Catalytic characteristics of CYP3A22-dependent mequindox detoxification

doi:10.1016/j.catcom.2010.12.029

Catalysis Communications

Volume 12, Issue 7, 10 March 2011, Pages 637-643

https://doi.org/10.1016/j.catcom.2010.12.029 Get rights and content

Abstract

The anti-bacterial agent mequindox is widely used as an animal feed additive in China. Its potential effect on host cells, however, remains largely unknown. Here, we report that pig CYP3A22 represents an important metabolic enzyme in mequindox catalysis. Interestingly, we found that ectopic expression of CYP3A22 reversed the mequindox-induced G2 cell cycle accumulation in cultured pig hepatocytes. To further probe the catalytic mechanism of CYP3A22 in mequindox metabolism, we identified 2′-acetyl-hydroxylation as a major catalytic step of CYP3A22-dependent mequindox metabolism using LC–MS/MS spectrometry. Together, our study suggests that CYP3A22 plays an important role in mequindox metabolism via 2′-acetyl-hydroxylation.

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).

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T-2 toxin induces the expression of porcine CYP3A22 via the upregulation of the transcription factor, NF-Y
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The porcine CYP3A protein subfamily consists of CYP3A22, CYP3A29, CYP3A39 and CYP3A46 [9]. Porcine CYP3A22, which is most highly expressed in the liver, has been identified as a key enzyme in the metabolism of exogenous compounds, such as T-2 toxin [10] and mequindox [11]. In addition, our previous research discovers that CYP3A22 detoxifies T-2 toxin by transforming T-2 toxin into 3′-OH-T-2 toxin in porcine liver.
T-2 toxin is one of the major pollutants in crops and feedstuffs. CYP3A22, one of hCYP3A4 homologs, detoxifies T-2 toxin in pigs. We investigated the mechanisms of expression activation of CYP3A22 under basal and induced conditions.
Based on MatInspector analysis, several mutations in the CYP3A22 promoter were assayed by dual luciferase reporter to identify the function of cis elements in the region. EMSA experiments were used to assess the binding of transcription factors to the cis elements. The mRNA and protein levels of CYP3A22 and the transcription factors were measured by RT-qPCR and Western blot. The enhancement of NF-Y binding to the CYP3A22 promoter was assayed by ChIP.
As predicted, two cis DNA elements in the CYP3A22 promoter, a CCAAT box and GC box, were confirmed to be crucial in the activation of CYP3A22 transcription. These two DNA motifs recruited two transcription factors, NF-Y and Sp1, which are involved in the activation of the basal transcription of CYP3A22. More interestingly, CYP3A22 expression was induced in porcine primary hepatocytes by the treatment with 0.1 μg/mL T-2 toxin. This induction of transcription by T-2 toxin was dominantly regulated by the binding of NF-Y to the CCAAT box, rather than GC box, which recruits Sp1 and functions only in the constitutive expression of CYP3A22.
Our study reveals the regulatory mechanisms of both basal and inducible transactivation of CYP3A22 in pigs. In particular, we identified that the mechanism by which T-2 toxin induces CYP3A22 expression is mediated by the upregulation of NF-YA.
Although porcine CYP3A22 is homologous to hCYP3A4, the regulation of basal and induced expression of CYP3A22 occurred via distinct mechanisms. This may account for the variety of CYP3A expression in animals and humans.
Hydroxylation of quinocetone and carbadox is mediated by CYP1As in the chicken (Gallus gallus)
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These results suggested that CYP1A (s) had no detectable hydroxylation activity toward mequindox and cyadox in the present reconstituted CYP1A monooxygenase system. Metabolism of quinoxaline drugs has been studied intensively in the past years and many metabolites have been identified (Liu et al., 2009, 2010a,b; J. Liu et al., 2011; Z.Y. Liu et al., 2011). However, most of previous metabolism studies were conducted in vivo or using microsomes (Liu et al., 2009, 2010 a,b; Z.Y. Liu et al., 2011), the enzymes taking part in quinoxaline transformation have not been clearly identified.
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Pharmacokinetics of mequindox and its metabolites in rats after intravenous and oral administration
