降低,并表现出体重、丰满系数的下降,血清生化指标的变化和组织的病理学损伤。随着毒素浓度的升高,毒素的毒性作用显著增加,具有明显的剂量-效应关系。和毒素单独作用相比,两种毒素联合作用后毒性明显大于一种毒素单独作用,具有一定的协同作用。
试验Ⅲ锦鲤原代肝细胞的分离及原代培养条件的优化
在本试验中,选用三种不同的细胞分离方法(机械分离法、胶原酶灌注法和胰酶-EDTA消化法)来获得肝细胞,并将其置于三种不同的培养基中(DMEM, M199和L-15),在不同的培养条件下(17℃、27℃和37℃,5%CO2供应和无CO2供应)进行培养,细胞培养时间为7天,筛选出最方便、实用的锦鲤肝细胞的原代分离培养方法。结果表明：采用胰酶-EDTA消化法来分离细胞,并将得到的肝细胞用L-15培养基(无C02供应)或M199培养基(有5%C02供应)于27℃培养可获得状态良好的肝细胞,满足进行毒理学试验的要求。
试验Ⅳ黄曲霉毒素B1和脱氧雪腐镰刀菌烯醇对锦鲤原代培养肝细胞的毒性作用的研究
在试验Ⅲ的基础上,对分离得到的肝细胞进行培养,分别设置AFB1组(0.01μgmL-1和0.02μg ml-1),DON组(125ng ml-1、250ngmL-1、500ngmL-1、1000ngmL-1和2000 ngmL1)，联合毒性组(0.01+125、0.01+250、0.01+500、0.02+125、0.02+250和0.02+500,单位分别为μgmL-1和ng mL-1),试剂对照组(DMSO:2，μLmL-1)及空白对照组,每隔2h,从细胞形态学、酶活性和细胞毒性三个方面研究这两种真菌毒素对锦鲤原代培养肝细胞的单独和联合毒性作用,试验时间为24h。试验结果表明：光学显微镜下发现两种毒素均能在短时间内引起细胞的大量死亡,各试验组均与对照组之间差异显著。在电镜下,和对照组相比,低浓度联合毒性组和DON组均能发现数量不等的凋亡细胞,而低浓度的AFB1组则仅见个别凋亡的肝细胞。
试验V黄曲霉毒素B1、脱氧雪腐镰刀菌烯醇和两种毒素联合作用对锦鲤原代肝细胞DNA损伤的研究
在本试验中,分别采用单细胞凝胶电泳和DNA ladder两种方法对毒素引起的细胞凋亡情况进行了研究。用单细胞凝胶电泳进行研究的结果是,肝细胞凋亡率最高的为联合毒素组[AFB1+DON:0.02(μg mL-1)+125(ngm-1)]组,其次为DON 500ng mL-1组,再次为AFB1+DON:0.01 (μg mL-1)+125 (ng mL-1)组。用DNA ladder进行研究时发现,DON(<500ng mL-1)和低浓度的联合毒素[AFB1+DON:0.01 (μg mL-1)+125 (ng mL-1)和AFB1+DON:0.02 (μgmL-1)+125 (ngmL-1)]可以引起锦鲤肝细胞的明显凋亡,在2.5%琼脂糖电泳凝胶上呈梯状,其它剂量的毒素则不能检测到凋亡的存在。
Mycotoxins were toxic secondary metabolites produced by fungi(molds), wildly contaminated in raw material and finished product. It would result in mycotoxins poisoning when animals were exposed the feed contaminated the mycotoxins.
Aflatoxin B1 is a compound that produced during the growth of Aspergillus flavus and Aspergillus parasiticus. As we know it is the most power toxi city and stabization toxin in the world. Deoxynivalenol(DON)was a metabolism produced during the growth of Fusarium graminearum. It was a member of trichothecene family. DON is less toxicity than Aflatoxin B1.
The aim of our research is to study the toxic effect of AFB1 and DON to cyprinus carpio at normal dosage.
Test I The level which contaminated the aquatic animal feeds of AFB1 and DON in Jiangsu province.
