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Background: Zuotai (mainly β-HgS)-containing 70 Wei-Zhen-Zhu-Wan (70W, Rannasangpei ) is a famous Tibetan medicine for treating cardiovascular and gastrointestinal diseases.  We have shown that 70W protected against CCl 4 hepatotoxicity.  CCl 4 is metabolized via cytochrome P450 (CYP) to produce reactive metabolites. Whether 70W has any effect on CYPs is unknown and such effects should be compared with mercury compounds for safety evaluation.   Methods: Mice were given clinical doses of 70W (0.15-1.5 g/kg, po), Zuotai (30 mg/kg, po), and compared to HgCl 2 (33.6 mg/kg, po) and MeHg (3.1 mg/kg, po) for seven days. Liver RNA and protein were isolated for qPCR and Western-blot analysis. Results: 70W and Zuotai had no effects on hepatic mRNA expression of Cyp1a2, Cyp2b10, Cyp3a11, Cyp4a10 and Cyp7a1, and corresponding nuclear receptors [aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor-α (PPARα); farnesoid X receptor (FXR)]. In comparison, HgCl 2 and MeHg increased mRNA expression of Cyp1a2, Cyp2b10, Cyp4a10 and Cyp7a1 except for Cyp3a11, and corresponding nuclear receptors except for PXR. Western-blot confirmed mRNA results, showing increases in CYP1A2, CYP2B1, CYP2E1, CYP4A and CYP7A1 by HgCl 2 and MeHg only, and all treatments had no effects on CYP3A. Conclusions: Zuotai and Zuotai-containing 70W at clinical doses had minimal influence on hepatic CYPs and corresponding nuclear receptors, while HgCl 2 and MeHg produced significant effects.  Thus, the use of total Hg content to evaluate the safety of HgS-containing 70W is inappropriate., Competing Interests: No competing interests were disclosed., (Copyright: © 2021 Nie Y et al.)

Background: Zuotai (mainly β-HgS)-containing 70 Wei-Zhen-Zhu-Wan (70W, Rannasangpei ) is a famous Tibetan medicine for treating cardiovascular and gastrointestinal diseases.  We have shown that 70W protected against CCl 4 hepatotoxicity.  CCl 4 is metabolized via cytochrome P450 (CYP) to produce reactive metabolites. Whether 70W has any effect on CYPs is unknown and such effects should be compared with mercury compounds for safety evaluation.   Methods: Mice were given clinical doses of 70W (0.15-1.5 g/kg, po), Zuotai (30 mg/kg, po), and compared to HgCl 2 (33.6 mg/kg, po) and MeHg (3.1 mg/kg, po) for seven days. Liver RNA and protein were isolated for qPCR and Western-blot analysis. Results: 70W and Zuotai had no effects on hepatic mRNA expression of Cyp1a2, Cyp2b10, Cyp3a11, Cyp4a10 and Cyp7a1, and corresponding nuclear receptors [aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor-α (PPARα); farnesoid X receptor (FXR)]. In comparison, HgCl 2 and MeHg increased mRNA expression of Cyp1a2, Cyp2b10, Cyp4a10 and Cyp7a1 except for Cyp3a11, and corresponding nuclear receptors except for PXR. Western-blot confirmed mRNA results, showing increases in CYP1A2, CYP2B1, CYP2E1, CYP4A and CYP7A1 by HgCl 2 and MeHg only, and all treatments had no effects on CYP3A. Conclusions: Zuotai and Zuotai-containing 70W at clinical doses had minimal influence on hepatic CYPs and corresponding nuclear receptors, while HgCl 2 and MeHg produced significant effects.  Thus, the use of total Hg content to evaluate the safety of HgS-containing 70W is inappropriate., Competing Interests: No competing interests were disclosed., (Copyright: © 2021 Nie Y et al.)

