▼ Cinnabar is one of traditional Chinese medicines widely used in many Asian countries. It is also a medicine with potential toxicity especially when taking overdose. Up to date, studies on the mechanism of the biological activity of cinnabar were still insufficient. Aim of the study: To investigate the possible bioactive species from cinnabar after oral administration, which is the fundamental of biological effects of cinnabar. Materials and methods: Under mimetic intestinal and gastric conditions, the chemical components dissolved from cinnabar were analyzed by infrared spectroscopy (IR) and Raman spectroscopy. Furthermore, binding of mercuric species of cinnabar extractions to human serum protein (HSA) was characterized and their intestinal permeability was determined using the Caco-2 cell monolayer. The cytotoxicity of cinnabar extractions was assessed on human kidney-2 (HK-2) cell. Results: Major dissolved species included mercuric polysulfide (i.e. HgS₂(OH)^- and Hg₃S₂Cl₂). The apparent permeability coefficient (P_app) of mercuric polysulfides was (1.6+/-0.3)x10^-^6cm/s, which is slightly lower than that of mercuric chloride (HgCl₂). Unlike HgCl₂, mercuric polysulfides exhibited two tightly binding sites to HSA and had little effect on viability of HK-2 cells. Conclusion: Mercuric polysulfides, as the major dissolved components, may serve as the active species of cinnabar exhibiting pharmacological and/or toxicological effects.

▼ Ethnopharmacological relevance: Cinnabar (Cin), a naturally occurring mercuric sulfide (HgS), is a mineral widely used in traditional Chinese medicine throughout history. As for the toxicity of cinnabar, one important assumption is that cinnabar may be transformed into highly toxic methylmercury by gastrointestinal flora. There is no evidence in humans to support this assumption. Aim of the study: To investigate the biotransformation of cinnabar (HgS) in the human intestinal bacteria with modern analytical techniques. Materials and methods: A gas chromatograph, equipped with electron capture detection (GC-ECD) and mass spectrometry (GC-MS), were used to detect the formation of methylmercury after incubation of cinnabar with human intestinal bacteria. The content of soluble mercury in the bacteria media was determined by cold vapor-atomic absorption spectrometry (CV-AAS). In addition, X-ray absorption near-edge structure spectroscopy (XANES) was used to confirm the possible transformation of cinnabar in the bacteria media, and under mimetic intestinal condition by measuring the species of sulfur and mercury in the reaction extraction of cinnabar and Na₂S mixture. Results: No methylmercury was detected by both GC-ECD and GC-MS, which suggest that cinnabar (HgS) is not methylated in the human intestine. A small amount of soluble mercury was found to be released in the flora medium of HgS or cinnabar by CV-AAS. The XANES analyses revealed that polysulfides exist in the flora medium, and the simulated results showed that the products by incubating cinnabar with Na₂S were mercuric polysulfides. Conclusion: These results showed that under gut flora conditions cinnabar would be transformed into mercuric polysulfides rather than methylmercury. Our work provides evidences of nontoxic transformation of cinnabar in the human intestinal bacteria.

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New experiments have been conducted to determine the speciation of dissolved mercury (Hg) over wide pH (1–12) and sulfide concentration ranges (0.5–30 mM) and in the presence of elemental sulfur (S⁰) or Hg⁰, conditions that encompass those of near-bottom and pore waters of sediments. Samples containing synthetic red mercuric sulfide (HgS, cinnabar), buffer solution, aliquots of bisulfide (HS⁻¹) solution, and, in special cases, S⁰ or Hg⁰ were prepared anaerobically and allowed to equilibrate for several months. Filtered samples were analyzed for pH, total sulfide (ΣS²⁻), and total mercury [Hg]_tot. Plots of [Hg]_tot values vs. pH at varying ΣS²⁻ verified the formation of three previously known mercury-sulfide complexes (HgS₂H_n ⁿ⁻²) and revealed that a new Hg₂SOH⁺ complex is important at low pH and low ΣS²⁻. Our constants for ionic strength (I) 0.7 and 25⁰ C are as follows: K₁=10^{−5.76(+0.71, −1.02)} for HgS_cinn+H₂S ↔ HgS₂H₂ ⁰; K₂=10^{−4.82(+0.72, −1.10)} for HgS_cinn+HS⁻ ↔ HgS₂H⁻; K₃=10^{−13.41(+0.76, −0.93)} for HgS_cinn+HS⁻ ↔ HgS₂ ²⁻+H⁺; K₄=10^{−8.36(+0.71, −0.93)} for 2HgS_cinn+H⁺+H₂O ↔ Hg₂SOH⁺+H₂S. With decreasing pH, below 1, Hg solubility decreased sharply, indicating the formation of a new solid phase, inferred to be corderoite (Hg₃S₂Cl₂). From our solubility data, we calculated the free energy of formation (ΔG_f ^o) of Hg₃S₂Cl₂ to be −396 (+3, −11) kJ/mol. In experiments where excess S⁰(s) was present, a new mercury-polysulfide dimer was identified; its formation constant is K₅=10^{−1.99(+0.69, −1.27)} for 2HgS_cinn+2HS⁻ + nS⁰ ↔ Hg₃S₄ ^IIS_n ^oH₂ ²⁻. Data from experiments where Hg⁰(aq) was added confirmed the reversibility of HgS dissolution. An application of our mercury-sulfide speciation model to a natural anoxic basin, Saanich Inlet, British Columbia, is discussed.

