Zuotai (β-HgS)-containing 70 Wei Zhen-Zhu-Wan differs from mercury chloride and methylmercury on hepatic cytochrome P450 in mice

Reagents

70W and Zuotai was provided by Tibetan Medicine Manufacture Factory as described previously⁸, based on the 2015 edition of Pharmacopoeia of China for QA/QC control (Lot number Z20110561). 70W was prepared by grinding the pill into powder, adding distilled water to prepare the suspension for oral administration. Mercury chloride (HgCl₂ Cat# M1136) and methylmercury (MeHgCl Cat# 442534) were from Sigma (St. Louis, MO, USA). All other chemicals were commercially available reagents.

Animals

Male Kunming mice (20 ± 2 g) were purchased from Animal Experimental Center of theThird Military Medical University (Chongqing, China). Animals were maintained in the SPF-grade facilities at Zunyi Medical University, with a controlled environment (22 ± 1°C, 50 ± 2% humidity and a 12 h: 12 h light: dark cycle) and free access to purified water and standard laboratory feed. Efforts were made to ameliorate distress and harm to animals by daily monitoring and humane treatment of the animals. To reduce the use of animals, the minimal number of mice (n=5)/group according to the experiment requirement was used which are sufficient for statistical analysis. All animal care and experimental protocols are complied with the Animal Management Guidelines of the Chinese Ministry of Health and approved by Animal Use and Care Committee of Zunyi Medical University (2015-07).

Animal treatments

Mice were randomly divided into seven groups of five mice each (Total number n=35), respectively as the control, 70W (0.15, 0.5, 1.5g/kg), Zuotai (30 mg/kg, the amount contained in 70W), HgCl₂ (33.6 mg/kg, equivalent Hg as HgS) and MeHgCl (MeHg, 3.1 mg/kg, 1/10 of Hg). Mice were given oral administration for seven consecutive days. The dose regimen selection was based on our prior publications for 70W (at clinical dose)⁸ or for zuotai and mercury compounds²⁰. Twenty-four hours after the last dose, the animals were euthanized and the livers were collected and stored at 80°C prior to analysis.

Liver toxicity evaluation

The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by commercial kits (Jaingcheng, Nanjing, China)²¹. Liver samples were fixed in 10% formalin prior to routine processing and paraffin embedding. Liver sections (4 µm) were dewaxed in xylene, rehydrated in different concentrations of alcohol (100%, 95%, 80%, 75%) and stained with hematoxylin, followed by counterstaining with eosin. After rinsing, the slides were rehydrated with series of alcohol (75%, 95%. 100%) and mounted with cover glass slip. The slides were examined in nine random fields under a light microscope (Leica Microsystems Ltd., Wetzlar, Germany)^21,22．

Real-time PCR

Approximately 50–100 mg of tissue was homogenized in 1 ml TRIzol (TakaRa Biotechnology, Dalian, China) and the total RNA was extracted according to manufacturer’s instructions. The quality and quantity of RNA were determined by the Nanodrop (Thermo Scientific, ND-2000, USA), with 260/280 ratio >1.8. Total RNA was reverse transcribed with a High Capacity Reverse Transcriptase Kit (Applied Biosystems, Foster City, CA, USA). The primers were designed with Primer3 software and listed in Table 1．

The 15 µL PCR reaction mix contained 3 µL of cDNA (10 ng/µL), 7.5 µL of iQ^TM SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA), 0.5 µL of primer mix (10 µM each), and 4 µL of ddH₂O. After 5 min denature at 95°C, 40 cycles were performed: annealing and extension at 60°C for 45 seconds and denature at 95°C for 10 seconds. Dissociation curve was performed after finishing 40 cycles to verify the quality of primers and amplification. Relative expression of genes was calculated by the 2^-ΔΔCt method and normalized to the house keeping gene β-actin or expressed as a percentage of controls^8,21.