We collected the aquatic animal feed of Jiangsu province (Yangzhou, Yancheng et al.,).Among these 15 samples are powder feed and 15 samples are Pellet diet. We used the HPLC to detect the level of the toxin after they have been extracted and purified.
The detection rate of AFB1 is 40% in the power feed and 26.7% in the pellet diet, and the detection rate of DON is 100% whatever in anyone feed. The maximum content of AFB1 is 16.4μg kg-1 which is less than the government standard of AFB1 in animal feed(20μg kg-1),and the maximum content of DON is 7046ug kg-1 which is more higher than the government standard(1000μg kg-1).
Test II Toxic effect of AFB1 and DON to cyprinus carpio
Two hundred and seventy cyprinus carpio(middle weight was 18±0.1g) were devided into 9 groups and each group has 10 fishes. Three parallel were designed for every group. One parallel work for the anatomy during the study and two parallel group for the observation and analysis. Basic feed, basic feed and AFB1(0.05mg kg-1), basic feed and AFB1(0.1mg kg-1), basic feed and DON (1mg kg-1), basic feed and DON(5mg kg-1), basic feed and AFB1(0.05mg kg-1)+DON(1 mg kg-1), basic feed and AFB1(0.05mg kg-1)+DON(5 mg kg-1), basic feed and AFB1(0.1 mg kg-1)+DON(1 mg kg-1),basic feed and AFB1(0.1 mg kg-1)+DON(5mg kg-1)were designed for 9 groups. The experiment lasted for 40 days. The body length, body weight, fullness, liver weight/body weight, blood biochemical index and the pathologic histology of the liver, spleen and intestinal tract were checked every 20 days. It showed that whatever the toxin was separated or combined, it could depress the performance of the fish. And this injury is more serious with the higher concentration of the toxin. This toxic effect showed the significant dose-effect relationship.
TestⅢTo Optimize the Isolation and Cultivation of cyprinus carpio primary hepatocytes
In this study, the cyprinus carpio hepatocytes were isolated by mechanical separation, two-step collagenase perfusion and pancreatin digestion, respectively. The hepatocytes or parenchymal cells could be separated from cell debris and from non-parenchymal cells by low-speed centrifugation (Percoll grade centrifugation). The harvesting hepatocytes were suspended in DMEM, M199(cultured in 5%CO2) and L-15 (cultured without 5%CO2) medium, then cultured at 17℃,27℃and 37℃, respectively. The cell yield was counted by hemocytometer, and the viability of the cells were assessed by Trypan blue exclusion test. Results from these studies showed that the best isolation method was pancreatin digestion [the cell yield was 2.7×108 per g(liver weight) and the viability was 98.4%] and the best medium was M199(cultured in 5%CO2) or L-15(cultured without 5%CO2), respectively. The optimal culture temperature was 27℃. The primary hepatocytes culture of cyprimus carpio grew well and satisfied the requirement of most of toxicological experiments in this condition.
Test IV Toxic effect of AFB1 and DON to cyprinus carpio primary culture hepatocytes
Isolation and culture the hepatocytes base on the test III,design the experiment group as AFB1(0.01μg mL-1 and 0.02μg mL-1),DON(125ng mL-1、250 ng mL-1、500 ng mL-1、1000 ngmL-1and2000ngmL-1), conbination group(0.01+125、0.01+250、0.01+500、0.02+125、0.02+250 and 0.02+500,μg mL-1, ng mL-1), reagent controI(DMSO:2μL mL-1)and the control. Check the cell shape, enzyme activity and the cytotoxicity every 2 hours to study the toxic effect of the toxin. The experiment lasted for 24 hours. From the experiment it showed that all of the toxin could induce a great quality of cells death. Alsothere was no significant difference between the experimental groups, but the difference between each experimental groups and the control is significant (P<0.05).We could saw the inequality apoptosis cells under the TEM in experimental groups contrast to the control. And the results of enzyme activity and the cytotoxicity support the viewpoint.