Mercury (Hg) is generally considered as a toxic metal; yet the biological outcomes of Hg-containing compounds are highly dependent upon their chemical forms. We hypothesize that mercury sulfide (HgS) is different from HgCl 2 and methylmercury (MeHg) in producing intestinal Hg absorption and disruption of gut microbiome. To test this hypothesis, mice were given orally with HgS (α-HgS, 30 mg/kg), Zuotai (β-HgS, 30 mg/kg), HgCl 2 (33.6 mg/kg, equivalent Hg as HgS), or MeHg (3.1 mg/kg, 1/10 Hg as HgS) for 7 days. Accumulation of Hg in the duodenum and ileum after HgCl 2 (30-40 fold) and MeHg (10-15 fold) was higher than HgS and Zuotai (~2-fold). HgCl 2 and MeHg decreased intestinal intake peptide transporter-1 and Ost-β, and increased ileal bile acid binding protein and equilibrative nucleoside transporter-1. The efflux transporters ATP-binding cassette sub-family C member-4 (Abcc4), Abcg2, Abcg5/8, and Abcb1b were increased by HgCl 2 and to a lesser extent by MeHg, while HgS and Zuotai had minimal effects. Bacterial DNA was extracted and subjected to 16S rDNA sequencing. Operational taxonomic unit (OTU) results showed that among the 10 phyla, HgS increased Firmicutes, Proteobacteria, while HgCl 2 increased Bacteroidetes, Cyanobacteria and decreased Firmicutes; among the 79 families, HgS increased Rikenellaceae, Lactobacillaceae, Helicobacteraceae, and decreased Prevotellaceae, while HgCl 2 increased Odoribacteraceae, Porphyromonadaceae, and decreased Lactobacillaceae; among the 232 genus/species, HgS and Zuotai affected gut microbiome quite differently from HgCl 2 and MeHg. qPCR analysis with 16S rRNA confirmed sequencing results. Thus, chemical forms of mercury are a major determinant for intestinal Hg accumulation, alterations in transporters and disruption of microbiome., (Copyright © 2019. Published by Elsevier Inc.)

Ethnopharmacological Relevance: Zuotai (gTso thal) has a long history in the treatment of cardiovascular disease, liver and bile diseases, spleen and stomach diseases as a precious adjuvant in Tibetan medicine. However, Zuotai is a mercury preparation that contains 54.5% HgS. Its application has always been controversial., Aim of the Study: To evaluate the toxicological effects of Zuotai in hepatocytes and in zebrafish., Materials and Methods: MTT was used to determine the survival rate of hepatocytes; Hoechst and TUNEL staining were used to detect the apoptosis cells; Western blot and RT-qPCR assay were used to determine the expression levels of the protein and mRNA; Liver morphology observation and H&E staining were used to evaluate the hepatotoxicity of Zuotai in Zebfrafish., Results: The survival rate of L-02 cells, HepG2 cells and RBL-2A cells reduced by Zuotai (10 -4 -0.1 mg/mL) in a dose and time-dependent manner. Zuotai (0.1 mg/mL) induced HepG2 cells shrinkage, condensation and fragmentation and increased the number of apoptosis cells. The protein expression levels of cleaved Caspase-3 and Bax were increased and the expression levels of Bcl-2 were reduced after HepG2 cells exposed to Zuotai (10 -4 -0.1 mg/mL) for 24 h. In addition, Zuotai (0.2 mg/mL) induced the darker liver color of the larval zebrafish and changed the liver morphologic of adult zebrafish. Zuotai (0.2 mg/mL) also increased the mRNA levels of CYP1A1, CYP1B1 and MT-1 in the liver of adult zebrafish. However, no significantly hepatotoxicity was observed after hepatocytes and zebrafish exposed to HgS at the same dose., Conclusions: Results showed that Zuotai induced hepatotoxicity effectively under a certain dose but its hepatotoxicity likely occurs via other mechanisms that did not depend on HgS., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ethnopharmocological Relevance: Herbo-metallic preparations have a long history in the treatment of diseases, and are still used today for refractory diseases, as adjuncts to standard therapy, or for economic reasons in developing countries., Aim of the Review: This review uses cinnabar (HgS) and realgar (As 4 S 4 ) as mineral examples to discuss their occurrence, therapeutic use, pharmacology, toxicity in traditional medicine mixtures, and research perspectives., Materials and Methods: A literature search on cinnabar and realgar from PubMed, Chinese pharmacopeia, Google and other sources was carried out. Traditional medicines containing both cinnabar and realgar (An-Gong-Niu-Huang Wan, Hua-Feng-Dan); mainly cinnabar (Zhu-Sha-An-Shen Wan; Zuotai and Dangzuo), and mainly realgar (Huang-Dai Pian; Liu-Shen Wan; Niu-Huang-Jie-Du) are discussed., Results: Both cinnabar and realgar used in traditional medicines are subjected to special preparation procedures to remove impurities. Metals in these traditional medicines are in the sulfide forms which are different from environmental mercurials (HgCl 2 , MeHg) or arsenicals (NaAsO 2 , NaH 2 AsO 4 ). Cinnabar and/or realgar are seldom used alone, but rather as mixtures with herbs and/or animal products in traditional medicines. Advanced technologies are now used to characterize these preparations. The bioaccessibility, absorption, distribution, metabolism and elimination of these herbo-metallic preparations are different from environmental metals. The rationale of including metals in traditional remedies and their interactions with drugs need to be justified. At higher therapeutic doses, balance of the benefits and risks is critical. Surveillance of patients using these herbo-metallic preparations is desired., Conclusion: Chemical forms of mercury and arsenic are a major determinant of their disposition, efficacy and toxicity, and the use of total Hg and As alone for risk assessment of metals in traditional medicines is insufficient., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Zuotai and cinnabar(96%HgS) are contained in many traditional medicines. To examine their potential effects on drug metabolism genes, mice were orally given Zuotai or HgS at doses of 10, 30, 100, 300 mg•kg⁻¹ for 7 days. HgCl2(33.6 mg•kg⁻¹) was gavaged for control. Twenty-four hour later after the last administration, livers were collected, and expressions of genes related to metabolic enzymes and transporters were examined. Zuotai and HgS had no effects on major phase-1, phase-2 and transporter genes; HgCl2 increased the expressions of CYP2B10, CYP4A10, OATP1A4, UGT1A1, UGT2A3, SULT1A1, SULT2A1, MRP1, MRP3 and MRP4; expression of OATP1A1 was decreased by HgCl2, but not by Zuotai and HgS. Therefore, Zuotai and HgS have different adverse effects on drug-metabolizing genes from HgCl2., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Mercury sulfides are used in Ayurvedic medicines, Tibetan medicines, and Chinese medicines for thousands of years and are still used today. Cinnabar (α-HgS) and metacinnabar (β-HgS) are different from mercury chloride (HgCl 2 ) and methylmercury (MeHg) in their disposition and toxicity. Whether such scenario applies to weanling and aged animals is not known. To address this question, weanling (21d) and aged (450d) rats were orally given Zuotai (54% β-HgS, 30mg/kg), HgS (α-HgS, 30mg/kg), HgCl 2 (34.6mg/kg), or MeHg (MeHgCl, 3.2mg/kg) for 7days. Accumulation of Hg in kidney and liver, and the toxicity-sensitive gene expressions were examined. Animal body weight gain was decreased by HgCl 2 and to a lesser extent by MeHg, but unaltered after Zuotai and HgS. HgCl 2 and MeHg produced dramatic tissue Hg accumulation, increased kidney (kim-1 and Ngal) and liver (Ho-1) injury-sensitive gene expressions, but such changes are absent or mild after Zuotai and HgS. Aged rats were more susceptible than weanling rats to Hg toxicity. To examine roles of transporters in Hg accumulation, transporter gene expressions were examined. The expression of renal uptake transporters Oat1, Oct2, and Oatp4c1 and hepatic Oatp2 was decreased, while the expression of renal efflux transporter Mrp2, Mrp4 and Mdr1b was increased following HgCl 2 and MeHg, but unaffected by Zuotai and HgS. Thus, Zuotai and HgS differ from HgCl 2 and MeHg in producing tissue Hg accumulation and toxicity, and aged rats are more susceptible than weanling rats. Transporter expression could be adaptive means to reduce tissue Hg burden., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Mercury sulfides (α-HgS, β-HgS) are frequently included in traditional medicines. Mercury is known for nephrotoxicity, their safety is of concern. To address this question, mice were orally administrated with Zuotai (54% β-HgS, 30mg/kg), α-HgS (HgS, 30mg/kg), HgCl 2 (33.6mg/kg), or MeHgCl (3.1mg/kg) for 7days, and nephrotoxicity was examined. Animal body weights were decreased by HgCl 2 and to a lesser extent by MeHg, but unaltered after Zuotai and HgS. HgCl 2 and MeHg produced renal tubular vacuolation, interstitial inflammation and cell degeneration with protein cysts in the tubular lumen, while these pathological lesions were mild in Zuotai and HgS-treated mice. Electron microscopy showed that HgCl 2 and MeHg produced spotted swelling endothelium reticulum, while these lesions were mild or absent in Zuotai and HgS-treated mice. Renal Hg contents reached 250-300ng/mg kidney in HgCl 2 and MeHg groups as compared to 2-3ng/mg in Zuotai and HgS groups. The expression of kidney injury biomarkers, kidney injury molecule-1 (Kim-1) and neutrophil gelatinase-associated lipocalin (Ngal), were increased after HgCl 2 and MeHg, but unaltered after Zuotai and HgS. The expression of renal influx transporters Oat3 and Oatp4c1 was decreased, while the expression of renal efflux transporter such as Mrp2, Mrp4, and Mate2 was increased following HgCl 2 and MeHg. These gene expressions were unchanged after Zuotai and HgS. In summary, both α-HgS and β-HgS are less nephrotoxic than HgCl 2 and MeHg, indicating that chemical forms of mercury are a major determinant of mercury disposition and toxicity., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

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, Nad(p)h: quinone oxidoreductase 1 (Nqo1) and Glutamate-cysteine ligase, catalytic subunit (Gclc). In conclusion, the Tibetan medicine Zuotai and HgS are less hepatotoxic than HgCl2 and MeHg, and differ from HgCl2 and MeHg in hepatic Hg accumulation and toxicological responses., (Copyright © 2016 Elsevier Inc. All rights reserved.)

α-HgS is the main component of traditional Chinese medicine cinnabar, while β-HgS is the main component of Tibetan medicine Zuotai. However, there was no comparative study on the dissolution and absorption in gastrointestinal tract and bioaccumulation in organs of mercury in Cinnabar, Zuotai, α-HgS and β-HgS. In this study, the dissolution process of the four compounds in the human gastrointestinal tract was simulated to determine the mercury dissolutions and compare the mercury dissolution of different medicines and the dissolution-promoting capacity of different solutions. To explore the absorption and bioaccumulation of cinnabar and Zuotai in organisms, mice were orally administered with clinical equivalent doses cinnabar and Zuotai. Meanwhile, a group of mice was given α-HgS and β-HgS with the equivalent mercury with cinnabar, while another group was given β-HgS and HgCl2 with the equivalent mercury with Zuotai. The mercury absorption and bioaccumulation capacities of different medicines in mice and their mercury bioaccumulation in different tissues and organs were compared. The experimental results showed a high mercury dissolutions of Zuotai in artificial gastrointestinal fluid, which was followed by β-HgS, cinnabar and α-HgS. As for the mercury absorption and bioaccumulation in mice, HgCl2 was the highest, β-HgS was the next, and a-HgS was slightly higher than cinnabar. The organs with the mercury bioaccumulation from high to low were kidney, liver and brain. This study is close to clinical practices and can provide reference for the clinical safe medication as well as a study model for the safety evaluation on heavy metal-containing medicines by observing the mercury dissolution, absorption, distribution and accumulation of mercury-containing medicines cinnabar and zuotai.

Objective: To establish the method of quality control for traditional Tibetan Medicine Zsuotai., Methods: Collecting the samples of Tsuotai from Qinghai, Tibet, Sichuan, and Gansu province, to detect Hg2+ by Zsuotai reacted with HCl-HNO3 (3:1), and to determine the quantity of HgS in Zsuotai by sulfocyanate volumetric method., Results: The method for the determination of HgS in Zsuotai was in good reproducibility (RSD = 0.68%). The calibration curve was linear (r = 0.9999) within -0.0002 - 0.2123 g of mercuric sulfide. The recovery was 100.94% (RSD = 0.66%)., Conclusions: This method is convenient and accurate, so it can be used to establish quality control of the medicinal material.