Chemical Form and Bioaccessibility of Mercury in Traditional Tibetan

Aug 20, 2018 - A substantial amount of Hg could be released from TTM after ingestion. In total, an average of 12 μg of Hg (mostly inorganic)/g (range...
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Letter Cite This: Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX

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Chemical Form and Bioaccessibility of Mercury in Traditional Tibetan Medicines Maodian Liu,†,‡ Yipeng He,†,‡ Shidong Ge,† Menghan Cheng,† Han Xie,† Chenghao Yu,† and Xuejun Wang*,† †

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Ministry of Education Laboratory of Earth Surface Process, College of Urban and Environmental Sciences, Peking University, Beijing 100871, China ‡ Department of Marine Sciences, University of Connecticut, 1080 Shennecossett Road, Groton, Connecticut 06340, United States ABSTRACT: Extremely high mercury (Hg) concentrations have been identified in traditional Tibetan medicine (TTM) products, but the chemical form and bioaccessibility of Hg remain unknown. We conducted experiments to explore whether the Hg contained in TTM is toxic. We determined that HgS is not the exclusive form of Hg in TTM; other compounds could be present in substantial quantities, ranging from 2 to 52% of the total Hg in commonly used TTMs. A substantial amount of Hg could be released from TTM after ingestion. In total, an average of 12 μg of Hg (mostly inorganic)/g (range of 0.41−25 μg of Hg/g) in TTMs was released into the liquid phase in simulated human gastrointestinal environments. However, different from the results from ingestion by fish, the release of Hg from TTM in the intestinal environment is larger than that in the gastric environment by a factor of 2. In the case of joint ingestion with protein-rich products, releases of methyl Hg and inorganic Hg from TTM in gastrointestinal environments could be significantly enhanced by factors of 9 and 6, respectively. Our efforts further highlight that the Hg contained in TTMs could be harmful to human health, and the clinical safety of different TTM products should be thoroughly evaluated.

1. INTRODUCTION The toxic element mercury (Hg) is considered to be one type of chemical that can cause serious public health problems worldwide through biomagnification in food webs. 1−4 Although Hg occurs naturally, human activities have altered its global biogeochemical cycle.5−7 In many countries, such as the United States, seafood consumption is the nearly exclusive dietary source of human Hg exposure,8,9 while in some inland areas in Asia, rice consumption could be a significant source because of soil pollution.10−12 A highly elevated Hg concentration was found in municipal sewage in Tibet, which was among the highest levels in China.13 This is in contrast to the notion that the Tibetan Plateau is rarely impacted by chemical pollution because of its remoteness. Our recent study determined that the high Hg concentration in sewage in Tibet could be explained by the ingestion of traditional Tibetan medicine (TTM) products, which contain 5600 μg of Hg/g and 16 μg of methylmercury (MeHg)/g on average.14 Regular TTM ingestion, a novel Hg exposure pathway distinct from traditional seafood consumption, has resulted in high Hg exposure levels in Tibetan people that are tens to thousands of times higher than the levels from any other known dietary sources.14−17 TTM is produced using complex herbal and mineral pharmacopeia, and Hg has been considered an important therapeutic ingredient in TTM for more than 1000 years.18 © XXXX American Chemical Society

