Compatibility of Niuhuang Jiedu Tablets Results in Attenuated Arsenic Bioaccumulation and Consequent Protection against Realgar-Induced Toxicity in Mice

Niuhuang Jiedu Tablets (NJT) is a popular over-the-counter traditional Chinese medicine (TCM) preparation. It is composed of realgar (As2S2) and seven other TCMs. The safety of NJT is of growing concern because arsenic (As) is carcinogenic to humans. The toxicity of realgar in vivo can mainly be attributed to the absorbed and accumulated As. This study investigated the correlation between the detoxification effects of the other TCMs in NJT on realgar and their influences on arsenic accumulation of realgar in mice. Histopathological examination, clinical biochemical test, and metabolic profiling analysis were used to evaluate the toxicity of realgar. The concentration of arsenic in mice whole blood as the hazard indicator was determined by inductively coupled plasma mass spectrometry (ICP-MS). The compatibility of NJT could decrease arsenic bioaccumulation of realgar in mice whole blood and consequently reduce the toxicity of realgar, which could be considered as one detoxification mechanism to realgar in NJT. The combination of Rhei Radix et Rhizoma, Scutellariae Radix, Platycodonis Radix, and Glycyrrhizae Radix et Rhizoma exhibited almost the same effects as NJT in regulating the serum biochemical parameters and metabolic profiles disturbed by realgar and in reducing arsenic accumulation of realgar in mice whole blood.


Introduction
Realgar (As 2 S 2 ) is the sulphide mineral of arsenic (As) [1]. It has been used to treat external infectious dermatoses, convulsive epilepsy, malaria, and abdominal pain caused by parasitoses for hundreds of years. In recent years, realgar has also been shown to cure acute promyelocytic leukemia and other human malignancies effectively [2][3][4] It is a kind of antipyretic and detoxicate drug used for sore swollen throat, periodontitis, gingivitis, and mouth ulcer [5]. As is a widely distributed metalloid on the Earth. Epidemiological and clinical studies have indicated that As is associated with a variety of human cancers and noncancerous diseases. e Agency for Toxic Substances and Disease Registry (ATSDR) has placed As at the top of its Substance Priority List since 1997 [6]. Arsenic compounds have been identified as a carcinogenic hazard to humans by the International Agency for Research on Cancer (IARC) [7]. However, realgar is less toxic than soluble arsenicals and is even considered relatively safe depending on its median lethal dose. But exposure to realgar remains a toxicological risk and presents a safety concern of the public [8].
Realgar is poorly soluble in aqueous solutions, and it is an overestimate to evaluate the toxicity of realgar only by total arsenic content unless bioavailability is considered [9,10]. e bioavailable portion of realgar can be absorbed and transported into blood. e absorbed arsenicals have a strong affinity with proteins containing thiol groups such as hemoglobin, metallothionein, and tubulin, so they accumulate with proteins in the form of stable complexes [11][12][13]. erefore, compared with serum or plasma arsenic concentration, it is more reliable and scientific to assess the health risk of realgar and realgar-containing TCMs by whole blood arsenic concentration [11].
Realgar has been shown to cause acute or chronic toxic reactions in animals by some toxicological experiments [8,[14][15][16][17]. However, it became relatively safer after compatibility with NJT [11,[18][19][20][21]. In our previous study, the toxicity-attenuating effect of the compatibility of NJT with realgar was confirmed by metabonomics study [20,21]. But the reason why the detoxification effect of each single TCM in NJT on realgar was not obvious puzzled us from then on. e attenuation of arsenic bioaccumulation of realgar by the synergistic effect of the other TCMs was considered to be one of the detoxification mechanisms [11,18]. However, it remains unknown which of the other seven TCMs in NJT has this detoxification effect on realgar. e present study was designed to investigate the influences of the other TCMs in NJT on arsenic bioaccumulation of realgar and the relationship with their detoxification effects on realgar. Inductively coupled plasma mass spectrometry (ICP-MS) is a promising technique for trace element analysis in multiple research areas [22][23][24], and it has become a valuable tool for arsenic determination due to its low LOD, wide dynamic linear range, rapid detection, and few mass interferences [25][26][27][28][29]. In this experiment, whole blood arsenic accumulation of realgar was determined by ICP-MS. e toxicity of realgar was evaluated by histopathological examination, clinical biochemical test, and metabolic profiling analysis.

