三氧化二砷雌激素样效应及生殖发育毒性的研究
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摘要
环境砷暴露对机体的健康损害是全身性、多系统的,可以引起皮肤损害、心脑血管疾病、神经系统效应及生殖毒性效应,同时也是已被肯定的人类致癌源。然而其确切的致病机制一直没有共识。最近的研究提出砷化物可能是一类环境雌激素样物质,然而砷是否具有雌激素样效应,砷的健康损伤是否与其雌激素样效应有关等问题尚需进一步探讨。本研究着重从细胞培养及动物实验两个方面探讨砷的雌激素样效应及雌激素效应在生殖发育毒性中的可能机制。
1、三氧化二砷雌激素样效应及对Hela细胞生长的影响
通过在培养基中分别添加雌二醇(1nM)、三氧化二砷(0.5μM、1.0μM、5.0μM)、雌二醇拮抗剂(500nM)、雌二醇(1nM)与雌二醇拮抗剂(500nM)、三氧化二砷(1.0μM)与雌二醇拮抗剂(500nM),采用形态学观察细胞生长状况、MTT法检测细胞存活率的变化及流式细胞计量术检测细胞生长周期等技术手段研究砷的雌激素效应及对细胞生长的影响。结果显示0.5μM三氧化二砷与1nM雌二醇的作用效果相似,促进细胞增殖,促使细胞进入DNA合成期(S期),且该作用可被雌二醇拮抗剂所拮抗。1.0μM三氧化二砷则使细胞生长由促进转为抑制,其它各组对Hela细胞的生长均呈现抑制作用。因此认为小剂量三氧化二砷具有雌激素样效应,1.0μM As_2O_3是对Hela细胞生长影响的分界点。
2、三氧化二砷对Hela细胞ERα及PRmRNA表达的影响
通过在培养基中分别添加雌二醇(1nM)、三氧化二砷(0.5μM、1.0μM、5.0μM)、雌二醇拮抗剂(500nM)、雌二醇(1nM)与雌二醇拮抗剂(500nM)、三氧化二砷(1.0μM)与雌二醇拮抗剂(500nM),采用RT-PCR法检测细胞经各种处理后ERα及PRmRNA的表达。结果显示1nM雌二醇和0.5μM三氧化二砷对Hela细胞ERα及PRmRNA表达的影响相似,且其作用都可被雌二醇拮抗剂所拮抗,具体表现为Hela细胞经小剂量(0.5μM)三氧化二砷作用后,其ERαmRNA表达降低,PRmRNA表达升高,进一步证实了三氧化二砷具有雌激素样效应。
3、慢性砷暴露对小鼠动情周期的影响
采用宫颈黏液结晶法对不同浓度慢性砷暴露小鼠动情周期进行检查,结果显示不同浓度的三氧化二砷均使小鼠动情周期发生紊乱,但与对照组无显著差异,也无明显规律(或缩短、或延长),尤其是动情期。动情周期的紊乱是反应性激素代谢失调的敏感指标,因此本研究初步证明砷具有影响机体内分泌的作用,当属环境内分泌干扰物。
4、慢性砷暴露对雌性小鼠生殖器官ERα、PRmRNA表达的影响
采用等离子质谱方法检测了慢性砷暴露雌鼠血砷的变化并通过RT-PCR的方法检测了慢性砷暴露雌鼠生殖器官子宫、卵巢ERα及PRmRNA的表达情况。结果显示:①随着血砷浓度增加,ERαmRNA表达总体呈下降趋势。砷暴露各组子宫和卵巢的ERαmRNA表达显著降低,并与对照组之间差异显著(P＜0.05)。②砷暴露小鼠子宫的PRmRNA表达总体也呈下降趋势,砷暴露浓度为0.1-0.2μg/ml时PRmRNA表达水平最低,且此两组PRmRNA表达与对照组比较差异显著(P＜0.05),卵巢中PRmRNA表达虽有降低但与对照组无显著差异(P＞0.05)。
5、慢性砷暴露对雄性小鼠生殖器官ERα、PRmRNA表达的影响
采用等离子质谱方法检测了慢性砷暴露雄鼠血砷的变化,并通过RT-PCR的方法检测了慢性砷暴露雄鼠生殖器官睾丸ERα及PRmRNA的表达情况。结果显示:①砷暴露各组小鼠睾丸ERαmRNA表达水平高于对照组,且随着血砷浓度增加ERαmRNA表达增加,但在高浓度时ERαmRNA表达又有所下降,但总体变化差异不显著(P＞0.05)。②砷暴露各组睾丸PRmRNA与对照组比较表达增强,但总体变化同样差异不显著(P＞0.05)。
6、慢性砷暴露对怀孕雌鼠生殖器官及胚胎ERα、PRmRNA表达的影响
采用等离子质谱方法检测了慢性砷暴露孕鼠血砷的变化,并通过RT-PCR的方法检测了慢性砷暴露孕鼠生殖器官及胚胎ERα、PRmRNA的表达情况。结果显示:①孕鼠子宫ERαmRNA表达随砷暴露浓度增高而增高,PRmRNA表达随砷暴露浓度增高反而降低;②孕鼠卵巢随着血砷浓度升高,ERαmRNA表达降低,PRmRNA表达增强。③小鼠胚胎ERαmRNA表达随着血砷浓度升高而增强,与对照组比较,差异显著(P＜0.05),PRmRNA的表达水平也高于对照组,但无显著差异(P＞0.05)。