Mercurous chloride (HgCl2) has a long history of medicinal uses as a laxative and infant teething powder until the last century but is now believed to cause acrodynia or pink disease.19 Previous studies have stated that Hg used in TTM is a black fine mixture containing cubic crystal mercuric sulfide (β-HgS), which has an assimilation rate (99%). The first four steps of the extraction procedure, except for the final (fraction 5) step, included a rinse (deionized water; ρ = 18.2 MΩ cm) between two consecutive extractions.27 We conducted the first four steps of the extraction procedure by shaking the samples on an orbital shaker at 200 rpm and room temperature for 24 h.27 Other details of the extraction process were provided in the study by Bloom et al.27 2.3. In Vitro-Simulated Digestion System. In vitro gastrointestinal extractions were created to simulate the bioaccessibility of Hg contained in different TTM products within mammalian gastric and intestinal environments. Simulated gastrointestinal solutions were prepared according to the methods described in the U.S. Pharmacopeia.30 These extraction procedures have been widely used to understand the bioaccessibility of Hg in the human body.23,31,32 The bioaccessibility results of the sequential extraction and in vitro gastric extraction were compared. We further compared the different bioaccessibility values of Hg among the TTM samples, β-HgS, and bluefin tuna. The tuna meat was sampled from a market in Beijing in 2017, with a sample size of 4. The simulated gastric solution was prepared by diluting 7.0 mL of a 38% HCl solution and 2 g of NaCl with deionized water in a 1000 mL volumetric flask; then, we added 3.2 g of pepsin (from porcine gastric mucosa, Sigma-Aldrich Co.). Finally, deionized water was added to the mark, and we mixed the solution thoroughly.30 The pH of the final solution was approximately 1. The simulated intestinal solution was prepared by dissolving 6.8 g of KH2PO4 in 750 mL of deionized water and adding 77 mL of a 0.2 M NaOH solution and 10.0 g of trypsin (Sigma-Aldrich Co.).30 Then, we diluted the solution to 1 L with deionized water, and the pH of the final solution was approximately 6.8.30 The Hg contents in the finely powdered TTM products, β-HgS, and fish samples were sequentially incubated with 100 mL of simulated gastric and intestinal solutions. To facilitate the comparison, we adopted a solid:liquid ratio of 1:100 (solid vs solution) in the release experiments with the different samples, i.e., 1.0 g in dry weight. We selected Renqing Mangjue to evaluate the potential risk of the increased level of assimilation of Hg from TTM due to the ingestion of protein-rich products by adding different amounts of tuna tissue. Each gastrointestinal extraction was performed in triplicate. The samples were then tumbled end over end at 150 rpm for 4 h in 125 mL high-density polyethylene bottles at 37 ± 1.0 °C.32 After extraction, the mixture was centrifuged at 3000 rpm for 15 min and syringe-filtered through 0.45 μm cellulose acetate filters (Whatman, 10462100). After gastric extraction, the pH of the residual mixture was adjusted to 6.8 with a 0.2 M NaOH solution, 100 mL of the intestinal solution was added, and the same extraction procedure that was used for the gastric extraction was adopted.32 We measured the Hg concentration in the filtrates immediately after extraction.23,32 2.4. Analytical Methodology. The Hg concentrations were analyzed by a DMA-80 instrument following U.S. EPA method 7473.13,33 For the MeHg concentrations in the fish

2. MATERIALS AND METHODS 2.1. Sample Collection. Details of the sampling process were provided in our previous study.14 Briefly, 10 common TTM products were sampled in 2016: Renqing Mangjue (from two different companies), Qishiwei Zhenzhu Wan (from two different companies), Ershiwuwei Zhenzhu Wan (from two different companies), Zuozhu Daxi, Renqing Changjue, Zhenzhu Qishi Wan, and Shiliu Jianwei San. All of the TTM products evaluated in this study were sampled in triplicate from local Tibetan pharmacies and represented local Tibetan products. Hg and MeHg concentrations were measured, and the results were provided in our previous study.14 2.2. Sequential Selective Extractions. A variety of approaches have been used to identify the speciation of Hg in environmental samples. The traditional sequential extraction procedure reported by Tessier et al. is commonly used to study metals (e.g., Fe, Mn, Cu, and Zn) in environmental samples (e.g., soil, plants, and minerals) due to the low detection limit and wide availability of this method.26−28 Nevertheless, this traditional method is not appropriate for Hg because of the numerous and diverse Hg species with unique physical and chemical properties.29 The five-step sequential extraction procedure refined by Bloom et al. has been the most widely used method for Hg in many studies.27,29 The five fractions were defined as water soluble (fraction 1), “human stomach acid” soluble (fraction 2), organo-chelated (fraction 3), elemental Hg (fraction 4), and mercuric sulfide (fraction 5). The sum of the Hg contents in the water and “human stomach acid” soluble fractions correlated well with the results from the in vitro stomach bioaccessibility tests, and the recovery of HgS in the mercuric sulfide fraction was nearly 100%.27,29 These extractions were conducted at high Hg concentrations (1000− 10000 μg/g).27 TTM is produced using herbs and minerals. Hence, we applied the procedure reported by Bloom et al. to different TTM samples.27 B

DOI: 10.1021/acs.estlett.8b00371 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX

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Figure 1. Results for the Hg compounds in 10 commonly used traditional Tibetan medicine products. Panel a shows the percentages of Hg in different extraction solutions. Panel b shows the concentrations of Hg in different extraction solutions. Panel c shows the relations between MeHg concentrations and Hg concentrations in different extraction solutions. The error bars in panel b represent the standard deviations in the measurements. The statistical significance was determined at the p < 0.05 (one asterisk) and p < 0.01 (two asterisks) levels. The sizes of the dots in panel c represent the MeHg concentrations of different TTM products, which were referenced from our previous study.14 Legend: 1, Renqing Mangjue from company A; 2, Renqing Mangjue from company B; 3, Qishiwei Zhenzhu Wan from company C; 4, Qishiwei Zhenzhu Wan from company D; 5, Ershiwuwei Zhenzhu Wan from company E; 6, Ershiwuwei Zhenzhu Wan from company F; 7, Zuozhu Daxi; 8, Renqing Changjue; 9, Zhenzhu Qishi Wan; 10, Shiliu Jianwei San.