Test V The DNA injury of cyprinus carpio primary cultured hepatocytes by AFB1 and DON
We have used SCGE and DNA ladder to detect the cell apoptosis induced by the toxin. When chose the SCGE to do the research, we found that the group of the highest rate of the apoptosis is the combination group[AFB1+DON:0.02 (μg mL-1)+125 (ng mL-1)], the second is DON 500ng mL-1.and when we choose the DNA ladder to do the research, it showed that DON(≤500ng mL-1)and the low concentration of combination [AFB,+DON:0.01(μg mL-1)+125(ng mL-1)和AFB1+DON:0.02(μg mL-1)+125(ng mL-1)] could induce significant the hepatocyte apoptosis ly, and showed the obvious "ladder" on 2.5% agarose gel electrophoresis.
本论文主要研究这两种毒素在常规剂量下对锦鲤的毒性作用,分别从体内试验和体外试验两个方面对两种毒素单独和联合作用对锦鲤的毒性作用进行了研究。
试验Ⅰ江苏省水产饲料及饲料原料中黄曲霉毒素B,和脱氧雪腐镰刀菌烯醇的污染情况调查
分别从江苏省各地(扬州、盐城、苏州和徐州等)收集的水产饲料及饲料原料,其中粉状料15份,颗粒料15份。毒素经提取净化后,HPLC检测含量。
测定结果表明,在粉状料和颗粒料中,AFB1的阳性检出率分别为40%和26.7%,DON的阳性检出率均为100%,从真菌毒素的绝对含量来看,AFB1的最高含量为16.4μg kg-1,低于国家规定的饲料及饲料原料最大允许量kg-1);而DON的最高含量为7046μg kg-1,高于国家配合饲料允许含量标准(1000μg kg-1).
试验Ⅱ黄曲霉毒素B1和脱氧雪腐镰刀茵烯醇对锦鲤生长性能、血液生化和组织病理学的影响
锦鲤270尾(均重为18±0.1g),分为9组,每组10尾,每组设三个平行,2个平行组进行试验观察和统计,1个平行组作为试验过程中解剖检查用(在第1、20、40天各取样3尾剖检)。分别饲喂基础日粮、基础日粮+AFB1 (0.05mg kg-1)基础日粮+AFB1 (0.1mg kg-1)、基础日粮+DON (lmg kg-1)、基础日粮+DON (5mg kg-1)、基础日粮+AFB1 (0.05mg kg-1)+DON (1mg kg-1)、基础日粮+AFB1(0.05mg kg-1)+DON (5 mg kg-1)、基础日粮+AFB1(0.1 mg kg-1)+DON (1 mg kg-1)、基础日粮+AFB1(0.1mg kg-1)+DON (5mg kg-1).试验为期40天。每20天进行体重、体长、丰满系数、肝胰脏重／体重比、血清生化指标检测(ALP、ALB、ALT、AST、TP)以及肝胰脏、脾脏和肠道的组织学检查。试验结果表明：不管是毒素单独作用还是联合作用,均能不同程度的引起锦鲤生长性能的降低,并表现出体重、丰满系数的下降,血清生化指标的变化和组织的病理学损伤。随着毒素浓度的升高,毒素的毒性作用显著增加,具有明显的剂量-效应关系。和毒素单独作用相比,两种毒素联合作用后毒性明显大于一种毒素单独作用,具有一定的协同作用。
试验Ⅲ锦鲤原代肝细胞的分离及原代培养条件的优化
在本试验中,选用三种不同的细胞分离方法(机械分离法、胶原酶灌注法和胰酶-EDTA消化法)来获得肝细胞,并将其置于三种不同的培养基中(DMEM, M199和L-15),在不同的培养条件下(17℃、27℃和37℃,5%CO2供应和无CO2供应)进行培养,细胞培养时间为7天,筛选出最方便、实用的锦鲤肝细胞的原代分离培养方法。结果表明：采用胰酶-EDTA消化法来分离细胞,并将得到的肝细胞用L-15培养基(无C02供应)或M199培养基(有5%C02供应)于27℃培养可获得状态良好的肝细胞,满足进行毒理学试验的要求。
试验Ⅳ黄曲霉毒素B1和脱氧雪腐镰刀菌烯醇对锦鲤原代培养肝细胞的毒性作用的研究
