Regul Toxicol Pharmacol. 2016 Jul;78:1-7.

The Tibetan medicine Zuotai differs from HgCl2 and MeHg in producing liver injury in mice.

Wu Q1, Li WK1, Zhou ZP2, Li YY1, Xiong TW1, Du YZ3, Wei LX3, Liu J1,4.

  • 1Key Laboratory for Basic Pharmacology of Ministry of Health, Zunyi Medical College, China.
  • 2Medical Research Center, Zunyi Medical College, Zunyi, China.
  • 3Northwest Plateau Institute of Biology of Chinese Academy of Sciences, Xining, China.
  • 4Electronic address: JieLiu@zmc.edu.cn.

Abstract

Zuotai is composed mainly of β-HgS, while cinnabar mainly contains α-HgS. Both forms of HgS are used in traditional medicines and their safety is of concern. This study aimed to compare the hepatotoxicity potential of Zuotai and α-HgS with mercury chloride (HgCl2) and methylmercury (MeHg) in mice. Mice were orally administrated with Zuotai (30 mg/kg), α-HgS (HgS, 30 mg/kg), HgCl2 (33.6 mg/kg), or CH3HgCl (3.1 mg/kg) for 7 days, and liver injury and gene expressions related to toxicity, inflammation and Nrf2 were examined. Animal body weights were decreased by HgCl2 and to a less extent by MeHg. HgCl2 and MeHg produced spotted hepatocyte swelling and inflammation, while such lesions are mild in Zuotai and HgS-treated mice. Liver Hg contents reached 45-70 ng/mg in HgCl2 and MeHg groups; but only 1-2 ng/mg in Zuotai and HgS groups. HgCl2 and MeHg increased the expression of liver injury biomarker genes metallothionein-1 (MT-1) and heme oxygenase-1 (HO-1); the inflammation biomarkers early growth response gene (Egr1), glutathione S-transferase (Gst-mu), chemokine (mKC) and microphage inflammatory protein (MIP-2), while these changes were insignificant in Zuotai and HgS groups. However, all mercury compounds were able to increase the Nrf2 pathway genes

PMID: 27032305

 

Supplement:

Minerals have been used in traditional medicines since ancient times, and are still in use today. In Ayurveda, 8% of the recipes contain 15 kinds of minerals (metals) altogether; in traditional Chinese medicines, minerals are a kind of ingredients used to assist the presumed therapeutic effects.  For example, in the 2015 edition of Pharmacopeia of China (Vol 1), 1493 traditional recipes were listed, and approximately 7% of the recipes contain cinnabar (Hg) and/or realgar (As).  In Siddha Medicine, nearly half of the preparations contain inorganic preparations, including cinnabar and realgar.  Among these minerals, cinnabar (96% α-HgS) and Zuotai, 54% β-HgS) received increasing public concerns of their safety.

 

Mercury sulfide-based traditional medicines are of concern because Hg is Class 1 element of human toxicity that has limited or no use in the manufacture of pharmaceuticals.  In Pharmacopeia of China, the limit of Hg is 1mg/kg for crude herbal materials. However, in cinnabar purposely added traditional medicines, Hg contents are usually much higher over the limit.

 

Zuotai (gTso thal), a mixture of metal ash, mainly contains β-HgS (54%), and other trace elements.  Scanning electron microscopy (SEM) and atomic force microscopy (AFM) observations suggest that Zuotai is a kind of ancient nanodrug (1). Zuotai is included in many Tibetan medicines, like cinnabar (HgS) is included in hundreds of Chinese medicines, in an attempt to produce the assumed therapeutic effects, or to assist known therapeutics in the treatment of various diseases.

 

To assess the toxicity potential of Zuotai and HgS, mice were orally given Zuotai at 4.5-fold of clinical dose (30 mg/kg, po) and HgS (30 mg/kg, po) for consecutive 7 days, and equivalent HgS dose of HgCl2 (33.6 mg/kg, po) and 1/10 equivalent HgS dose of MeHg (3.1 mg/kg, po) were used for comparison (Figure 1).  Hg accumulation and toxicity in the liver (2) and kidneys (3) were examined.  Mercury accumulation in liver and kidney was increased 100- and 500-fold, respectively, after HgCl2 and MeHg, while only 1-2 fold after Zuotai and HgS.  More severe pathological lesions were observed by H&E and electromicroscopy in HgCl2 and MeHg-treated animals, but these lesions were mild or absent after Zuotai and HgS.  Toxicity-sensitive gene expressions in the liver (MT-1 and Ho-1) and in the kidneys (Kim-1 and Ngal) were dramatically increased by HgCl2 and MeHg, but not by Zuotai and HgS.  In addition, stress protein gene expressions in the liver and the uptake and efflux transporter in the kidney were also altered by HgCl2 and MeHg, but are not or minimally affected by Zuotai and HgS (2-3).

 

Figure 1. A graphic illustration of experimental approach to examine Zuotai and HgS effects on liver and kidney toxicity.