综合上述六个方面的实验研究,从而可知砷(As_2O_3)具有雌激素样效应,且该效应具有一定的剂量反应;而其生殖发育毒性与雌激素样效应有关,慢性砷暴露可以使雌性小鼠生殖器官子宫、卵巢ERαmRNA表达显著下降,PRmRNA表达总体也呈下降趋势,使怀孕小鼠子宫ERαmRNA表达显著增高,PRmRNA表达减低。实验结果同时表明慢性砷暴露对小鼠卵巢功能的影响不是主要的,指示慢性砷暴露造成的早期生殖毒性主要是由于子宫功能状态改变引起的。慢性砷暴露对雄鼠睾丸的影响也有待于进一步研究。
The health risks of body exposured to environmental As is systemic and multi-system.As exposure has been link to skin damages,cardiovascular diseases, nervous system and reproductive and developmental problems.Also As has been considered a definitely human carcinogen.However,the exact mechanism has not been clear.Recently some studies suggested that As has estrogen-like effects and it is a candidate environmental endocrine disruptor(EEDs).To determine whether or not As has estrogen-like effects and if the estrogen-like effects is related to the health problems,The following studies were conducted.These studies focused on As estrogen-like effects and influence on reproductive and developmental toxicity from the cell culture and animal experiments.
1、Study on estrogen-like effects and influence on the Hela cell growth of As_2O_3
The Hela cells were cultured in improved RPMI-1640 supplemented respectively with E_2(lnM),Arsenic trioxide(0.5μM,1μM,5μM),ICI(500nM),E_2 (1nM) and ICI(500nM),As_2O_3(1μM) and ICI(500nM),respectively.The morphology was used to observe the situation of cell growth.The method of MTT was used to study the cell proliferation.The technique of flow cytometry was used to measure Hela DNA content.The results showed that the effects of 0.5μM As_2O_3 and 1nM E_2 was similar to the cell proliferation,it promote cell to enter the DNA synthesis phase(S),and the effect could be antagonized by ICI.As_2O_3 in 1.0μM affected the cell growth from proliferation to inhibition,Hela cells showed apoptosis in other groups.From the results it is suggested that As_2O_3 has estrogen-like effects and As_2O_3 in 1.0μM is a turning point for affected the growth of Hela cells.
