Short CommunicationCatalytic characteristics of CYP3A22-dependent mequindox detoxification
Graphical Abstract
Research Highlights
►Mequindox induced G2 arrest of pig hepatocytes. ►High expression of CYP3A22 reversed the drug-induced G2 arrest. ►The catalytic activity of CYP3A22 is crucial for the mequindox detoxification.
Introduction
Mequindox is a quinoxaline-N-dioxide derivative that possesses antibiotic activity against gram-negative bacteria, such as salmonella [1]. Subsequent to its discovery in the 1980s, mequindox has been widely used in animal feeds and as veterinary medication to treat livestock diseases, including swine dysentery and white diarrhea of piglet [2]. Mequindox shares structural and pharmacologic properties with other synthetic organic compounds in the quinoxaline family, including carbadox and olaquindox (Supplementary Figure 1). Due to their mutagenic activity, developmental and reproductive toxicity, and carcinogenicity [3], [4], [5], the use of carbadox and olaquindox as feed additives was prohibited by the European Union and Health Canada in 1999 and 2001, respectively [3], [4], [5]. In addition to preventing transmission of infectious diseases within pig populations, mequindox is routinely added to animal feeds in China. However, little effort has been made to evaluate the safety issues mequindox imposes on its hosts.
We previously reported that pig CYP3A22 mediates T-2 toxin biotransformation via hydroxylation [6]. Ten mequindox metabolites were observed in pig liver microsome incubation experiments by high performance liquid chromatography (HPLC) [7]. As one of the most abundant species in P450 enzymes in pig liver, we speculated that CYP3A22 might play a critical role in mequindox metabolism. In this study, we found that mequindox treatment resulted in substantial cell accumulation in primary hepatocyte cultures of piglets at the G2 cell cycle phase. This effect was reversed by the ectopic expression of CYP3A22. In addition, liquid chromatography–tandem mass spectrography (LC–MS/MS) analysis of mequindox metabolites revealed that 2′-acetyl-hydroxylation is the crucial catalytic pathway of CYP3A22 in mequindox metabolism.
Section snippets
Primary hepatocyte isolation and culture
Hepatocytes were isolated from the liver of 3-day-old domestic Chinese piglets, which were crossbred from Duroc, Large Yorkshire, and Landrace breeds, as previously described [1], [4], [8], [9]. The viability of the isolated hepatocytes was estimated using the trypan blue exclusion test. Afterwards, cells were seeded on 6-well plates at a density of 1 × 105 cells/cm2 in a complete medium (William's E medium supplemented with 8% FBS, 500 U/ml penicillin G, 0.5 mg/ml streptomycin, 10 μM dexamethasone
Mequindox results in G2 cell cycle arrest in pig hepatocytes
To explore the cellular effects of mequindox on its hosts, we prepared primary hepatocytes from pigs and evaluated changes in cell proliferation parameters. We monitored the cell cycle distribution of both control hepatocytes and those treated with mequindox. Cells were collected at 24 h intervals and subjected to flow cytometry analysis. Significantly, mequindox-treated hepatocytes displayed a prominent arrest at the G2/M cell cycle phase after 48 h of treatment, as compared to the control (
Discussion
In contrast to synthetic quinoxalines, such as carbadox and olaquindox, which have been prohibited due to their mutagenic and carcinogenic properties, mequindox remains a common feed additive in China [11]. Mequindox is an analogue of carbadox and olaquindox (Supplementary Figure 1) [12], but its metabolites are unknown because there are no available standards for metabolite measurement, and the metabolites are rapidly eliminated in vivo [13]. In this study, we used a recombinant adenoviruses
Conclusion
In summary, our results support a new catalytic pathway for mequindox and identify CYP3A22 as an important enzyme in mequindox metabolism via 2′-acetyl-hydroxylation. Our study suggests that CYP3A22 reduces the toxicity of mequindox, in part, by alleviating the mequindox-perturbed cell cycle in primary culture hepatocytes. All of these data will raise more concerns about the toxic effects of mequindox use and provide a new basis for evaluating the safety issues mequindox imposes on its hosts.
Acknowledgements
This work was supported by the National Basic Research Program of China (973 Program), No: 2009CB118802; the Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (2009); the Program for New Century Excellent Talents in University, No: NCET-08-0643; and the Guangdong Province Universities and Colleges Special Funds for Talents Introduction (2008).
References (19)
- et al.
Acute and subchronic toxicological evaluation of Mequindox in Wistar rats
Regulatory Toxicology and Pharmacology
(2010) - et al.
Investigation of the genotoxicity of quinocetone, carbadox and olaquindox in vitro using Vero cells
Food and Chemical Toxicology
(2009) Teratogenic assessment of carbadox in rats
Toxicology Letters
(2002)- et al.
The catalytic activity of cytochrome P450 3A22 is critical for the metabolism of T-2 toxin in porcine reservoirs
Catalysis Communications
(2010) - et al.
Development of a high-performance liquid chromatography method for the simultaneous quantification of quinoxaline-2-carboxylic acid and methyl-3-quinoxaline-2-carboxylic acid in animal tissues
Journal of Chromatography A
(2007) - et al.
Unexpected cytokinetic effects induced by puromycin include a G2-arrest, a metaphase-mitoticarrest, and apoptosis
Leukemia Research
(1992) - et al.
The metabolism and N-oxide reduction of olaquindox in liver preparations of rats, pigs and chicken
Toxicology Letters
(2010) - et al.
Recent developments in the use and abuse of growth promoters
Analytica Chimica Acta
(2002) - et al.
CAR and PXR: the xenobiotic-sensing receptors
Steroids
(2007)
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