在试验Ⅲ的基础上,对分离得到的肝细胞进行培养,分别设置AFB1组(0.01μgmL-1和0.02μg ml-1),DON组(125ng ml-1、250ngmL-1、500ngmL-1、1000ngmL-1和2000 ngmL1)，联合毒性组(0.01+125、0.01+250、0.01+500、0.02+125、0.02+250和0.02+500,单位分别为μgmL-1和ng mL-1),试剂对照组(DMSO:2，μLmL-1)及空白对照组,每隔2h,从细胞形态学、酶活性和细胞毒性三个方面研究这两种真菌毒素对锦鲤原代培养肝细胞的单独和联合毒性作用,试验时间为24h。试验结果表明：光学显微镜下发现两种毒素均能在短时间内引起细胞的大量死亡,各试验组均与对照组之间差异显著。在电镜下,和对照组相比,低浓度联合毒性组和DON组均能发现数量不等的凋亡细胞,而低浓度的AFB1组则仅见个别凋亡的肝细胞。
试验V黄曲霉毒素B1、脱氧雪腐镰刀菌烯醇和两种毒素联合作用对锦鲤原代肝细胞DNA损伤的研究
在本试验中,分别采用单细胞凝胶电泳和DNA ladder两种方法对毒素引起的细胞凋亡情况进行了研究。用单细胞凝胶电泳进行研究的结果是,肝细胞凋亡率最高的为联合毒素组[AFB1+DON:0.02(μg mL-1)+125(ngm-1)]组,其次为DON 500ng mL-1组,再次为AFB1+DON:0.01 (μg mL-1)+125 (ng mL-1)组。用DNA ladder进行研究时发现,DON(<500ng mL-1)和低浓度的联合毒素[AFB1+DON:0.01 (μg mL-1)+125 (ng mL-1)和AFB1+DON:0.02 (μgmL-1)+125 (ngmL-1)]可以引起锦鲤肝细胞的明显凋亡,在2.5%琼脂糖电泳凝胶上呈梯状,其它剂量的毒素则不能检测到凋亡的存在。
Mycotoxins were toxic secondary metabolites produced by fungi(molds), wildly contaminated in raw material and finished product. It would result in mycotoxins poisoning when animals were exposed the feed contaminated the mycotoxins.
Aflatoxin B1 is a compound that produced during the growth of Aspergillus flavus and Aspergillus parasiticus. As we know it is the most power toxi city and stabization toxin in the world. Deoxynivalenol(DON)was a metabolism produced during the growth of Fusarium graminearum. It was a member of trichothecene family. DON is less toxicity than Aflatoxin B1.
The aim of our research is to study the toxic effect of AFB1 and DON to cyprinus carpio at normal dosage.
Test I The level which contaminated the aquatic animal feeds of AFB1 and DON in Jiangsu province.
We collected the aquatic animal feed of Jiangsu province (Yangzhou, Yancheng et al.,).Among these 15 samples are powder feed and 15 samples are Pellet diet. We used the HPLC to detect the level of the toxin after they have been extracted and purified.
The detection rate of AFB1 is 40% in the power feed and 26.7% in the pellet diet, and the detection rate of DON is 100% whatever in anyone feed. The maximum content of AFB1 is 16.4μg kg-1 which is less than the government standard of AFB1 in animal feed(20μg kg-1),and the maximum content of DON is 7046ug kg-1 which is more higher than the government standard(1000μg kg-1).
