Metabolic Activation and Covalent Protein Binding of Berberrubine: Insight into the Underlying Mechanism Related to Its Hepatotoxicity

Abstract

Introduction: Berberrubine (BRB), an isoquinoline alkaloid, is a major constituent of medicinal plants Coptis chinensis Franch or Phellodendron chinense Schneid. BRB exhibits various pharmacological activities, whereas exposure to BRB may cause toxicity in experimental animals.

Methods: In this study, we thoroughly investigated the liver injury induced by BRB in mice and rats. To explore the underlying mechanism, a study of the metabolic activation of BRB was conducted. Furthermore, covalent modifications of cysteine residues of proteins were observed in liver homogenate samples of animals after exposure to BRB, by application of an exhaustive proteolytic digestion method.

Results: It was demonstrated that BRB-induced hepatotoxicities in a time- and dose-dependent manner, based on the biochemical parameters ALT and AST. H&E stained histopathological examination showed the occurrence of obvious edema in liver of mice after intraperitoneal (i.p.) administration of BRB at a single dose of 100 mg/kg. Slight hepatotoxicity was also observed in rats given the same doses of BRB after six weeks of gavage. As a result, four GSH adducts derived from reactive metabolites of BRB were detected in microsomal incubations with BRB fortified with GSH as a trapping agent. Moreover, four cys-based adducts derived from reaction of electrophilic metabolites of BBR with proteins were found in livers.

Conclusion: These results suggested that the formation of protein adducts originating from metabolic activation of BRB could be a crucial factor of the mechanism of BRB-induced toxicities.

Keywords: berberrubine; hepatotoxicity; metabolic activation; protein modification.

Figure 1

Chemical structure of berberrubine: Section (A) the methylenedioxyphenyl structure; section (B) the methylated catechol structure.

Figure 2

Acute hepatotoxicity in mice: Time-dependent manner of ALT (A) or AST (B), mice were treated with BRB (i.p.) at 50mg/kg (n=6); dose-dependent manner of ALT (C) or AST (D), mice were treated with BRB (i.p.) at 0, 25, 50 or 100mg/kg (n=6); representative histopathology of liver from mice in BRB-treated group at 2 h after administration (E) and control group (F), all processed livers were microscopically (Scale bar=50um) examined.*p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group.

Figure 3

Sub-chronic hepatotoxicity in rats: Time-dependent and dose-dependent manner of ALT (A) or AST (B), rats were treated with BRB (i.g.) at 0, 25, 50, or100mg/kg/d (n=8) for 6 weeks; representative histopathology of liver from rats in BRB-treated group at the sixth week after administration (C) and control group (D), all processed livers were microscopically (Scale bar=50um) examined.*p < 0.05, **p < 0.01 compared with the control group.

Figure 4

Identification of metabolite M1: BRB was incubated with mouse liver microsomes fortified with NADPH and GSH at 37 °C for 1h, followed by LC-MS/MS analysis. Extracted ion chromatogram of M1 obtained from LC-Q-TOF/MS analysis of MLM incubations containing BRB and GSH in the absence (A) or presence (B) of NADPH; MS/MS spectrum of M1 (C).

Figure 5

Identification of metabolite M2: BRB was incubated with mouse liver microsomes fortified with NADPH and GSH at 37 °C for 1h, followed by LC-MS/MS analysis. Extracted ion chromatogram of M2 obtained from LC-Q-TOF/MS analysis of MLM incubations containing berberrubine and GSH in the absence (A) or presence (B) of NADPH; MS/MS spectrum of M2 (C).

Figure 6

Identification of metabolite M4: BRB was incubated with mouse liver microsomes fortified with NADPH and GSH at 37 °C for 1h, followed by LC-MS/MS analysis. Extracted ion chromatogram of M4 obtained from LC-Q-TOF/MS analysis of MLMincubations containing berberrubine and GSH in the absence (A) or presence (B) of NADPH; MS/MS spectrum of M4 (C).

Figure 7

Identification of metabolites M5: BRB was incubated with mouse liver microsomes fortified with NADPH and GSH at 37 °C for 1h, followed by LC-MS/MS analysis. Extracted ion chromatogram of M5 obtained from LC-Q-TOF/MS analysis of MLM incubations containing berberrubine and GSH in the absence (A) or presence (B) of NADPH; MS/MS spectrum of M5 (C).

Figure 8

Identification of metabolites A1: BRB-treated mouse liver homogenates were proteolytically digested, followed by LC-MS/MS analysis. Extracted ion chromatogram of A1 obtained from LC-Q-TOF/MS before (A) or after exhaustive proteolytic digestion (B); MS/MS spectrum of A1 (C).

Figure 9

Identification of metabolites A3: BRB-treated mouse liver homogenates were proteolytically digested, followed by LC-MS/MS analysis. Extracted ion chromatogram of A3 obtained from LC-Q-TOF/MS before (A) or after exhaustive proteolytic digestion (B); MS/MS spectrum of A3 (C).

Scheme 1

Proposed metabolic activation pathways of BRB (M1-M6).

Scheme 2

Proposed pathways of protein and amino acid adduct formation as a result of BRB metabolism (A1-A6).

Figure 10

Identification of metabolites A5: BRB-treated mouse liver homogenates were proteolytically digested, followed by LC-MS/MS analysis. Extracted ion chromatogram of A5 obtained from LC-Q-TOF/MS before (A) or after exhaustive proteolytic digestion (B); MS/MS spectrum of A5 (C).

Figure 11

Identification of metabolites A6: BRB-treated mouse liver homogenates were proteolytically digested, followed by LC-MS/MS analysis. Extracted ion chromatogram of A6 obtained from LC-Q-TOF/MS before (A) or after exhaustive proteolytic digestion (B); MS/MS spectrum of A6 (C).