Animals and Treatments.
Male Kunming mice with a weight of 25-35 g were obtained from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China). All animals were housed under controlled conditions (temperature 22-28°C, humidity 50-65%, and 12 h/12 h light-dark cycle). e animals were allowed to acclimate for 7 days with free access to food and water prior to the study. All animals were fasted overnight before the study. Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of SPF (Beijing) Biotechnology Co., Ltd. and Scientific Research Department of Beijing Hospital (AEW2020010801).
Mice were randomly divided into 11 groups (12 mice/ group), as shown in Table 1. All animals were subjected to oral administration for seven consecutive days. e dosage of realgar was set based on previous studies [11] [5]. Blood samples were collected from the carotid artery of mice after they were anesthetized with ether on the 8th day. Six of the blood samples in each group were directly stored at −80°C until arsenic determination. e other six blood samples were centrifuged at 15000 × g for 10 min at 4°C to obtain serum samples, which were frozen at −80°C prior to analysis. Liver and kidney tissues were immediately removed, washed with 0.9% (w/v) NaCl solution, and soaked in 10% formalin solution.

Histopathology Analysis and Clinical Biochemistry.
All liver and kidney tissues fixed in 10% formalin solution were embedded in paraffin wax and sectioned and stained with haematoxylin and eosin (H&E) for histopathological assessment. e measurement of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), and creatinine (CREA) in the serum was performed using a clinical analyzer (Hitachi, Ltd., Tokyo, Japan).

Pattern Recognition
Analysis. Serum samples were thawed at room temperature and centrifuged at 15000 × g for 10 min. en, 300 μL of the supernatant was mixed with 100 μL TSP (1 mg/mL) and 200 μL D 2 O. e resulting solution was transferred into a 5 mm NMR tube. All NMR spectra were recorded on a Bruker AV 500 MHz spectrometer (Bruker GmbH, Germany) at 298 K. e Carr-Purcell-Meiboom-Gill (CPMG) sequence was employed to suppress the NMR signals from the macromolecules and allow the signals of micromolecules to be clearly presented. Sixty-four transients were collected into 64 K data points with the spectral width of 10 kHz. All free induction decays (FIDs) were zero-filled and multiplied by an exponential function with a 0.5 Hz line-broadening factor prior to Fourier transformation.
All spectra were manually phased, baseline adjusted, and referenced to TSP (δ 0.0) with MestReNova software (version 9.0.1, Mestrelab Research SL, Spain). e spectral region of δ 0.2-9.6 was integrated into segments with an equal spectral width of 0.04 ppm.
e regions contributed to citrate (δ 2.70-2.64 and δ 2.58-2.52) were combined into two signals δ 2.66 and δ 2.54, respectively. e region at δ 4.6-5.2 was removed to eliminate baseline distortion caused by residual water resonance. e binned data were normalized to the total sum of spectral integrals to compensate for the concentration differences for all metabolites. Multivariate statistical analysis was conducted for the above normalized data using SIMCA-P software package (version 11.5, Umetrics AB, Sweden). Principal component analysis (PCA) was initially used to obtain a general overview of the metabolic pattern. Partial least square discriminant analysis (PLS-DA) was implemented on the mean-centered data to discover similarities or dissimilarities among the experimental groups. R 2 X, R 2 Y, and Q 2 values can explain the quality of these models. R 2 X and R 2 Y indicate the robustness of the models, and Q 2 represents the model predictability.
e scores plots, in which each point represents an individual sample, were constructed to visualize the classification of the serum samples. e loadings plots, in which each point represents a single spectral region, were used to show NMR signals (metabolites) contributing to the clustering. Endogenous differential metabolites were subjected to MetaboAnalyst 4.0 (https://www.metaboanalyst.ca) to reveal disturbed biological pathways involved in the metabolism.
2.5. ICP-MS Analysis. 0.5 mL of whole blood sample and 2 mL of trace metal-grade nitric acid were added to a microwave digestion tube. e digestion and preparation of the samples were carried out following our previously established method [30].
Total arsenic in blood was determined by an Agilent 7900 ICP-MS (Agilent Technologies International Japan, Ltd., Tokyo, Japan). MassHunter 4.5 Workstation Software was used for data acquisition. e equipment tuning was performed daily to assure responses of at least 3000 counts for Li, 10000 counts for Y, and 6000 counts for Tl and at most 2% for CeO/ Ce and 3% for Ce 2+ /Ce. More operating details are given in Table 2. e samples and standard solutions were introduced into the ICP-MS nebulizer using a sample tube (1.02 mm, ID). e internal standard solution was obtained by diluting ICP-MS internal standard mix (Section 2.1) with 5% (v/v) HNO 3 to give a concentration of 0.01 mg/L as Bi, Ge, In, Li, Lu, Rh, Sc, and Tb and injected online via an internal standard tube (0.19 mm, ID) throughout the data acquisition process.
Arsenic calibration standards were prepared by serially diluting the As standard solution with 5% HNO 3 , resulting in concentrations of 0.1, 1, 2.5, 5, 10, and 20 ng/mL. Calibration standards were freshly prepared each day. Quality control (QC) samples were prepared by spiking blank mice whole blood with As standards to reach final As concentrations of 5, 10, 400, and 600 ng/mL.