2、Study on ERα,PR mRNA expression of Hela cells exposured to As_2O_3
The Hela cells were cultured in improved RPMI-1640 supplemented respectively with E_2(lnM),Arsenic trioxide(0.5μM,1μM,5μM),ICI(500nM),E_2 (1nM) and ICI(500nM),As_2O_3(1μM) and ICI(500nM).RT-PCR was used to detect the ERαand PRmRNA expression.The results showed that 1nM E_2 and 0.5μM As_2O_3 was similar to ER and PRmRNA expression of Hela cells,the effects could be antagonized by ICI.It demonstrated that ERαmRNA expression reduced and PRmRNA expression increased after Hela cells exposed to 0.5μtM As_2O_3,it further indicated that As_2O_3 has estrogen-like effects.
3、Study on estrous cycle of mice exposed to chronic arsenic
The method of cervical mucus crystallization was used to examine estrous cycle of mice exposed to chronic different As_2O_3 drinking water.The results showed that the estrous cycle of mice was disorder,but there was no significant differences compared with control group,also the cycle was not regular(shortening or extension),particularly in estrus.The estrous cycle disorder is a sensitive marker responsed to the sex hormone metabolic disorders.This study proved that As_2O_3 has impact on body's endocrine,so it should be environmental endocrine disruptors.
4、Study on genital uterus,ovary ERαand PR mRNA expression of female mice exposed to chronic arsenic
ICP-MS wad used to analysis blood arsenic and RT-PCR was used to detect genital uterus,ovary ERαand PRmRNA expression of female mice exposed to chronic different As_2O_3 drinking water.The results showed that:①The overall trend of ERαmRNA expression decreased with the blood As increasing.Uterus and ovary ERαexpression significantly lower in mice exposed to different As drinking water, the difference was significant(P<0.05) compared with control group.②In general, the trend of PRmRNA expression in uterus declined,PRmRNA expression in uterus was the lowest in mice exposed to 0.1-0.2μg/ml arsenic drinking water,and the difference of PRmRNA expression in these two groups was significant(P<0.05) compared with control group.Although PRmRNA expression in ovary was declined too but there was no significant difference(P>0.05) compared with control group.
5、Study on genital testicular ERαand PRmRNA expression of male mice exposed to chronic arsenic
ICP-MS wad used to analysis blood arsenic and RT-PCR was used to detect genital testicular ERαand PRmRNA expression of male mice exposed to chronic different As_2O_3 drinking water.The results showed:①Testicular ERαmRNA expression was higher of mice exposed to arsenic drinking water than control group. ERαmRNA expression increased with blood arsenic increasing,but the ERαmRNA expression declined in high As.Overall the changes were no significant differences (P>0.05).②Testicular PRmRNA expression increased of mice exposed to different arsenic drinking water,but in gerenal,the changes were not significant difference (P>0.05).
6、Study on genital uterus,ovary and embryo ERαand PR mRNA expression of pregnant mice exposed to chronic arsenic
ICP-MS wad used to analysis blood arsenic and RT-PCR was used to detect genital uterus,ovary and embryo ERαand PRmRNA expression of pregnant mice exposed to chronic different As_2O_3 drinking water.The results showed that:①ERαmRNA expression in pregnant uterus increased with the increased arsenic exposure,PRmRNA expression in pregnant uterus decreased with the increased arsenic exposure;②ERαmRNA expression declined and PRmRNA expression increased in pregnant ovary with the increased arsenic in blood.③ERαmRNA expression increased in embryo with maternal blood As increasing,there was significant differences(P<0.05) compared with control group;PRmRNA expression is also higher than the control group,but there was no significant difference(P>0.05).