3. RESULTS AND DISCUSSION

and gastrointestinal solutions, 4.5 M HNO3 was added and extracted in a water bath at 60 °C for 12 h, following the methods used in previous studies.14,34 The extracted solutions were neutralized with KOH, buffered with acetate, derivatized into methylethylmercury with sodium tetraethyl borate, and quantified using gas chromatography−atomic fluorescence spectrometry (GC−AFS).13,34 We analyzed each sample in triplicate. The detection limits for Hg and MeHg were 0.1−2 ng/mL (for different extraction solutions) and 0.1 ng/L, respectively, and were calculated on the basis of the average concentrations of the method blanks plus triple the standard deviation of the blanks.13 The spike recoveries for the ambient Hg and MeHg were 110 ± 12 and 81 ± 16%, respectively.

In this study, we determined that HgS is not the exclusive form of Hg in TTM products, and a substantial amount of Hg may be released from TTMs after its ingestion on the basis of both the sequential extraction procedure and in vitro gastrointestinal extraction (Figures 1 and 2). HgS was not leached by any of the solutions, except aqua regia, according to the extraction fingerprint suggested by Bloom et al.27 In the study presented here, we also verified that ∼100% of the Hg in HgS was stabilized in the mercuric sulfide fraction (Figure 1a). The percentages of Hg in the mercuric sulfide fraction compared to the total Hg concentrations in the different TTM products were low [average of 79%, range of 48−99% (Figure 1a)]. The C

DOI: 10.1021/acs.estlett.8b00371 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX

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1b). Bloom et al. found that samples with a high humic matter content could typically color the extractant liquid dark brown in the organo-chelated fraction and have high Hg concentrations,27 20 μg/g on average (range of 0.16−63 μg/g; n = 30) in the study presented here (Figure 1b). MeHg was identified in the organo-chelated fraction of sediment samples and biota in a previous study, which presented the strongest correlation with sediment methylation potential.27 Similar to Bloom et al., we determined that the fraction of MeHg in the different TTMs showed a strong positive correlation with the total Hg concentrations in the organo-chelated fraction [R2 = 0.67; **p < 0.001 (Figure 1c)], suggesting that the mixed water and human stomach acid soluble extractions may represent the stomach bioaccessibility of inorganic Hg (IHg) but not MeHg in TTMs. In addition to the mercuric sulfide fraction, the elemental Hg fraction represents a major component of Hg in TTMs [range of 0.67−62% (Figure 1a)], which was mostly composed of Hg0, Hg2Cl2, and Hghumic.27 Although we were unable to identify all specific compounds in the TTM based on the selective extractions, the results could provide needed information about the biogeochemical behavior of the classes of different Hg compounds in various TTM products. The sum of the Hg concentrations in the water and human stomach acid soluble fractions favorably correlates with the results from the gastric bioaccessibility test in the study presented here [R2 = 0.47; *p = 0.02 (Figure 2a)]. Nevertheless, we observed that the average release of Hg in the intestinal solution (7.9 μg/g; range of 0.29−16 μg/g) was higher than that in the gastric solution by a factor of 2 (Figure 2b). This result was different from the Hg bioaccessibility in tuna meat (averages of 3.5 ± 1.2 and 0.44 ± 0.15 μg/g in the simulated gastric and intestinal solutions in this study, respectively) simulated in this study and was also different from those of the traditional Chinese patent medicines included in a previous study.32 These results suggested that Hg could be significantly released from TTM in the intestinal environment in the presence of trypsin; therefore, selective extractions and gastric tests could not make assertions about the Hg bioaccessibility of TTM. The mechanism of the high Hg bioaccessibility of TTM in the intestinal environment is not clear. One explanation is the dissolution of solid-state HgS in the intestinal solution.35 The percentage of Hg released from HgS in the intestinal solution (100:1 liquid:HgS) was 0.0078% (average of 78 ± 32 μg/g), which was 24 times higher than that in the gastric solution [average of 3.3 ± 0.15 μg/g (Figure 2b)]. An increased amount of Hg released from HgS in the intestinal solution was observed in a previous study.23 The solubility of Hg increased at high pH values and S 2+ concentrations, and HgS2H− or HgS22− was generated.35 Although the level of the soluble product of HgS was low, Hg released from medicinal products may nevertheless pose a risk to the human body because of their extremely high HgS concentrations. The bioaccessibility of Hg in TTM is high in simulated gastrointestinal environments (average of 12 μg/g, range of 0.41−25 μg/g), but the average MeHg concentration in TTM is 16 μg/g (range of 0.12−37 μg/g).14 Additionally, to the best of our knowledge, there is no study focusing on the potential risk of the increased level of assimilation of Hg from TTMs due to the ingestion of protein-rich products. We selected Renqing Mangjue, which contains averages of 12000 ± 3000 and 37 ± 19 μg/g for Hg and MeHg, respectively, to answer

