Elsevier

Toxicology

Volume 280, Issue 3, 27 February 2011, Pages 126-134
Toxicology

Metabolites and JAK/STAT pathway were involved in the liver and spleen damage in male Wistar rats fed with mequindox

https://doi.org/10.1016/j.tox.2010.12.001Get rights and content

Abstract

Mequindox (MEQ) is a novel synthetic quinoxaline 1,4-dioxides antibacterial agent and growth promoter in animal husbandry. This study was to investigate whether reactive oxygen species (ROS), the Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway, suppressors of cytokine signaling (SOCS) and inflammatory cytokines were involved in toxicities of MEQ. Our data demonstrated that high dose of MEQ (275 mg/kg) apparently led to tissue impairment combined with imbalance of redox in liver. In liver and spleen samples, hydroxylation metabolites and desoxymequindox were detected, directly confirming the potential link of N  O group reduction metabolism with its organ toxicity. Moreover, up-regulation of JAK/STAT, SOCS family, tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) were also observed in the high-dose group. Meanwhile, significant changes of oxidative stress indices in liver were observed in the high-dose group. As for NADPH subunit, the mRNA levels of many subunits were significantly up-regulated at low doses but down-regulated in a dose-dependent manner in liver and spleen, suggesting an involvement of NADPH in MEQ metabolism and ROS generation. In conclusion, we reported the dose-dependent long-term toxicity as well as the discussion of the potential mechanism and pathways of MEQ, which raised further awareness of its toxicity following with the dose change.

Introduction

Since 1970s, quinoxaline 1,4-dioxide derivatives (QdNOs) are one of the useful classes of synthetic agents with wide range of biological activities like growth promoter, antibacterial, anti-candida, antitubercular, anticancer and antiprotozoal properties (Vicente et al., 2009). Owing to their worthwhile effects in animal husbandry, these compounds have been added to cattle, swine, and poultry feeds to improve the growth at subtherapeutic levels. Our previous studies in vitro have demonstrated that the N  O group reduction was found to be the main metabolic pathway of QdNOs metabolism (Liu et al., 2008, Liu et al., 2009, Liu et al., 2010a, Liu et al., 2010b) and the reduction of QdNOs may lead to the formation of reactive oxygen species (ROS) and cell apoptosis (Huang et al., 2010).

Mequindox (MEQ), a new synthetic QdNOs antibacterial agent, was found to cause liver and adrenal toxicity by acute and sub-chronic oral administration at high doses (Huang et al., 2009, Ihsan et al., 2010). In vitro research revealed that more than 10 metabolites of MEQ were characterized and observed in rat, chicken and pig liver microsomes (Liu et al., 2010a). In addition, NADPH oxidase was considered as a major source of ROS generation in both physiologic and pathophysiologic situations (Abid et al., 2007, Cave et al., 2006, Ying et al., 2009, Teufelhofer et al., 2003). From the information obtained above, we may hypothesize that the metabolism of MEQ could also be happened in vivo through deoxidize reaction with the help of NADPH enzyme, finally leading to the cell and tissue damage. But up till now, there is no attempt to testify if a direct linkage of the metabolism and toxic mechanism of MEQ existed in vivo.

Sub-chronic study in our lab has shown that MEQ can result in cellular swelling, centrilobular liver cell necrosis and disorganized hepatic cord pattern in liver in Wistar rats (Ihsan et al., 2010). In vitro study revealed that the prooxidant properties and imbalance in the redox metabolism of QdNOs would lead to dose and time-dependent cell damage via the mitochondria-dependent pathway (Huang et al., 2010). However, the mechanism of MEQ results in liver damage in vivo still remains unclear.

The Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway was assumed to be involved in the ROS mediated tissue dysfunction. It was shown as an important mediator of ROS-induced apoptosis in Jurkat T cells and mouse splenocytes (Oh et al., 2009). In addition, suppressors of cytokine signaling (SOCS) may act as part of a negative feedback loop to attenuate signal transduction from cytokines that act through JAK/STAT signaling pathway. Prolonged activation of STAT1 and STAT3 induced by interleukin-6 (IL-6) were found in SOCS3-deficient cells (Lang et al., 2003, Croker et al., 2003). The SOCS family was also involved in liver damage (Naka et al., 1998, Starr et al., 1998) and negatively regulates IL-6 signaling in vivo (Croker et al., 2003). Moreover, inflammatory factors such as tumor necrosis factor (TNF-α) and IL-6 may play important roles in hepatic steatosis (Sánchez-Garrido et al., 2009, Garcia-Ruiz et al., 2003, Naugler et al., 2007).