Statistical Analysis.
All numerical data are expressed as mean ± SD. e data were statistically analyzed by one-way analysis of variance (one-way ANOVA) and Dunnett's t-test using SPSS 17.0 software (SPSS Inc., Chicago, USA). Values of p < 0.05 were considered statistically significant.

3.1.
Histopathology and Clinical Biochemistry. Histopathological examination of the liver and kidney samples showed no pathological changes among all the treated groups ( Figures S1 and S2). e experimental dose of realgar did not possess the toxicity that could result in visible pathological changes. Serum biochemical parameters of all mice are displayed in Table 3. AST, ALT, and ALP levels increased significantly to varying degrees in groups R, RBC, RBS, and RGF compared to the control group. No significant difference in any biochemical parameter was observed among all the other experimental groups.

Metabolic Profiling Analysis.
e endogenous metabolites were characterized according to the literature and our previous studies [15,17,20,21]. e metabolic profiles among control and experimental groups were further illustrated by pattern recognition analysis. e PCA scores plots based on 1 H NMR spectra of the serum from the 11 groups are shown in Figure 1(a). It is observed that group R is separated distinctly from the control group. e points of groups RBC, RBS, RGF, RRR, RSR, RPR, and RGR cluster in Evidence-Based Complementary and Alternative Medicine the area of group R, while the points of groups NJT and RFH are distributed in the range of control group. e PLS-DA scores plot (Figures 1(b) and 1(d)) was used to sharpen the already observed separation. e corresponding loadings plot (Figures 1(c) and 1(e)) and one-way ANOVA were utilized for identification of potential differential metabolites that contributed to different metabolic profiles from comparative samples. e variable importance for projection (VIP) values of peak integral data in the loadings plot reflect the influences of the metabolites on the classification. Integral data with VIP values > 1 are considered to have aboveaverage influence on the classification. One-way ANOVA was used to test the significance of differences in metabolites among groups, with p < 0.05 as the standard.
From the PLS-DA scores plot, the obvious separation of group R from the control group is observed (Figure 1(b)).  54, d, and δ 2.66, d), and trimethylamine-N-oxide (TMAO, δ 3.27, s) in group R compared with the control group. e metabolic profiles of groups RBC, RBS, RGF, RRR, RSR, RPR, and RGR were in line with that of group R. As shown in Figure 1(d), groups NJTand RFH could be easily distinguished from group R. According to the results of the loadings plot (Figure 1(e)) and statistical analysis, the changes in the concentrations of the above endogenous metabolites in group R were modulated to be close to the normal state in groups NJT and RFH.
Metabolic profiling analysis provides a comprehensive and sensitive map of endogenous metabolites associated with toxic reactions to realgar. e biological pathways involved in the metabolism of the above-mentioned metabolites were determined by pathway analysis. e results are presented graphically (Figure 2) and in a detailed table (Table 4). e pathways with the impact value > 0.1 were considered closely related to realgar-induced toxicity [31]. Consequently, valine, leucine, and isoleucine biosynthesis, synthesis and degradation of ketone bodies, glyoxylate and dicarboxylate metabolism, pyruvate metabolism, citrate cycle, glycolysis or gluconeogenesis, and butanoate metabolism were recognized as the influenced metabolic pathways associated with the toxicity of realgar.