From above six experiments,we suggested that As_2O_3 has estrogen-like effects and the effects according to dose-response relationship.The reproductive and developmental toxicity of arsenic is related to its estrogen-like effects.Uterus and ovary ERαmRNA expression significantly lower and PRmRNA expression also declined totally in non-pregnant mice,Uterus ERαmRNA expression increased and PRmRNA expression declined.At the same time,experiments results indicated that it is not major to influence the ovarian function of mice exposed to chronic arsenic, the early reproductive toxicity is mainly due to uterine function conditions exposed to arsenic.The impact of the male testes exposured to arsenic need to be further study.
1、三氧化二砷雌激素样效应及对Hela细胞生长的影响
通过在培养基中分别添加雌二醇(1nM)、三氧化二砷(0.5μM、1.0μM、5.0μM)、雌二醇拮抗剂(500nM)、雌二醇(1nM)与雌二醇拮抗剂(500nM)、三氧化二砷(1.0μM)与雌二醇拮抗剂(500nM),采用形态学观察细胞生长状况、MTT法检测细胞存活率的变化及流式细胞计量术检测细胞生长周期等技术手段研究砷的雌激素效应及对细胞生长的影响。结果显示0.5μM三氧化二砷与1nM雌二醇的作用效果相似,促进细胞增殖,促使细胞进入DNA合成期(S期),且该作用可被雌二醇拮抗剂所拮抗。1.0μM三氧化二砷则使细胞生长由促进转为抑制,其它各组对Hela细胞的生长均呈现抑制作用。因此认为小剂量三氧化二砷具有雌激素样效应,1.0μM As_2O_3是对Hela细胞生长影响的分界点。
2、三氧化二砷对Hela细胞ERα及PRmRNA表达的影响
通过在培养基中分别添加雌二醇(1nM)、三氧化二砷(0.5μM、1.0μM、5.0μM)、雌二醇拮抗剂(500nM)、雌二醇(1nM)与雌二醇拮抗剂(500nM)、三氧化二砷(1.0μM)与雌二醇拮抗剂(500nM),采用RT-PCR法检测细胞经各种处理后ERα及PRmRNA的表达。结果显示1nM雌二醇和0.5μM三氧化二砷对Hela细胞ERα及PRmRNA表达的影响相似,且其作用都可被雌二醇拮抗剂所拮抗,具体表现为Hela细胞经小剂量(0.5μM)三氧化二砷作用后,其ERαmRNA表达降低,PRmRNA表达升高,进一步证实了三氧化二砷具有雌激素样效应。
3、慢性砷暴露对小鼠动情周期的影响
采用宫颈黏液结晶法对不同浓度慢性砷暴露小鼠动情周期进行检查,结果显示不同浓度的三氧化二砷均使小鼠动情周期发生紊乱,但与对照组无显著差异,也无明显规律(或缩短、或延长),尤其是动情期。动情周期的紊乱是反应性激素代谢失调的敏感指标,因此本研究初步证明砷具有影响机体内分泌的作用,当属环境内分泌干扰物。
4、慢性砷暴露对雌性小鼠生殖器官ERα、PRmRNA表达的影响
采用等离子质谱方法检测了慢性砷暴露雌鼠血砷的变化并通过RT-PCR的方法检测了慢性砷暴露雌鼠生殖器官子宫、卵巢ERα及PRmRNA的表达情况。结果显示:①随着血砷浓度增加,ERαmRNA表达总体呈下降趋势。砷暴露各组子宫和卵巢的ERαmRNA表达显著降低,并与对照组之间差异显著(P＜0.05)。②砷暴露小鼠子宫的PRmRNA表达总体也呈下降趋势,砷暴露浓度为0.1-0.2μg/ml时PRmRNA表达水平最低,且此两组PRmRNA表达与对照组比较差异显著(P＜0.05),卵巢中PRmRNA表达虽有降低但与对照组无显著差异(P＞0.05)。
5、慢性砷暴露对雄性小鼠生殖器官ERα、PRmRNA表达的影响
采用等离子质谱方法检测了慢性砷暴露雄鼠血砷的变化,并通过RT-PCR的方法检测了慢性砷暴露雄鼠生殖器官睾丸ERα及PRmRNA的表达情况。结果显示:①砷暴露各组小鼠睾丸ERαmRNA表达水平高于对照组,且随着血砷浓度增加ERαmRNA表达增加,但在高浓度时ERαmRNA表达又有所下降,但总体变化差异不显著(P＞0.05)。②砷暴露各组睾丸PRmRNA与对照组比较表达增强,但总体变化同样差异不显著(P＞0.05)。
6、慢性砷暴露对怀孕雌鼠生殖器官及胚胎ERα、PRmRNA表达的影响