▼ An-Gong-Niu-Huang Wan (AGNH) is a famous traditional Chinese medicine used for brain trauma, hemorrhage, and coma. AGNH contains 10% realgar (As₄S₄) and 10% cinnabar (HgS). Both As and Hg are well-known for their toxic effects, and the safety of AGNH is of concern. To address this question, the acute toxicity of AGNH, realgar and cinnabar were compared to sodium arsenite (NaAsO₂) and mercuric chloride (HgCl₂). Mice were administrated orally AGNH at 1, 3 and 6g/kg. AGNH at 3g/kg contains 2.8mmol As/kg as realgar and 1.18mmol Hg/kg as cinnabar. Realgar, cinnabar, arsenite (0.28mmol/kg, 10% of realgar) and HgCl₂ (0.256mmol/kg, 20% of cinnabar) were orally given to mice for comparison. Blood and tissues were collected 8h later for toxicity evaluation. Serum alanine aminotransferase was increased by arsenite and blood urea nitrogen was increased by HgCl₂. Total As accumulation after arsenite in liver (100-fold) and kidney (13-fold) was much higher than that after realgar. The accumulation of Hg after HgCl₂ in liver was 400-fold higher and kidney 30-fold higher than after cinnabar. Histopathology showed moderate liver and kidney injuries after arsenite and HgCl₂, but injuries were mild or absent after AGNH, realgar, and cinnabar. The expression of metallothionein-1, a biomarker of metal exposure, was increased 4-10-fold by arsenite and HgCl₂, but was unchanged by AGNH, realgar and cinnabar. Thus, AGNH, realgar and cinnabar are much less toxic acutely than arsenite and HgCl₂. The chemical forms of As and Hg are extremely important factors in determining their disposition and toxicity.

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An-Gong-Niu-Huang Wan (AGNH) is a patent traditional Chinese medicine for brain disorders. It contains 10% cinnabar (HgS). Hg is known to produce toxicity to the kidney, brain and liver. Is AGNH safe? Liver is a major organ for drug metabolism, whether the long-term use of AGNH would affect hepatic P450 enzymes is unknown. To address these concerns, mice were given orally cinnabar (300mg/kg), cinnabar-containing AGNH daily for 44days, and liver toxicity was examined and compared with that of methylmercury (MeHg, 2.6mg/kg) and mercuric chloride (HgCl₂, 32mg/kg). Serum aminotransferases were increased by MeHg and HgCl₂ only. Histopathology showed more severe liver damage in MeHg- and HgCl₂-treated mice than in the cinnabar and AGNH groups. Accumulation of Hg in MeHg- and HgCl₂-treated mice was 96- and 71-fold higher than controls, respectively, but was only 2-fold after cinnabar and AGNH administration. Expressions of metallothionein-1 and heme oxygenase-1, biomarkers for Hg toxicity, were increased by MeHg and HgCl_2, but were not altered in cinnabar- and AGNH-treated mice. Expression of hepatic cytochrome P450 genes, such as Cyp1a1, Cyp1b1 and Cyp4a10 was increased only after MeHg and HgCl₂, and the expressions of Cyp3a11and Cyp3a25 were increased by all treatments, indicating the potential Hg-drug interactions after long-term use of cinnabar-containing traditional medicines. Taken together, the results demonstrate that AGNH is much less hepatotoxic than common mercurials, and that the use of total Hg content to evaluate the toxicity of cinnabar-containing traditional Chinese medicines appears to be inappropriate.

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The toxicity of cinnabar, a naturally occurring mercury sulphide (HgS), has long been referred to soluble mercury chloride (HgCl₂). To investigate whether the speciation of mercury plays a role in its disposition and toxicity, we hereby investigated and compared cinnabar with soluble HgCl₂ and pure insoluble HgS in mice on mercury absorption, tissue distribution and in relation to the biological effects. The male C57BL/6J mice were treated by oral administration of various doses of cinnabar, with 0.01 g/kg of HgCl₂ for comparison, or the same dose of cinnabar or pure HgS (0.1 g/kg), once a day for 10 consecutive days. The total mercury contents in serum and tissue (brain, kidney, liver) were measured by atomic fluorescence spectrometer (AFS). The biological effects investigated involved monoamine neurotransmitters (serotonin, 5-HT) in brain as an indicator of therapeutic function, and serum alanine transaminase (ALT) as a marker of hepatic damage, blood urea nitrogen (BUN) and serum creatinine as markers for renal function. The mercury absorption of cinnabar or HgS was much less than that of HgCl₂. The mercury levels in brains of the cinnabar group were only slightly changed and kept in a steady-state with the dose elevated. Cinnabar or HgS suppressed brain 5-HT levels. HgCl₂ could not cause any changes in brain 5-HT although the mercury level increased considerably. The results revealed that cinnabar or HgS is markedly different from HgCl₂ in mercury absorption, tissue distribution and influence on brain 5-HT levels, which suggests that the pharmacological and/or toxicological effects of cinnabar undertake other pathways from mercuric ions.