Method Validation Results.
e method was validated in detail based on our previous experiment [30]. e calibration curve and linearity parameters over the range of 0.1-20 ng/mL for As was automatically generated with MassHunter 4.5 Workstation Software. e calibration curve was Y � 0.3185X + 0.0143 with a correlation coefficient (r) of 0.9999, and the LOQ was 0.043 ng/mL. e precision and accuracy results are summarized in Table 5. Good precision and accuracy were acquired for As at the four different levels. e RSD values of intrabatch and interbatch precision were between 4.17% and 9.89% and 3.80% and 8.35%, respectively. e relative error (RE) of the four selected concentrations were −9.00%, −7.40%, −3.81%, and −1.32%, respectively. e recoveries which were investigated at each QC level were between 93.15% and 101.02%.

Determination of Whole Blood Arsenic Accumulation.
e total blood arsenic concentration of mice is shown in Figure 3. e whole blood arsenic concentrations of mice in groups RBC, RBS, and RGF were not significantly different from those of group R. e amounts of As in mice whole blood of groups RRR, RSR, RPR, and RGR did not increase as much as that of group R. e As accumulated in mice whole blood of group NJT decreased by about 60% compared with the realgar solo group. e result of group RFH was similar to that of group NJT, which indicated that Rhei Radix et Rhizoma, Scutellariae Radix, Platycodonis Radix, and Glycyrrhizae Radix et Rhizoma synergistically reduced the amount of arsenic accumulated in mice as the whole prescription.