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In the present study, pentobarbital sodium associating with the induction of cytochrome P450 (CYP450) enzymes was used to anesthetize animals prior to jugular vein catheterization, which might have some effect on the dispositions of MEQ and its metabolites. Liu et al. (2011b) reported that CYP3A22 was an important enzyme in MEQ metabolism in pig via 2′-acetyl-hydroxylation. By means of comparisons of the metabolic rates of MEQ which was incubated in the liver microsomes systems of rats (unpublished data), chicken (Liang, 2008) and pigs (Si, 2009) with CYP450 enzymes inhibitors or not, we also found that CYP3A22, CYP2D6, CYP2E1 could be the subtypes of CYP450 enzymes which were involved with the metabolisms of MEQ in these three species.
Pharmacokinetics of mequindox (MEQ) and its metabolites were determined in rats after intravenous (i.v.) and oral (p.o.) administration of MEQ at a single dose of 10 mg kg⁻¹ bodyweight. After both administrations, MEQ and five of its metabolites were quantified, except M4, whereas M1 and M2 were the predominant ones. The areas under the concentration–time curves (h ng mL⁻¹) of MEQ, M1, M2, M3, M5 and M10 after i.v. administration were 7559 ± 495, 6354 ± 2761, 5586 ± 2337, 1034 ± 160, 2370 ± 791 and 1813 ± 622, respectively, whereas after p.o. administration, remained as 2809 ± 40, 4361 ± 3544, 4351 ± 1046, 1444 ± 814, 3864 ± 305 and 1213 ± 569, respectively. The elimination half-lives (h) of these compounds after i.v. administration were 3.48 ± 0.80, 4.20 ± 0.76, 6.25 ± 2.41, 4.77 ± 1.54, 4.69 ± 1.62 and 16.89 ± 5.15, respectively, and were 3.21 ± 0.40, 3.66 ± 1.06, 4.20 ± 1.03, 8.91 ± 5.99, 4.20 ± 2.02 and 20.84 ± 10.85 after p.o. administration, respectively. After p.o. administration, the bioavailability of MEQ was 37.16%. The results showed that MEQ was extensively metabolized in rats and rapidly absorbed after p.o. administration.
Simultaneous determination of four synthesized metabolites of mequindox in urine samples using ultrasound-assisted dispersive liquid-liquid microextraction combined with high-performance liquid chromatography
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Mequindox (2-methyl-3-acetylquinoxaline-1,4-dioxide; C11H10N2O3; MEQ) is a novel synthetic QdNOs derivative that was developed by the Lanzhou Institute of Animal Husbandry and Veterinary Drugs, Chinese Academy of Agriculture Sciences. MEQ has been applied to the treatment of diseased piglets, including those with white diarrhea and swine dysentery [7]. Although MEQ was considered to have better growth-enhancing activity and to be safer than carbadox and olaquindox when administered to animals [2], very little information was available about the cytotoxicity and genotoxicity, and the metabolomics of mequindox are quite poorly understood.
A novel pretreatment method termed ultrasound-assisted dispersive liquid–liquid microextraction (UADLLME) coupled with high-performance liquid chromatography-ultraviolet detector (HPLC-UV) was applied for the detection of four synthesized metabolites of mequindox in pig urine samples. A total volume of 200 μL of methanol (dispersant) and 60 μL of 1,1,2,2-tetrachloroethane (extract) were injected into 5.0 mL of urine sample and then emulsified by ultrasound treatment for 4 min to form a cloudy solution. The effect of several factors on the recovery of each metabolite was investigated by a fitting derivation method for the first time. Under optimum conditions, the method yields a linear calibration curve in the concentration range from 0.5 to 500 μg/L and a limit of detection (LOD) of 0.16–0.28 μg/L for target analytes. The recoveries ranged from 72.0% to 91.3% with a relative standard deviation (RSD) of less than 5.2%. The enrichment factors for the four compounds ranged from 75 to 95. Two pig urine samples were successfully analyzed using the proposed method.
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Short CommunicationCatalytic characteristics of CYP3A22-dependent mequindox detoxification

Abstract

Graphical Abstract

Research Highlights

Introduction

Section snippets

Primary hepatocyte isolation and culture

Mequindox results in G2 cell cycle arrest in pig hepatocytes

Discussion

Conclusion

Acknowledgements

Regulatory Toxicology and Pharmacology

Food and Chemical Toxicology

Toxicology Letters

Catalysis Communications

Journal of Chromatography A

Leukemia Research

Toxicology Letters

Analytica Chimica Acta

Steroids

Short Communication
Catalytic characteristics of CYP3A22-dependent mequindox detoxification