采用等离子质谱方法检测了慢性砷暴露孕鼠血砷的变化,并通过RT-PCR的方法检测了慢性砷暴露孕鼠生殖器官及胚胎ERα、PRmRNA的表达情况。结果显示:①孕鼠子宫ERαmRNA表达随砷暴露浓度增高而增高,PRmRNA表达随砷暴露浓度增高反而降低;②孕鼠卵巢随着血砷浓度升高,ERαmRNA表达降低,PRmRNA表达增强。③小鼠胚胎ERαmRNA表达随着血砷浓度升高而增强,与对照组比较,差异显著(P＜0.05),PRmRNA的表达水平也高于对照组,但无显著差异(P＞0.05)。
综合上述六个方面的实验研究,从而可知砷(As_2O_3)具有雌激素样效应,且该效应具有一定的剂量反应;而其生殖发育毒性与雌激素样效应有关,慢性砷暴露可以使雌性小鼠生殖器官子宫、卵巢ERαmRNA表达显著下降,PRmRNA表达总体也呈下降趋势,使怀孕小鼠子宫ERαmRNA表达显著增高,PRmRNA表达减低。实验结果同时表明慢性砷暴露对小鼠卵巢功能的影响不是主要的,指示慢性砷暴露造成的早期生殖毒性主要是由于子宫功能状态改变引起的。慢性砷暴露对雄鼠睾丸的影响也有待于进一步研究。
The health risks of body exposured to environmental As is systemic and multi-system.As exposure has been link to skin damages,cardiovascular diseases, nervous system and reproductive and developmental problems.Also As has been considered a definitely human carcinogen.However,the exact mechanism has not been clear.Recently some studies suggested that As has estrogen-like effects and it is a candidate environmental endocrine disruptor(EEDs).To determine whether or not As has estrogen-like effects and if the estrogen-like effects is related to the health problems,The following studies were conducted.These studies focused on As estrogen-like effects and influence on reproductive and developmental toxicity from the cell culture and animal experiments.
1、Study on estrogen-like effects and influence on the Hela cell growth of As_2O_3
The Hela cells were cultured in improved RPMI-1640 supplemented respectively with E_2(lnM),Arsenic trioxide(0.5μM,1μM,5μM),ICI(500nM),E_2 (1nM) and ICI(500nM),As_2O_3(1μM) and ICI(500nM),respectively.The morphology was used to observe the situation of cell growth.The method of MTT was used to study the cell proliferation.The technique of flow cytometry was used to measure Hela DNA content.The results showed that the effects of 0.5μM As_2O_3 and 1nM E_2 was similar to the cell proliferation,it promote cell to enter the DNA synthesis phase(S),and the effect could be antagonized by ICI.As_2O_3 in 1.0μM affected the cell growth from proliferation to inhibition,Hela cells showed apoptosis in other groups.From the results it is suggested that As_2O_3 has estrogen-like effects and As_2O_3 in 1.0μM is a turning point for affected the growth of Hela cells.
