Minerals (metals) in traditional medicines are the matter of debate on their efficacy and toxicity potentials [7, 20]. In the present research, we examined the effects of β-HgS-containing Zuotai and Zuotai-containing 70W on hepatic CYP 1-4 and CYP-7 families, and their corresponding nuclear receptors, compared to HgCl2 and MeHg at both mRNA and protein levels. Briefly, 70W at 1 to 5-fold clinical doses and Zuotai (30 mg/kg, po) for 7 days did not produce significant effects on the liver CYP450 gene and protein expressions; while HgCl2 at equivalent Hg dose increased the expression of CYP1A, CYP2B, CYP2E1, and CYP7A at the transcription and/or protein levels, MeHg at 1/10 Hg dose produced similar effects, but to a lesser extent. These results further demonstrated that chemical forms of metals are a major determinant of their biological effects [24-27, 29-31].
Zuotai in Tibetan Medicines
The Tibetan medicine has thousand years of history and is still used in the world today for a variety of diseases, including liver diseases [1-6]. Herbal-metallic preparations are believed to assist the delivery of drugs to the target, contribute to therapeutic effects, and reduce toxicity [7, 20]. 70W is one of famous Tibetan medicines listed in the 2015 Edition of Chinese Pharmacopoeia in the treatment of various diseases [3-7]. We have shown that 70W is effective against CCl4-induced liver injury [11] and protected LPS plus MPTP-induced neurotoxicity in mice [8], and the present study further demonstrated that the hepatoprotective effects of 70W is not due to the inhibition of CYP450 to reduce CCl4 bioactivation, rather than due to activation of the Nrf2 antioxidant pathway [11].
Zuotai is included in 70W in a small amount [2-4, 8]. The chemical speciation, spatial distribution of mercury from Zuotai are different from that of HgCl2 [7, 18], resulting in differential toxicity when compared to environmental mercury compounds (HgCl2 and MeHg) in experimental animals and cultured human cells [24-27, 29-31]. In patients taking Zuotai-containing Tibetan medicines, mercury toxicity to the liver and kidney was mild, tolerable and reversible [10, 21-23]. 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 [19, 21-23].
Mercury effects on CYP-1 family
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 family [32]. 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 expression [33, 34]. However, in rats, Zuotai at higher doses decreased CYP1A2 activity [35]. In comparison, the effects of HgCl2 on CYP1A expression were more dramatic. In Zebrafish, low dose (0.1 LC50) of HgCl2 increased CYP1A1, but at higher doses (0.4 and 0.8 LC50), the expression of CYP1A1 was suppressed [36]. Zuotai at the concentration of 0.2 mg/mL (below LC50) could increase CYP1A1 and CYP1B1 in Zebrafish [36]. In the mouse heart, kidney and lung, HgCl2 (2.5 mg/kg, ip) increased CYP1A1, along with other CYP450 isoforms [37]. However, in the interactions with AhR ligand TCDD, TCDD induction of Cyp1a1, Cyp1a2, and Cyp1b1 was suppressed by HgCl2 (2.5 mg/kg, ip) in livers of mice [38]. In the present study, HgCl2 at 33.6 mg/kg increased CYP1A2 at mRNA and protein levels and tended to increase AhR mRNA, largely in agreement with the above literature. In another study, mice chronically (6 weeks) received HgCl2 (32 mg/kg) and MeHg (2.6 mg/kg), expression of hepatic Cyp1a1 and Cyp1b1 was increased by MeHg, and tended to increase by HgCl2, while cinnabar (HgS, 300 mg/kg) and cinnabar-containing An-Gong-Niu-Huang Wan were ineffective [39]. Thus, Effects of mercury on CYP1 family are dependent on the mercury forms, the dose, route, and duration of administration.
Mercury effects on CYP-2 and CYP-3 family
CYP-2 family is easily induced by many xenobiotics such as phenobarbital. The CAR is shown to play a crucial role in the activation of CYP2B genes by xenobiotics [17]. CYP-2 family mainly includes 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 mitochondria [40]. HgCl2-induced hepatotoxicity and oxidative stress is partially mediated through its effects on CYP2E1 [41]. HgCl2 (2.5 mg/kg, ip) increased Cyp2b9, Cyp2b10 in mouse heart [38], and HgCl2 (33.6 mg/kg, po) increased Cyp2b10 expression in the livers of mice [34]. Similar to CYP1A2, higher doses of systemic HgCl2 decreased hepatic Cyp2e1 in livers of rats [35]. 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 [14, 15].CYP3A can be induced or inhibited by a variety of substances, influence factors and individual differences. This enzyme is an activator of aflatoxin B, can be transformed into liver cancer cells induced by carcinogens. CYP3A can catalyze the structurally different substrates, including calcium channel blockers and antiarrhythmic drugs. In the present study conditions, 70W and mercury compounds had minimal effects on Cyp3a11 mRNA and CYP3A protein expression. However, the polymorphisms in human CYP3A genes (CYP3A4. CYP3A5 and CYP3A7) may modify the response to dietary MeHg exposure during early life development [42]. In mice chronically dosed with HgCl2 (32 mg/kg) and MeHg (2.6 mg/kg), cinnabar (HgS, 300 mg/kg) and cinnabar-containing An-Gong-Niu-Huang Wan, expression of hepatic Cyp3a11 and 3a25 were increased [39]. The length of Hg compound administration makes a difference as compared to the present study.
Mercury effects on CYP-4 and CYP-7 family
CYP4A is involved in lipid metabolism and is regulated by PPARα, their dysregulations are implicated in xenobiotics induced adverse effects leading to various human diseases [17]. Researchers found mercury 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, alone with CYP4A genes (Cyp4a10, Cyp4a12, Cyp4a14) [37]. In the present study, 70W and Zuotai at 1-5 fold clinical doses do not have appreciable effects on PPARα and Cyp4a10. HgCl2 produced a slight increase in PPARα and Cyp4a10in previous studies [33], and in mice chronically dosed with HgCl2 (32 mg/kg) and MeHg (2.6 mg/kg), Expression of Cyp4a10 was increased, but cinnabar (HgS, 300 mg/kg) and cinnabar-containing An-Gong-Niu-Huang Wan was ineffective [39]. However, in the present study, only the trends of increase were evident.
CYP7A1 is a rate-limiting enzyme for bile acid synthesis and is regulated by FXR [28]. Little is known on the effects of mercury compounds on FXR and CYP7A1 expression. This is the first reports to show that HgCl2 and MeHg had capability of induction of Cyp7a1 mRNA and CYP7A1 protein. The biological effects of CYP7A1 induction by HgCl2 and MeHg warrant further investigation.