Awais Ihsan

Long-term dose-dependent response of Mequindox on aldosterone, corticosterone and five steroidogenic enzyme mRNAs in the adrenal of male rats.

Mequindox (MEQ) is a synthetic quinoxaline 1,4-dioxides (QdNOs) derivative which can effectively improve growth and feed efficiency in animals. This study was to investigate the dose-dependent long-term toxicity in the adrenal of male rats exposed to 180 days of MEQ feed. Our data demonstrated that high doses of MEQ in the diet for 180 days led to adrenal damage and steroid hormone decrease, combined with sodium decrease and potassium increase in rat plasma. Significant changes of GSH and SOD in plasma were observed in the high doses (110, 275 mg/kg) groups. At the same doses, MEQ treatment down-regulated the mRNA levels of CYP11A1, CYP11B1 and CYP11B2 which located in mitochondria, but up-regulated mRNA levels of CYP21 and 3beta-HSD which located in endoplasmic reticulum. In conclusion, we reported the dose-dependent long-term toxicity of MEQ on adrenal gland in male rats, which raise awareness of its toxic effects to animals and consumers, and its mechanism may involve in oxidative stress and steroid hormone biosynthesis pathway.

JAK/STAT Pathway Plays a Critical Role in the Proinflammatory Gene Expression and Apoptosis of RAW264.7 Cells Induced by Trichothecenes as DON and T-2 Toxin

Deoxynivalenol (DON) and T-2 toxin commonly affect cells of the immune system and cause inflammation and apoptosis. Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is highly associated with inflammatory process and apoptosis and is worth investigating its role when cells were exposed to trichothecenes. The results showed that DON and T-2 upregulated the messenger RNA (mRNA) expressions of interleukin (IL)-6, IL-1β, tumor necrosis factor-α, JAK1-2, STAT1-3, and suppressors of cytokine signaling members and activated the tyrosine phosphorylation of STAT1 and STAT3 with a dose-dependent manner in RAW264.7 cells. AG490 and Stattic, the specific inhibitors of JAK/STAT pathway, blocked the STAT1 and STAT3 tyrosine phosphorylation and decreased the gene expressions of proinflammatory cytokines induced by trichothecenes. Interestingly, the time when the mRNA levels of STAT1 and STAT3 were significantly upregulated was at 12 h, which was much later than the time when mitogen-activated protein kinase was activated, indicating that STATs might be the downstream targets of the trichothecenes. With the intervention of AG490 and Stattic, DON and T-2 toxin induced apoptosis in a strengthened way, with the loss of mitochondrial membrane potential and the decrease ratios of the B-cell leukemia/lymphoma 2 (Bcl-2)/bcl-2-associated X (Bax) and B-cell lymphoma-extra large (Bcl-xL)/Bax. After exposing to DON and T-2 toxin, cells exhibited G2/M and G0/G1 phase arrest, respectively. The increased mRNA expressions of STAT target genes p21 and cyclin D1 for DON and the increases in p21 mRNA and the decreases in cyclin D1 for T-2 toxin were observed. These results demonstrated for the first time that the activation of JAK/STAT might be a critical mediator to induce the inflammatory response and apoptosis in macrophage in response to trichothecenes.

Genotoxicity of quinocetone, cyadox and olaquindox in vitro and in vivo

Quinocetone (QCT) and Cyadox (CYA) are important derivative of heterocyclic N-oxide quinoxaline (QdNO), used actively as antimicrobial feed additives in China. Here, we tested and compared the genotoxic potential of QCT and CYA with olaquindox (OLA) in Ames test, HGPRT gene mutation (HGM) test in V79 cells, unscheduled DNA synthesis (UDS) assay in human peripheral lymphocytes, chromosome aberration (CA) test, and micronucleus (MN) test in mice bone marrow. OLA was found genotoxic in all 5 assays. In Ames test, QCT produced His(+) mutants at 6.9 μg/plate in Salmonella typhimurium TA 97, at 18.2 μg/plate in TA 100, TA 1535, TA 1537, and at 50 μg/plate in TA 98. CYA produced His(+) mutants at 18.2 μg/plate in TA 97, TA 1535, and at 50 μg/plate in TA 98, TA 100 and TA 1537. QCT was found positive in HGM and UDS assay at concentrations ≥10 μg/ml while negative results were reported in CA test and MN test. Collectively, we found that OLA was more genotoxic than QCT and CYA. Genotoxicity of QCT was found at higher concentration levels in Ames test, HGM and UDS assays while CYA showed weak mutagenic potential to bacterial cells in Ames test.