2、Study on ERα,PR mRNA expression of Hela cells exposured to As_2O_3
The Hela cells were cultured in improved RPMI-1640 supplemented respectively with E_2(lnM),Arsenic trioxide(0.5μM,1μM,5μM),ICI(500nM),E_2 (1nM) and ICI(500nM),As_2O_3(1μM) and ICI(500nM).RT-PCR was used to detect the ERαand PRmRNA expression.The results showed that 1nM E_2 and 0.5μM As_2O_3 was similar to ER and PRmRNA expression of Hela cells,the effects could be antagonized by ICI.It demonstrated that ERαmRNA expression reduced and PRmRNA expression increased after Hela cells exposed to 0.5μtM As_2O_3,it further indicated that As_2O_3 has estrogen-like effects.
3、Study on estrous cycle of mice exposed to chronic arsenic
The method of cervical mucus crystallization was used to examine estrous cycle of mice exposed to chronic different As_2O_3 drinking water.The results showed that the estrous cycle of mice was disorder,but there was no significant differences compared with control group,also the cycle was not regular(shortening or extension),particularly in estrus.The estrous cycle disorder is a sensitive marker responsed to the sex hormone metabolic disorders.This study proved that As_2O_3 has impact on body's endocrine,so it should be environmental endocrine disruptors.
4、Study on genital uterus,ovary ERαand PR mRNA expression of female mice exposed to chronic arsenic
ICP-MS wad used to analysis blood arsenic and RT-PCR was used to detect genital uterus,ovary ERαand PRmRNA expression of female mice exposed to chronic different As_2O_3 drinking water.The results showed that:①The overall trend of ERαmRNA expression decreased with the blood As increasing.Uterus and ovary ERαexpression significantly lower in mice exposed to different As drinking water, the difference was significant(P<0.05) compared with control group.②In general, the trend of PRmRNA expression in uterus declined,PRmRNA expression in uterus was the lowest in mice exposed to 0.1-0.2μg/ml arsenic drinking water,and the difference of PRmRNA expression in these two groups was significant(P<0.05) compared with control group.Although PRmRNA expression in ovary was declined too but there was no significant difference(P>0.05) compared with control group.
5、Study on genital testicular ERαand PRmRNA expression of male mice exposed to chronic arsenic
ICP-MS wad used to analysis blood arsenic and RT-PCR was used to detect genital testicular ERαand PRmRNA expression of male mice exposed to chronic different As_2O_3 drinking water.The results showed:①Testicular ERαmRNA expression was higher of mice exposed to arsenic drinking water than control group. ERαmRNA expression increased with blood arsenic increasing,but the ERαmRNA expression declined in high As.Overall the changes were no significant differences (P>0.05).②Testicular PRmRNA expression increased of mice exposed to different arsenic drinking water,but in gerenal,the changes were not significant difference (P>0.05).
6、Study on genital uterus,ovary and embryo ERαand PR mRNA expression of pregnant mice exposed to chronic arsenic
ICP-MS wad used to analysis blood arsenic and RT-PCR was used to detect genital uterus,ovary and embryo ERαand PRmRNA expression of pregnant mice exposed to chronic different As_2O_3 drinking water.The results showed that:①ERαmRNA expression in pregnant uterus increased with the increased arsenic exposure,PRmRNA expression in pregnant uterus decreased with the increased arsenic exposure;②ERαmRNA expression declined and PRmRNA expression increased in pregnant ovary with the increased arsenic in blood.③ERαmRNA expression increased in embryo with maternal blood As increasing,there was significant differences(P<0.05) compared with control group;PRmRNA expression is also higher than the control group,but there was no significant difference(P>0.05).
From above six experiments,we suggested that As_2O_3 has estrogen-like effects and the effects according to dose-response relationship.The reproductive and developmental toxicity of arsenic is related to its estrogen-like effects.Uterus and ovary ERαmRNA expression significantly lower and PRmRNA expression also declined totally in non-pregnant mice,Uterus ERαmRNA expression increased and PRmRNA expression declined.At the same time,experiments results indicated that it is not major to influence the ovarian function of mice exposed to chronic arsenic, the early reproductive toxicity is mainly due to uterine function conditions exposed to arsenic.The impact of the male testes exposured to arsenic need to be further study.
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