 

Liver is the major organ of drug metabolism. Metal-drug interactions involve the phase-1 and phase-2 drug metabolizing enzymes as well as transporters (phase-3). We further examined the potential Hg-drug interactions by examining the expression of genes encoding liver phase-1 (cytochrome P450), phase-2 (conjugation reaction), and phase-3 (uptake and efflux transporters). Although mercurials at the dose and duration used in the study did not have significant effects on the expression of cytochrome P450 1-4 family genes except for a slight increase in PPARα and Cyp4a10 by HgCl2. The expressions of UDP-glucuronosyltransferase and sulfotransferase were increased by HgCl2 and MeHg, but not by Zuotai and HgS. HgCl2 decreased the expression of organic anion transporter (Oatp1a1), but increased Oatp1a4. Both HgCl2 and MeHg increased the expression of multidrug resistance-associated protein genes (Mrp1, Mrp2, Mrp3, and Mrp4). Zuotai and HgS had little effects on these transporter genes (4). Thus, Zuotai and HgS differ from HgCl2 and MeHg in affecting hepatic drug-processing gene expressions.

 

Exposure of Medaka fish (Oryzias latipes) embryos to MeHg, HgCl2, HgS, and Zuotai from stage 10 (6-7 hpf) to 10 days post fertilization (dpf) showed that  MeHg is the most toxic, followed by HgCl2, both producing embryo developmental toxicity and induced stress gene MT-1 and Ho-1, while Zuotai and HgS did not show overt toxicity (5).

 

Thus, chemical forms of mercury compounds is a major determinant of Zuotai and HgS  disposition and toxicity, quite different from that observed for the environmental HgCl2 and MeHg.  Thus, the use of total Hg content for risk assessment of HgS-based traditional medicines seems to be insufficient and inappropriate.

 

Figure 2 is a graphic summary indicating that Zuotai and HgS used in traditional Tibetan medicines and Chinese medicines, their current applications and the future research needs to determine the potential drug-drug interactions, the pharmacological basis, and risk assessments to balance their benefits and risks.

 

Figure 2. An illustration of HgS-based preparations and the research needs.

 

Future perspectives

Chemical forms of mecury are an important consideration. The well-known environmental mercury (HgCl2, MeHg) are not used in traditional medicines, as they do not have known medicinal values but are highly toxic.  In the traditional medicines, minerals are subjected to processing procedures and in sulfide forms suitable for medication.   To characterize the final herbo-metallic products by advanced technology is essential for understanding the physiochemical basis of their disposition in the body.

 

Clinical efficacy and toxicity monitoring.  The values of traditional medicine reside in the treatment of refractory diseases, difficult and complicated diseases such as cancer, stroke, diabetes, etc.  In this regard, the clinical efficacy and safety is of utmost important.

 

Toxicity of herbo-metallic mixtures  HgS is not used alone, rather as mixtures with herbs, animal-based products, and/or other minerals.  The mixtures, rather than individual metals, should be considered to define the biological effects and risks.

 

Pharmacology of herbo-metallic preparations.  The pharmacological basis of including minerals in traditional medicines remains elusive.  In this regard, “Herbogenomic” approaches could be used to profile biological responses, as “program the liver” at appropriate doses could differentiate the beneficial effects from toxic outcomes.

 

Benefits and risks often go hand by hand.  To cope with the refractory medical conditions such as brain emergency, the efficacy of cinnabar-containing traditional medicines overweighs its toxicity.  Any medications given at the high dose and long-term, toxicity is inevitable.  To balance the benefits and risks is important.

 

References

  1. Li, C., Yang, H., Du, Y., Xiao, Y., Zhandui, Sanglao, Wang, Z., Ladan, D., Bi, H., Wei, L., 2016. Chemical Species, Micromorphology, and XRD Fingerprint Analysis of Tibetan Medicine Zuotai Containing Mercury. Bioinorg Chem Appl. 2016:7010519.
  2. Wu, Q., Li, W.K, Zhou, Z. P., Li, Y.Y., Xiong, T.W., Du, Y.Z., Wei L.X., Liu, J., 2016. The Tibetan medicine Zuotai differs from HgCl2 and MeHg in producing liver injury in mice. Regul. Toxicol. Pharmacol. 78, 1-7
  3. Liu, J., Lu, Y.F., Li, W.K., Zhou, Z.P., Li, Y.Y., Yang, X., Li, C., Du, Y.Z., Wei, L.X., 2016. Mercury sulfides are much less nephrotoxic than mercury chloride and methylmercury in mice. Toxicol. Lett.  262, 253-160.
  4. Dong, W., Liu, J., Wei, L., Jingfeng, Y, Chernick, M., Hinton, D.E., 2016. Developmental toxicity from exposure to various forms of mercury compounds in medaka fish (Oryzias latipes) embryos. PeerJ. 2016 Aug 23;4:e2282.
  5. Xu, S.F., Wu, Q., Zhang, B.B., Li, H., Xu, Y.S., Du, Y.Z., Wei, L.X., Liu, J., 2016. Comparison of mercury sulfides with mercury chloride and methylmercury on hepatic P450, Phase-2 and transporter gene expression in livers of mice. J. Trace Elem. Med. Biol. 37, 37-43.

 

Acknowledgements:  This work was supported by National Key Technology R&D Program in the 12th Five Year Plan of China (2012BAI27B05); National Natural Science Foundation (81374063, 81460632); and Guizhou Science and Technology (2011-7020).

 

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