Test II Toxic effect of AFB1 and DON to cyprinus carpio
Two hundred and seventy cyprinus carpio(middle weight was 18±0.1g) were devided into 9 groups and each group has 10 fishes. Three parallel were designed for every group. One parallel work for the anatomy during the study and two parallel group for the observation and analysis. Basic feed, basic feed and AFB1(0.05mg kg-1), basic feed and AFB1(0.1mg kg-1), basic feed and DON (1mg kg-1), basic feed and DON(5mg kg-1), basic feed and AFB1(0.05mg kg-1)+DON(1 mg kg-1), basic feed and AFB1(0.05mg kg-1)+DON(5 mg kg-1), basic feed and AFB1(0.1 mg kg-1)+DON(1 mg kg-1),basic feed and AFB1(0.1 mg kg-1)+DON(5mg kg-1)were designed for 9 groups. The experiment lasted for 40 days. The body length, body weight, fullness, liver weight/body weight, blood biochemical index and the pathologic histology of the liver, spleen and intestinal tract were checked every 20 days. It showed that whatever the toxin was separated or combined, it could depress the performance of the fish. And this injury is more serious with the higher concentration of the toxin. This toxic effect showed the significant dose-effect relationship.
TestⅢTo Optimize the Isolation and Cultivation of cyprinus carpio primary hepatocytes
In this study, the cyprinus carpio hepatocytes were isolated by mechanical separation, two-step collagenase perfusion and pancreatin digestion, respectively. The hepatocytes or parenchymal cells could be separated from cell debris and from non-parenchymal cells by low-speed centrifugation (Percoll grade centrifugation). The harvesting hepatocytes were suspended in DMEM, M199(cultured in 5%CO2) and L-15 (cultured without 5%CO2) medium, then cultured at 17℃,27℃and 37℃, respectively. The cell yield was counted by hemocytometer, and the viability of the cells were assessed by Trypan blue exclusion test. Results from these studies showed that the best isolation method was pancreatin digestion [the cell yield was 2.7×108 per g(liver weight) and the viability was 98.4%] and the best medium was M199(cultured in 5%CO2) or L-15(cultured without 5%CO2), respectively. The optimal culture temperature was 27℃. The primary hepatocytes culture of cyprimus carpio grew well and satisfied the requirement of most of toxicological experiments in this condition.
Test IV Toxic effect of AFB1 and DON to cyprinus carpio primary culture hepatocytes
Isolation and culture the hepatocytes base on the test III,design the experiment group as AFB1(0.01μg mL-1 and 0.02μg mL-1),DON(125ng mL-1、250 ng mL-1、500 ng mL-1、1000 ngmL-1and2000ngmL-1), conbination group(0.01+125、0.01+250、0.01+500、0.02+125、0.02+250 and 0.02+500,μg mL-1, ng mL-1), reagent controI(DMSO:2μL mL-1)and the control. Check the cell shape, enzyme activity and the cytotoxicity every 2 hours to study the toxic effect of the toxin. The experiment lasted for 24 hours. From the experiment it showed that all of the toxin could induce a great quality of cells death. Alsothere was no significant difference between the experimental groups, but the difference between each experimental groups and the control is significant (P<0.05).We could saw the inequality apoptosis cells under the TEM in experimental groups contrast to the control. And the results of enzyme activity and the cytotoxicity support the viewpoint.
Test V The DNA injury of cyprinus carpio primary cultured hepatocytes by AFB1 and DON
We have used SCGE and DNA ladder to detect the cell apoptosis induced by the toxin. When chose the SCGE to do the research, we found that the group of the highest rate of the apoptosis is the combination group[AFB1+DON:0.02 (μg mL-1)+125 (ng mL-1)], the second is DON 500ng mL-1.and when we choose the DNA ladder to do the research, it showed that DON(≤500ng mL-1)and the low concentration of combination [AFB,+DON:0.01(μg mL-1)+125(ng mL-1)和AFB1+DON:0.02(μg mL-1)+125(ng mL-1)] could induce significant the hepatocyte apoptosis ly, and showed the obvious "ladder" on 2.5% agarose gel electrophoresis.
引文
Beardall J M, Miller J D. Diseases in humans with mycotoxins as possible causes. Mycotoxins in Grains: Compounds Other than Aflatoxins[M]. St Paul, Minnesota: Eagan Press,1994:487-539
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