Long-term mequindox treatment induced endocrine and reproductive toxicity via oxidative stress in male Wistar rats

Mequindox (MEQ) is a synthetic antimicrobial chemical of quinoxaline 1, 4-dioxide group. This study was designed to investigate the hypothesis that MEQ exerts testicular toxicity by causing oxidative stress and steroidal gene expression profiles and determine mechanism of MEQ testicular toxicity. In this study, adult male Wistar rats were fed with MEQ for 180days at five different doses as 0, 25, 55, 110 and 275mg/kg, respectively. In comparison to control, superoxide dismutase (SOD), reduced glutathione (GSH) and 8-hydroxydeoxyguanosine (8-OHdG) levels were elevated at 110 and 275mg/kg MEQ, whereas the malondialdehyde (MDA) level was slightly increase at only 275mg/kg. Furthermore, in LC/MS-IT-TOF analysis, one metabolite 2-isoethanol 4-desoxymequindox (M11) was found in the testis. There was significant decrease in body weight, testicular weight and testosterone at 275mg/kg, serum follicular stimulating hormone (FSH) at 110 and 275mg/kg, while lutinizing hormone (LH) levels were elevated at 110mg/kg. Moreover, histopathology of testis exhibited germ cell depletion, contraction of seminiferous tubules and disorganization of the tubular contents of testis. Compared with control, mRNA expression of StAR, P450scc and 17β-HSD in testis was significantly decreased after exposure of 275mg/kg MEQ while AR and 3β-HSD mRNA expression were significantly elevated at the 110mg/kg MEQ group. Taken together, our findings provide the first and direct evidence in vivo for the formation of free radicals during the MEQ metabolism through N→O group reduction, which may have implications to understand the possible mechanism of male infertility related to quinoxaline derivatives.

Acute and subchronic toxicological evaluation of Mequindox in Wistar rats

We studied an acute and subchronic oral toxicity of Mequindox (MEQ), a quinoxaline 1,4-dioxide antimicrobial promoter, in Wistar rats according to OECD guidelines. For acute toxicity study, single doses of MEQ at 175, 550 and 2000 mg/kg b.w. were administered to rats by oral gavage. The calculated LD(50) was 550 mg/kg b.w. In subchronic study, rats were fed diets containing 0, 55, 110 or 275 mg MEQ/kg. There was a reduction in body weight of rats fed 275 mg MEQ/kg diet. At 90 days autopsy, a significant decrease in the kidney weight was observed in males while an increase in relative liver and adrenal weights were observed in females fed 275 mg MEQ/kg diet. There was a significant increased in alanineaminotransferase (ALT) and malondialdehyde (MDA) concentrations in males, superoxide dismutase (SOD) activities in females, and aspartateaminotransferase (AST) levels in serum of both genders fed 275 mg MEQ/kg diet. Other toxic effects of 275 mg MEQ/kg diet included significant decrease in sodium and significant increase in potassium concentrations in serum in both genders. We may conclude that MEQ can induce hepatic and adrenal histological changes as well as leaking of different serum constituents in Wistar rats.

Acute and sub-chronic oral toxicological evaluations of quinocetone in Wistar rats