Western blot analysis

Approximately 80 mg of liver tissue was homogenized with RIPA lysis buffer containing 1 mM PMSF and freshly prepared proteinase inhibitors. The homogenates were centrifuged at 12,000 g at 4⍛C for 10 min, and the protein concentration in the supernatants was determined by the BCA assay, and denatures at 90⍛C for 10 min with Nupage loading buffer. Approximately 30 µg proteins were separated in the 10% Nupage gel and transferred to the PVDF membrane. The membranes were blocked in 5% of the skim milk for 1 hour at room temperature, followed by incubation with primary antibodies (CYP1A2 (1:500), CYP2B1 (1:500), CYP2E1 (1:500), CYP3A4 (1:500), CYP4 (1:500), CYP7A1 (1:500), and GAPDH (1:2000)) at 4°C overnight. After washing the membranes with TBST four times, the secondary horseradish peroxidase (HRP) labelled anti-rabbit, or anti-mouse antibodies were added (1:5000) (Beyotime, Shanghai, China), and incubated at room temperature for 1 hour. The enhanced chemiluminescent reagents (ECL) were used to detect the intensity of protein-antibody complexes, and intensity was semi-quantified with Quantity One software (Bio-Rad, USA)¹⁸.

Statistical analysis

Data were expressed as mean and standard error. SPSS 19 was used for statistical analysis. Data were analyzed using a one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test, and a p value < 0.05 was considered significant.

Animal general conditions

At the doses of 70W and Zuotai used in the present study, animals were healthy, without body weight loss and no mortality occurred. No significant elevations of serum ALT and AST were evident, and histology did not reveal overt lesions^12,20–22. HgCl₂ and MeHg groups showed body weight loss and mild histology lesions, consistent with prior publications^12,21,22.

mRNA expression of nuclear receptors and cytochrome P450 genes

Figure 1 illustrates mRNA expression of nuclear receptors (left side) and cytochrome P450 isozyme genes (right side). The aryl hydrocarbon receptor (AhR) mainly mediates the expression of CYP1A enzymes such Cyp1a2; Constitutive androstane receptor (CAR) mediates CYP2 enzymes such as Cyp2b10; Pregnane X receptor (PXR) plays an important role in the regulation of CYP3A enzymes such as Cyp3a11; Peroxisome proliferator-activated receptor α (PPARα) regulates induction of CYP4A enzymes such as Cyp4a10¹⁹. The Farnesoid X receptor (FXR) regulates cholesterol 7α hydroxylase (Cyp7a1) induction²³. The results show that compared with the controls, 70W at 0.15, 0.5, and 1.5 g/kg doses and Zuotai (β-HgS, 30 mg/kg) had no effects on these nuclear receptor and CYP gene expressions. In contrast, HgCl₂ (33.6 mg/kg) and MeHg (3.1 mg/kg) significantly increased AhR, Cyp1a2, CAR, Cyp2b10, PPARα, Cyp4a10, FXR, and Cyp7a1, while having no significant effects on PXR, Cyp3a11 (Figure 1).

Figure 1. Effect of 70W and mercury compounds on nuclear receptor and corresponding CYP gene expression.

Mice were given 70W 0.15, 0.5, and 1.5 g/kg, po. Zuotai (30 mg/kg, po), HgCl₂ (33.6 mg/kg, po), and MeHg (3.1 mg/kg, po) daily for seven days, and hepatic total RNA and protein were extracted for RT-PCR analysis. Data are mean ± SE, n = 5. *Significantly different from control, p< 0.05.

Protein expression of cytochrome P450 isozymes

Figure 2 illustrates protein expression of P450 isozymes. Figure 2A shows representative western-blots for CYP1A2, CYP2B1, CYP2E1, CYP3A, CYP4A, and CYP7A1; Figure 2B shows the statistical analysis of 3-5 replicates. Consistent with mRNA expression, 70W at 0.15, 0.5, and 1.5 g/kg doses and Zuotai (β-HgS, 30 mg/kg) had no apparent effects on cytochrome P450 isozyme protein expressions. In contrast, HgCl₂ (33.6 mg/kg) and MeHg (3.1 mg/kg) significantly increased CYP1A2, CYP2B1, CYP4A, and CYP7A1, while having no effects on CYP3A (Figure 2).