Based on the above studies, we may hypothesize that ROS generated by MEQ metabolism, JAK/STAT, SOCS family and inflammatory factors may be involved in MEQ-induced oxidative stress and hazard effects on organ damage at high dose. Since the liver has been documented to be the most common toxicological target of all toxicants and the spleen is the most important lymphoid organ, the present study was to evaluate the hepatic and lienal toxicity in male Wistar rats fed with MEQ for 180 days. In order to find direct evidence for the linkage of MEQ metabolism and its toxicity, MEQ and its metabolites in liver and spleen samples were detected by using high-performance liquid chromatography combined with ion trap/time-of-flight mass spectrometry (LC/MS–ITTOF). Five NADPH enzymes in liver and spleen were monitored to further explore the relationship between drug metabolism and oxidative damage. Morphological changes and oxidative stress indices as well as IL-6 and TNF-α in liver and spleen were examined. Meanwhile, real-time reverse transcriptional polymerase chain reaction (RT-PCR) was used to estimate the dose–response interaction of gene expression. Components of JAK/STAT pathway and SOCS family were detected to further elucidate the crosstalk among JAK/STAT pathways, SOCS family and inflammatory factors involved in organ impairment originated by ROS.

Section snippets

Materials

Mequindox (C11H10N2O3, mol. wt. 218.21, purity 98%) was purchased from Beijing Zhongnongfa Pharmaceutical Co. Ltd. (Huanggang, PR China). All chemicals were purchased from Sigma (St. Louis, USA) unless otherwise stated.

Animals and diets

Male Wistar rats (4 W of age, n = 75) were purchased from Center of Laboratory Animals of Hubei Province (Wuhan, PR China). Prior to initiation of dosing, all rats were quarantined for 13 days and evaluated for weight gain and any gross signs of disease or injury. After quarantine,

Effect of MEQ on pathological changes in liver and spleen

There were significant increases in the relative organ to body weight ratios of liver and spleen in male rats when compared with controls (Table 2). The result showed important evidence of drug-related toxicity in the high-dose group.

In order to see whether MEQ diet cause any direct pathological alterations in rats, H & E was used to stain the liver and spleen tissues of rats as described above. As shown in Fig. 1, marked morphological changes were observed at high dose (275 mg/kg) when compared

Discussion

The main findings in this report are: (1) High dose of MEQ in the diet for 180 days led to liver and spleen impairment. (2) The N  O group reduction was the main metabolic pathway of MEQ metabolism in vivo, directly confirming the potential linkage of drug metabolism with its organ toxicity. (3) The mRNA levels of five components of JAK/STAT pathway as well as IL-6, TNF-α and SOCS family were up-regulated in liver and spleen, indicated they were involved in the dose-dependent long-term toxicity

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by grants from National Basic Research Program of China (Grant No. 2009CB118800) and Chinese Fundamental Research Funds for the Central Universities (Grant No. 2009QC001).

References (39)

  • M. Scheutwinkel-Reich et al.

    Sister-chromatid exchange in Chinese hamster V79 cells exposed to quindoxin, carbadox and olaquindox

    Mutat. Res.

    (1984)
  • E. Vicente et al.

    Selective activity against Mycobacteriumtuberculosis of new quinoxaline 1,4-di-N-oxides

    Bioorg. Med. Chem.

    (2009)
  • R.S. Arnold et al.

    Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase Nox1

    Proc. Natl. Acad. Sci. U. S. A.

    (2001)
  • H.J. Badham et al.

    In utero exposure to benzene disrupts fetal hematopoietic progenitor cell growth via reactive oxygen species

    Toxicol. Sci.

    (2010)
  • L. Beutin et al.

    Mutagenicity of quindoxin, its metabolites, and two substituted quinoxaline-di-N-oxides

    Antimicrob. Agents Chemother.

    (1981)
  • G. Byfield et al.

    The role of supplemental oxygen and JAK/STAT signaling in intravitreous neovascularization in a ROP rat model

    Invest. Ophthalmol. Vis. Sci.

    (2009)
  • A.C. Cave et al.

    NADPH oxidases in cardiovascular health and disease

    Antioxid. Redox Signal.

    (2006)
  • G. Chowdhury et al.

    Enzyme-activated, hypoxia-selective DNA damage by 3-amino-2-quinoxalinecarbonitrile 1,4-di-N-oxide

    Chem. Res. Toxicol.

    (2004)
  • B.A. Croker et al.

    SOCS3 negatively regulates IL-6 signaling in vivo

    Nat. Immunol.

    (2003)
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