Discussion
e dose of realgar used in this study did not cause pathological changes in the liver and kidney of mice. Significant increases in serum AST, ALT, and ALP levels were observed in groups R, RBC, RBS, and RGF, which proved that liver injury occurred. Although the changes were not statistically significant, the levels of AST, ALT, and ALP in groups RRR, RSR, RPR, and RGR were found to increase to different degrees compared with that of the control group. e elevated levels of AST, ALT, and ALP due to realgar injury could be decreased by NJT treatment, which suggested that NJT had hepatoprotective effects on realgar-induced liver injury. e combination of Rhei Radix et Rhizoma, Scutellariae Radix, Platycodonis Radix, and Glycyrrhizae Radix et Rhizoma exhibited the same effect as the whole prescription in regulating the serum biochemical parameters disturbed by realgar.
Conventional biological assessment of drug toxicity is based on targeted analysis of a small number of measured indicators, which provides limited opportunities to detect the trends and characterize mechanisms of toxicity [32]. e variations of endogenous metabolic networks reflect different pathological conditions and characterize the biochemical mechanisms of xenobiotics in humans or animals [33]. Metabolic profiling analysis makes recognition of the consistently varying endogenous metabolites and elucidation of changes in the associated metabolic regulatory networks come true [34]. As a convenient and effective tool, metabolic profiling analysis has been used to elucidate the toxicity of TCMs [35]. is study investigated the effects of the other TCMs in NJT on realgar-induced toxicity in mice.
e metabolic profiles of mice influenced by realgar could not be altered by combining realgar with any other single TCM in NJT. However, the compatibility of them could reduce the toxicity of realgar. e concentrations of leucine, isoleucine, valine, lactate, acetate, alanine, pyruvate, 2oxoglutarate, citrate, creatine, choline, and TMAO affected by realgar in mice were positively regulated toward normal  [20]. From the perspective of correlation to the influence on the toxic sources of realgar, the possible reason why each single TCM in NJT did not show the toxicity alleviation effect on realgar and the possible underlying mechanism of their synergistic detoxification effect on realgar were also explored in the present study. e toxicity of realgar can mainly be attributed to the absorbed and accumulated soluble As. Some herbal ingredients in TCM patent medicines containing toxic minerals showed detoxification effect by inhibiting the bioaccessibility or bioavailability of toxic elements [36,37]. e bioaccessibility of soluble As in NJT was significantly lower than that of realgar in simulated gastrointestinal juice in vitro [38][39][40]. Furthermore, in vivo experiment revealed that NJT could significantly inhibit the arsenic absorption and reduce the concentration of total As in tissues compared to realgar [11,18]. In this study, arsenic accumulation in whole blood of mice was measured to attempt to reveal the different toxic reactions of mice in each group to realgar. Bovis Calculus Artifactus, Borneolum Syntheticum, and Gypsum Fibrosum did not influence the arsenic accumulation and toxicity of realgar in mice, simultaneously. Rhei Radix et Rhizoma, Scutellariae Radix, Platycodonis Radix, and Glycyrrhizae Radix et Rhizoma could decrease the As accumulated in whole blood of mice to different degrees, respectively. However, the accumulated As in mice of these four groups could still result in toxic reactions as in group realgar. It was speculated that the amount of As accumulated in these four groups were higher than the normal tolerable amount of As in mice, which requires further study. e combination of Rhei Radix et Rhizoma, Scutellariae Radix, Platycodonis Radix, and Glycyrrhizae Radix et Rhizoma dramatically inhibited the arsenic accumulation of realgar in mice, which was consistent with the experimental result of group NJT. Meanwhile, the mice from groups RFH and NJT did not display the toxic responses as the mice from group realgar. e reduction of arsenic accumulation of realgar in mice by Rhei Radix et Rhizoma, Scutellariae Radix, Platycodonis Radix, and Glycyrrhizae Radix et Rhizoma in terms of decreasing absorption and increasing excretion of As will be investigated in our further studies, which are still ongoing and require more work.

Conclusion
In the present study, the correlation between the effects of the other TCMs in NJT on the toxicity of realgar and their influences on its arsenic accumulation was assessed in vivo. e toxicity of realgar was evaluated by histopathological examination, clinical biochemical test, and metabolic profiling analysis. e concentration of As accumulated in mice whole blood was determined by ICP-MS. NJT could decrease arsenic accumulation of realgar in whole blood of mice and consequently alleviate its toxicity. Rhei Radix et Rhizoma, Scutellariae Radix, Platycodonis Radix, and Glycyrrhizae Radix et Rhizoma were found to be the herbal basis of this detoxification effect on realgar in NJT.

Data Availability
e data used to support the findings of this study are available from the corresponding authors upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest associated with this publication.   Figure S1. Photomicrographs of representative sections of the liver from the control group (a), group R (b), group NJT (c), group RBC (d), group RBS (e), group RGF (f ), group RRR (g), group RSR (h), group RPR (i), group RGR (j), and group RFH (k). Figure S2. Photomicrographs of representative sections of the kidney from the control group (a), group R (b), group NJT (c), group RBC (d), group RBS (e), group RGF (f ), group RRR (g), group RSR (h), group RPR (i), group RGR (j), and group RFH (k). (Supplementary  Materials)