To provide a detailed toxicity with wide spectrum of doses for quinocetone, a new antimicrobial growth promoting agent, acute and sub-chronic toxicological studies were conducted. For acute study, quinocetone was administered singly by oral gavage to Wistar rats and Kunming mice. Calculated LD50 was 8687.31 mg/kg b.w./day in rats and 15848.93 mg/kg b.w./day in mice. In sub-chronic study, quinocetone was fed to Wistar rats at dietary levels of 0, 50, 300 and 1800 mg/kg or olaquindox (300 mg/kg), approximately equivalent to quinocetone 5, 30, 180 or olaquindox 30 mg/kg b.w./day. There was significant decrease in body weight in both genders, total protein and creatinine in females and alkaline phosphatase in males fed with 1800 mg/kg diet, while alkaline aminotransferase values decreased in all treated groups. Significant increase in relative weights of liver and kidneys in both genders and testis in male rats were noted at 1800 mg/kg diet. Histopathological observations revealed that 1800 mg/kg quinocetone diet and 300 mg/kg olaquindox diet could induce proliferation of bile canaliculi in the portal area. In conclusion, quinocetone can induce hepatic histological changes as well as leaking of different serum enzymes. The no-observed-adverse-effect level of quinocetone was considered to be 300 mg/kg diet.

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

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.

RepeatsDB: a database of tandem repeat protein structures.

RepeatsDB (http://repeatsdb.bio.unipd.it/) is a database of annotated tandem repeat protein structures. Tandem repeats pose a difficult problem for the analysis of protein structures, as the underlying sequence can be highly degenerate. Several repeat types haven been studied over the years, but their annotation was done in a case-by-case basis, thus making large-scale analysis difficult. We developed RepeatsDB to fill this gap. Using state-of-the-art repeat detection methods and manual curation, we systematically annotated the Protein Data Bank, predicting 10 745 repeat structures. In all, 2797 structures were classified according to a recently proposed classification schema, which was expanded to accommodate new findings. In addition, detailed annotations were performed in a subset of 321 proteins. These annotations feature information on start and end positions for the repeat regions and units. RepeatsDB is an ongoing effort to systematically classify and annotate structural protein repeats in a consistent way. It provides users with the possibility to access and download high-quality datasets either interactively or programmatically through web services.

Interactions of NADPH oxidase, renin-angiotensin-aldosterone system and reactive oxygen species in mequindox-mediated aldosterone secretion in Wistar rats

High doses of mequindox (MEQ) are associated with oxidative stress and pathological toxicity in the kidney. In this study, we demonstrated long term effects of MEQ on intra- or extra-adrenal renin-angiotensin-aldosterone system (RAAS) in vivo. RAAS plays a major role in aldosterone secretion. High doses of MEQ in the diet for 180 days in male rats led to inhibition of intra- and extra-adrenal RAAS, concident with down-regulation of Na(+)/K(+)-ATPase (NAKA) and mineralocorticoid receptor (MR), the downstream of aldosterone action. Significant changes of malondialdehyde (MDA), reduced glutathione (GSH), and superoxide dismutase (SOD) in kidney were also observed in the high doses (110, 275mg/kg) groups. The mRNA levels of most subunits of NADPH oxidase were significantly upregulated at low doses (25-110mg/kg) but the upregulation was diminished at higher doses in both kidney and adrenal gland, indicating a complicated and contradictory effect of MEQ on NADPH. These results highlight the complex interactions of drug metabolism, RAAS, NADPH oxidase and oxidative stress in response to MEQ-induced tissue toxicity and aldosterone secretion.

A chronic toxicity study of cyadox in Wistar rats

To investigate the chronic toxicity of cyadox, a growth promoting agent, five groups of Wistar rats (30 rats/group/sex) were fed with the diets containing cyadox (0, 100, 400 and 2000 mg/kg) or olaquindox (400 mg/kg) for 78weeks. There were significant decreases in body weights in both genders during most of the study period in 2000 mg/kg cyadox and 400 mg/kg olaquindox rats. Significant decreases in serum alkaline aminotransferase in the 2000 mg/kg cyadox rats at weeks 26, 52 and 78 were observed. Relative weights of liver and kidney were significantly increased in 2000 mg/kg cyadox and 400 mg/kg olaquindox rats at weeks 26, 52 and 78. A significant increase in relative brain and heart weights in 2000 mg/kg cyadox males was observed. The histopathological examinations revealed that 2000 mg/kg cyadox diet or 400 mg/kg olaquindox diet could induce proliferation of bile canaliculi in the portal area of liver and swelling and fatty degeneration of the proximal renal tubular epithelial cells in kidneys. In conclusion, the target organs of cyadox for rats were liver and kidney. The no-observed-adverse-effect level of cyadox in this study was estimated to be 400 mg/kg diet.