Figure 2. Releases of Hg from 10 commonly used traditional Tibetan medicine products and HgS in simulated gastrointestinal environments. Panel a shows the comparison of Hg concentrations in the mixed water and human stomach acid soluble fractions and stomach extraction solutions, and panel b shows the percentages of Hg released from samples in simulated gastric and intestinal solutions. In panel a, the red solid line represents the regression curve between the simulated gastric release and the mixture of water and human stomach acid soluble fractions. The dotted black line represents the 1:1 line. The error bars in panel a represent the standard deviations in the measurements. The statistical significance was determined at the p < 0.05 (one asterisk) and p < 0.01 (two asterisks) levels. The names of the medicines are provided in Figure 1.

extractions of the mixed water and “human stomach acid” soluble fractions were previously thought to be available in human stomachs.27 In the mixed water and “human stomach acid” soluble extractions, the Hg compounds with a relatively high water solubility (mostly HgCl2, HgSO4, and HgO) behaved as a general class.27 Nevertheless, the level of Hg in the mixed soluble fractions of water and human stomach acid in TTM was low, ranging from 0.23 to 12 μg/g (from 0.0093 to 0.96%, respectively) in the study presented here (Figure D

DOI: 10.1021/acs.estlett.8b00371 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX

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Environmental Science & Technology Letters these questions.14 The results show substantial increases in the release of total Hg and MeHg from TTMs, with increases in tuna tissue in the gastric solution and total Hg in the intestinal solution and a slight increase in the release of MeHg in the intestinal solution (Figure 3). The total release of MeHg from

proteins in solubilizing Hg and MeHg from TTM. The weight of Renqing Mangjue is ∼1 g/pill. Suppose a person takes one pill of Renqing Mangjue each day with sufficient tuna meat: the calculated probable daily intake of MeHg could reach 0.68 μg kg−1 day−1 (ingestion rate × MeHg concentration/body weight),14 which is 6.8 times higher than the U.S. Environmental Protection Agency recommended intake of MeHg. Because of cultural preferences, the fish consumption rate is low in Tibet but the yak meat and milk product consumption rates are high. Hence, further investigation of the risk of MeHg to the Tibetan population caused by the ingestion of local Tibetan foods combined with TTM products is needed. In summary, we found that HgS is not the exclusive form of Hg in TTM products, and a substantial amount of Hg might be released from TTM after its ingestion based on various experiments. More Hg could be released from TTMs in simulated intestinal environments than in gastric environments. The release of MeHg in Renqing Mangjue could be significantly enhanced by adding tuna tissue to the gastric solution, whereas IHg was significantly released in the intestinal solution. On the basis of the information provided above, we suggest that Hg-susceptible populations, such as pregnant women, should avoid the consumption of these TTM products, especially ingestion combined with protein-rich products, such as deep-sea fish. Further investigation of the behavior of the release of Hg from TTM products and possible human health effects would be highly desirable.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +86-10-62759190. E-mail: [email protected]. edu.cn. ORCID

Maodian Liu: 0000-0001-5059-0334 Xuejun Wang: 0000-0001-9990-1391 Notes

The authors declare no competing financial interest. Figure 3. Releases of Hg and MeHg from Renqing Mangjue in simulated gastrointestinal environments influenced by adding tuna tissue. The release of Hg from the added tuna tissue was deducted in the calculation. The error bars represent the standard deviations in the measurements.

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ACKNOWLEDGMENTS

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REFERENCES

The authors thank Robert P. Mason and Zofia Baumann for their helpful discussion of the experiments. This work was funded by the National Natural Science Foundation of China (41630748, 41571484, 41571130010, 41130535, and 41471403).

Renqing Mangjue was 41 ± 16 μg/g (110% of the total MeHg concentration in Renqing Mangjue) in the gastrointestinal solutions when 2 g of tuna tissue was added, which significantly increased from 4.5 ± 1.5 μg/g when no tuna tissue was added. The total release of Hg was 160 ± 52 μg/g (1.3% of the total Hg concentration in Renqing Mangjue). The majority of the increase in the Hg released from Renqing Mangjue in the gastric solution was in MeHg [42−76% of the total Hg (Figure 3)], which may be attributed to the release of amino acid carriers released from the tuna tissue.36−38 Here, we suggest that the solubilization of MeHg in Renqing Mangjue might be the effect of complexation rather than enzymatic processes in the gastric solution. By contrast, the majority of the increase in the Hg release in the intestinal solution was IHg (72−95%), which may be attributed to the enhanced dissolution of HgS in Renqing Mangjue by enzymes isolated from the tuna tissue. More research is needed to solidify these initial findings. Overall, these results suggest the potential importance of

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DOI: 10.1021/acs.estlett.8b00371 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX