硫化汞的生物效應及毒理機制的研究

THE STUDY OF BIOLOGICAL EFFECTS AND TOXIC MECHANISMS OF MERCURIC SULFIDE

巖正傑(Cheng-Chieh Yen)

指導教授 ： 蕭水銀

國立臺灣大學/醫學院/毒理學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.02418

摘要

汞是目前環境中污染較嚴重的重金屬之一，它以元素汞、無機汞及有機汞三種型式在環境中循環。雖然相關文獻針對於汞金屬化合物的毒性作用探討相當多，但對中醫用藥---硃砂之主成份，亦即硫化汞 (HgS)的毒性研究卻不多，根據記載硃砂又名辰砂，在神農本草經中列為上品，具有“清心鎮靜，安神解毒”的功能，隨著硃砂應用歷史的演變，《本草綱目》記述“入火則熱而有毒能殺人”即因為硫化汞加熱分解，使汞游離出來，而《本草從新》更有”獨用多用，令人呆悶”的敘述。近年來在大陸有臨床報告指出誤用硃砂的毒性反應，而臺灣亦有小兒八寶散的重金屬污染問題。因此，釐清硫化汞化合物藥效與毒性間相關性及其重要之毒性效應生物指標的建立是相當重要的。本研究將從硫化汞在生物組織及細胞樣品之分析技術建立與驗證、細胞毒性機制之探討與比較以及螯合劑在其毒性表現之影響等方向逐步架構出硫化汞之生物效應與毒理機制。
首先，生物樣品中的汞含量可以藉由流注分析系統結合冷蒸氣原子吸收光譜儀的技術加以定量。然而，在石英管內的水氣凝結卻會干擾訊號進而影響分析結果。本部份的研究主要探討在流注分析系統中多加一組氣－液分離器是否能有效處理上述汞分析之問題及其實用性。經過修改後之系統可以有效地降低管路及石英管內的水氣干擾問題。同時，將此技術應用於分析汞在經過5天連續餵食2 mg MeHg/KgBW甲基汞或0.1 g HgS/Kg BW 之硫化汞的鼷鼠體內主要器官的汞含量及分佈。硫化汞在器官內的累積程度約為甲基汞的五千分之ㄧ。此外，鼷鼠在經過28天連續餵食0.2mg 或 2 mg/ kg BW之甲基汞以及0.1 g 或1 g/ kg BW之硫化汞之後，結果顯示硫化汞在器官內的累積程度約為甲基汞的七千分之ㄧ(2 mg MeHg vs. 0.1 g HgS)到三十萬分之ㄧ(0.2mg MeHg vs. 0.1 g HgS; 2 mg MeHg vs. 1 g HgS)。證實非水溶性的硫化汞在腸胃道可以被微量吸收並分佈到其他的器官(包括大腦、小腦、肝、腎、肺、心、睪丸、胃等)。此外，經餵食0.2mg / kg BW之甲基汞以及0.1 g / kg BW之硫化汞的鼷鼠，收集並分析由糞便排出之汞含量分別佔其總餵食量之57.9% 及61.0%。
甲基汞及氯化汞對細胞毒性已有相當深入的研究，但是硫化汞的藥物動力學及毒性效應卻仍需更進一步的探討。在之前的研究發現硫化汞可以由動物的腸胃道吸收，並分佈到各主要器官包括肺。本部份的研究首先發現硫化汞對動物之生物效應與能降低鼷鼠肺、肝、腎組織之 GSH含量及增加肺部脂質過氧化產物、抑制肺、肝、腎組織之SOD酵素活性及增加氧化性物質(as H2O2)而刺激腎組織之catalase 酵素活性有關。進一步以倉鼠肺部纖維母細胞(V79 cell)為標的與甲基汞及氯化汞做比較。實驗數據顯示，硫化汞對V79 細胞存活率的IC 50濃度為795.6 µM明顯高於氯化汞(8.1µM)及甲基汞(5.9µM)。此外，硫化汞亦會造成細胞產生DNA傷害，增加氧化性物質及脂質過氧化物、降低粒線體膜電位、ATP及GSH含量，和氯化汞或甲基汞之作用類似，但毒性較緩和。
比較細胞外汞離子濃度較接近之1200µM硫化汞、10µM氯化汞及10µM甲基汞的細胞毒理作用機制。數據顯示，1200µMHgS在細胞暴露8-24小時產生time-dependent之毒性效應，而細胞內的氧化性物質含量在細胞暴露0.5小時就產生，顯著增加持續到暴露2小時，比10µM氯化汞或甲基汞更為顯著，而氧化性物質之產生亦比粒線體膜電位之下降更早發生，顯示是氧化性傷害導致粒線體之損傷。而更進一步探討GSH生合成之重要酵素γ-GCS之活性顯示三種汞化合物皆會抑制γ-GCS之活性，而其酵素活性IC 50之濃度分別為1650µM(HgS)，8.5µM(HgCl2)及5.1µM(MeHg)。此外，低濃度的汞化合物暴露導致細胞產生凋亡，但高濃度的氯化汞及硫化汞及會產生細胞壞死，而高濃度的甲基汞則是同時增加細胞凋亡及壞死。
PDTC(Pyrrolidine dithiocarbamate是 dithiocarbamate的穩定衍生物，具有抗氧化及金屬螯合劑之功用。因汞離子對硫氫基具有高度親和力，推論PDTC可能會與硫化汞產生交互作用，而增加游離汞離子的含量，並且以PDTC-Hg的形式進入細胞。為驗證此一假說，選用倉鼠肺部纖維母細胞(V79 cell)探討PDTC對硫化汞的吸收與細胞毒性之影響。實驗數據顯示，同時暴露5µM PDTC及硫化汞會明顯增加細胞內的汞含量達3.4-10.7倍，而細胞毒性亦較硫化汞單獨暴露增加8倍。細胞內氧化性物質明顯增加，降低粒線體膜電位及GSH含量，同時導致細胞凋亡。
實驗顯示，非水溶性的硫化汞在腸胃道可以被微量吸收並分佈到其他的器官，經過5及28天餵食實驗，其在器官內的累積程度約為甲基汞的五千分之ㄧ(2 mg MeHg vs. 0.1 g HgS；5 days)到約三十萬分之ㄧ(0.2mg MeHg vs. 0.1 g HgS; 2 mg MeHg vs. 1 g HgS；28 days)。在細胞毒性部份，硫化汞和氯化汞及甲基汞皆會藉由增加細胞內氧化性壓力導致粒線體膜電位下降，抑制γ-GCS活性並降低GSH含量而產生細胞毒性，但其作用機制略有不同，硫化汞造成較明顯之活性氧化物質，但對GSH之生合成抑制作用較小，減輕其傷害。此外，金屬螯合劑(PDTC)能增加硫化汞被細胞吸收而增加毒性。
綜合實驗結果證明硫化汞之生物毒性與累積性較甲基汞及氯化汞為低，而更進一步探討其訊息調控機制之差異與活性氧化物質在其生物效應所扮演之角色，將是未來重要之課題。

關鍵字

硫化汞 ； 器官分布 ； 細胞毒性 ； 螯合劑 ； 冷蒸氣原子吸收光譜

並列摘要

Althought the toxic effects of mercuric chloride (HgCl2) and methylmercury chloride (MeHg) have been extensively studied, the insoluble HgS (the main constituent of a Chinese mineral drug, cinnabar, used as a sedative) was subjected fewer studies, and it has not been given the attention it needs.
Mercury contents in biological samples can be measured by cold vapor atomic absorption spectroscopy (CVAAS) combined with the flow injection analysis system (FIAS). However, water vapor in the absorption cell attenuated and distorted the signals. This study described the strategy to overcome this problem by adding an additional gas/liquid separator after the mixing/separator assembly. This modification can efficiently minimize the moisture in the transfer line and in the absorption cell. This improved technique was adopted to study the differential tissue distribution of MeHg and HgS after oral administration to the mice for 5 or 28 consecutive days. The present study suggests that the insoluble HgS can still be absorbed from gastrointestinal tract and distributed to various tissues including the brain. As compared with MeHg, the total amount of HgS accumulated in the tissues ranging about one five thousandth of MeHg (2 mg MeHg vs. 0.1 g HgS；5 days) or about 0.3 million times of that of HgS (0.2mg MeHg vs. 0.1 g HgS; 2 mg MeHg vs. 1 g HgS；28 days). In addition, the total Hg excreted by the feces was 34.99 mg (0.1g Hg) and 0.13 mg (0.2 mg MeHg), which were about 57.9% (0.1g Hg) and 61.0% (0.2 mg MeHg) of total administration of Hg in the mice, respectively.
Although the cytotoxic effects of mercuric chloride (HgCl2) and methylmercury chloride (MeHg) have been extensively studied, the insoluble HgS was subjected fewer studies. Because the traditional Chinese mineral drug, cinnabar (containing >95% HgS) has been and still currently used as an indispensable ingredient of sedative for infants, the pharmacological and toxicological effects of HgS need to be elucidated. In our previous experiments, HgS and cinnabar can still be absorbed from G-I tract and distributed in various tissues including lung. Therefore, in this experiment, a preliminarily examination of whether HgS could exert any oxidative stress in the mouse lung. Indeed, HgS reduced GSH contents and increased lipid peroxidation in the lung. Further studies on the cytotoxic effects and the possible action mechanisms of HgS were compared with those of HgCl2 and MeHg in the cultured lung fibroblast V79 cells. The results showed that HgS produced cytotoxic effects in a concentration (400-1200 μM) dependent manner with IC50 of 795.6 μM, as compared with those of HgCl2 and MeHg, 8.1 μM and 5.9 μM, respectively. In addition, the events of DNA fragmentation, increasing reactive oxygen species and decreasing mitochondrial membrane potential followed by decreased levels of intracellular ATP and GSH as well as increasing lipid peroxidation levels, were induced by HgS similar to those induced by HgCl2 and MeHg but with different toxicokinetic properties.
In our previous experiments, we have found that HgS and cinnabar can still be absorbed from G-I tract and produce ototoxicity. In addition, we have also demonstrated that HgS can be distributed in various tissues including lung. Therefore, in this experiment, we have examined on the cytotoxic mechanisms of HgS which were compared with those of HgCl2 and MeHg in the cultured lung fibroblast V79 cells. The results obtained showed that 1200 μM HgS produced cytotoxic effects in a time (8-24hr) dependent manner. Analysis of the time course (0.5~2 hr) of cellular oxidative stress produced by the mercurial compounds, we found that a sequential events of increasing reactive oxygen species and 1200 μM HgS was more potential than 10 μM HgCl2 and MeHg. In addition, the adverse effect produced by HgS on the activity of γ-GCS with IC50 of 1650 μM, which was higher than those of HgCl2 (8.5 μM) and MeHg (5.1 μM), respectively. Furthermore, V79 cells died by the apoptotic pathway after exposure for 24 hr of lower concentration mercurial compounds.
Pyrrolidine dithiocarbamate (PDTC), a stable analog of dithiocarbamate(DC), possesses antioxidant and metal chelating properties. Based on its high affinity toward thiol-containing group of Hg ions, we proposed that PDTC may chelate HgS to form a complex and thus enhanced the production of free Hg ion in cell culture medium and then transported into cells as a complex form of PDTC-Hg. In order to verify this proposal, we attempted in this study to investigate the effects of PDTC on the uptake and cytotoxic effects of HgS in the cultured lung fibroblast V79 cells. The results indicated that mercury contents (6.29×10-2 -0.41µg/mg protein) of V79 cells after treatment with HgS plus 5µM PDTC were significantly higher by 3.4-10.7 fold than those(5.87×10-3 -0.12µg/mg protein) treated with HgS alone. The signaling pathways of the enhanced cytotoxic effects (by 8 fold) induced by PDTC-Hg complex is compared with HgS alone were studied which suggested that PDTC-Hg complex markedly produced reactive oxygen species (ROS) followed by decreased intracellular contents of GSH, ATP and induction of cell necrosis. These findings provide for the first time the cellular mechanisms of enhanced cytotoxic effects of PDTC-Hg complex. The impact of this information on the dramatic enhancement of the frequently used PDTC as on agricultural pesticide on the biological toxicity of the precipitated waste HgS in the environment merits for further investigation.
Our data suggesting the HgS similar to the well known HgCl2 and MeHg induced cytotoxic effects of cultured V79 cells major through evaluated oxidative stress, γ-GCS inhibition, GSH depletion and disruption of mitochondrial membrane potential, but with a different toxicokinetic property. In addition, insoluble HgS (the main constituent of a Chinese mineral drug, cinnabar) can still be absorbed from gastrointestinal tract and distributed to various tissues. These findings provide for the useful information for understanding the mechanisms of toxic effects of HgS and cinnabar used in Chinese traditional mineral medicine.

並列關鍵字

mercury ； tissue distribution ； V79 cells ； cytotoxicity ； pyrrolidine dithiocarbamate ； cold-vapor atomic absorption spectroscopy

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