Figure 2. Effect of 70W and mercury compounds on cytochrome P450 isoenzyme protein expression.

Mice were given 70W 0.15, 0.5, and 1.5 g/kg, po. Zuotai (30 mg/kg, po), HgCl₂ (33.6 mg/kg, po), and MeHg (3.1 mg/kg, po) daily for seven days, and hepatic proteins were extracted and pooled for Western-blot analysis. A, the representative western-blot; B, statistical analysis of P450 proteins. Data are mean ± SE of 3-5 replicates. *Significantly different from control, p< 0.05.

70W and Zuotai in Tibetan Medicines

Tibetan medicine has thousands of years of history and is still used in the world today to treat a variety of diseases, including liver diseases^1–4,6. Herbal-metallic preparations are believed to assist the delivery of drugs to the target, contribute to therapeutic effects, and reduce toxicity⁷. 70W is a famous Tibetan medicine listed in the 2015 Edition of Chinese Pharmacopoeia for the treatment of various diseases^3,10. The major ingredients in 70W and the mode of the protection against cerebral ischemia-reperfusion injury has recently been demonstrated¹¹. We have shown that 70W is effective against CCl₄-induced liver injury, protected LPS plus MPTP-induced neurotoxicity⁸, and modulated gut microbiota^8,12. The present study further demonstrated that the hepatoprotective effects of 70W is not due to the inhibition of CYP450 to reduce CCl₄ bioactivation, rather the activation of the Nrf2 antioxidant pathway¹³.

Zuotai is a mineral mixture, with 54% of β-HgS²⁵, and is included in a small amount to many valuable Tibetan medicines^1–3. Mercury (Hg) is a toxic metal; the safety of Hg-containing traditional medicines is of concern²⁴. The chemical speciation, spatial distribution of mercury from Zuotai are different from that of HgCl₂^7,25, resulting in differential toxicity. A recent human study revealed that Zuotai-containing Tibetan medicines are safe at clinical doses^26–28, including 70W²⁹. Indeed, Zuotai differs from HgCl₂ and MeHg in producing hepatotoxicity²¹, nephrotoxicity³⁰, and intestinal toxicity with gut microbiome disruptions²⁰. The present study demonstrated that Zuotai-containing 70W at clinical doses had minimal effects on hepatic CYP450, supporting the notion that Zuotai and 70W at clinical doses are safe^26–29.

Effects of mercury compounds on cytochrome P450

Cytochrome P450 1A1 (CYP1A1) is a hepatic and extrahepatic enzyme that is regulated by the AhR signaling pathway and is regarded as carcinogen activation CYP450 family³¹. CYP-1 family includes CYP1A1, CYP1A2, and CYP1B1，and CYP1A1/CYP1A2 has become a therapeutic tool for the bioactivation of prodrugs, particularly cytotoxic agents. Little is known about effects of 70W on CYP1A family. We have shown previously that oral Zuotai (β-HgS) and cinnabar (α-HgS) had minimal effects of hepatic P4501A family gene expression³². However, in rats, Zuotai at higher doses could decrease CYP1A2 activity³³. In comparison, the effects of HgCl₂ on CYP1A expression were more dramatic. In Zebra fish, a low dose (0.1 LC50) of HgCl₂ increased CYP1A1, but at higher doses (0.4 and 0.8 LC50), the expression of CYP1A1 was suppressed³⁴. In the mouse heart, kidney and lung, HgCl₂ (2.5 mg/kg, ip) increased CYP1A1, along with other CYP450 isoforms³⁵. In the present study, HgCl₂ at 33.6 mg/kg increased CYP1A2 at mRNA and protein levels, largely in agreement with the above literature^32–35 In another study, mice that chronically (6 weeks) received HgCl₂ (32 mg/kg) and MeHg (2.6 mg/kg), had increased expressions of hepatic Cyp1a1 and Cyp1b1, while cinnabar (HgS, 300 mg/kg) and cinnabar-containing An-Gong-Niu-Huang Wan were ineffective³⁶. Thus, the effects of mercury compounds on CYP1 family are dependent on the mercury forms, the dose, route, and duration of administration.

The CYP-2 family is easily induced by many xenobiotics such as phenobarbital. CAR is shown to play a crucial role in the activation of CYP2B genes by xenobiotics¹⁹. The CYP-2 family mainly includes the CYP2B subfamily and CYP2E1. CYP2E1 metabolizes an extensive array of pollutants, drugs, and other small molecules, often resulting in bioactivation to reactive metabolites, which in turn damage mitochondria³⁷. HgCl₂-induced hepatotoxicity and oxidative stress is partially mediated through its effects on CYP2E1³⁸. HgCl₂ (2.5 mg/kg, ip) increased the expression of Cyp2b9 and Cyp2b10 in mice hearts³⁹ and HgCl₂ (33.6 mg/kg, po) increased Cyp2b10 expression in the livers of mice³². Under the present experimental conditions, Cyp2b10 mRNA and CYP2B protein expression were increased by HgCl₂ and MeHg only.

CYP3A is the most abundant subfamily of CYP450, with the highest content in the liver and intestines, and is involved in the metabolism of clinical drugs^17,18. CYP3A can be induced or inhibited by a variety of substances. In the present study conditions, 70W and mercury compounds had minimal effects on Cyp3a11 mRNA and CYP3A protein expression. The length of Hg compound administration could make a difference as compared to the present study.

CYP4A is involved in lipid metabolism and is regulated by PPARα, their dysregulations are implicated in xenobiotics induced adverse effects leading to various human diseases¹⁹. Researchers found that HgCl₂ exposure is associated with increased risk of cardiovascular disease and profound cardiotoxicity, and their results show that mercury treatment caused a significant induction of the cardiac hypertrophy markers, along with CYP4A genes (Cyp4a10, Cyp4a12, Cyp4a14)³⁵. In the present study, 70W and Zuotai at 1–5 times clinical doses do not have appreciable effects on PPARα and Cyp4a10 mRNA expression and CYP4A protein expression, while HgCl₂ and MeHg increased PPARα and Cyp4a10 mRNA, as well as CYP4A protein, consistent with our prior observation that HgCl₂ increased PPARα and Cyp4a10 in livers of mice after seven days of administration³². In mice chronically (6 weeks) dosed with HgCl₂ (32 mg/kg) and MeHg (2.6 mg/kg), the expression of Cyp4a10 was increased, but cinnabar (HgS, 300 mg/kg) and cinnabar-containing An-Gong-Niu-Huang Wan was ineffective³⁶. Increased expression of the CYP-4A family genes under the dose of HgCl₂ and MeHg used in the present study could impact lipid metabolism.

CYP7A1 is a rate-limiting enzyme for bile acid synthesis and is regulated by FXR²³. Little is known on the effects of mercury compounds on FXR and CYP7A1 expression. The present study showed that 70W and Zuotai did not affect CYP7A1, while HgCl₂ and MeHg increased Cyp7a1 mRNA and CYP7A1 protein. The biological effects of CYP7A1 induction by HgCl₂ and MeHg warrant further investigation.

Underlying data

Zenodo: Zuotai (β-HgS)-containing 70 Wei Zhen-Zhu-Wan differs from mercury chloride and methylmercury on hepatic cytochrome P450, http://doi.org/10.5281/zenodo.4403717⁴⁰

This project contains the following underlying data:

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).