The Effects Of Quinoxalines And Their Metabolites On Aldosterone Synthetase Expression In Adrenal Cortexs

Quinoxalines, quinoxaline-1,4-dioxides basic structure of drugs, are widely used in China as antimicrobial and feed additives, mainly including Carbadox (CBX), Olaquindox (OLA), Cyadox (CYA), Quinocetone (QCT) and Mequindox (MEQ). Studies had confirmed that most of the Quinoxalines could seriously damage animal adrenal syaterm, such as the CBX, OLA, CYA continued to the animals led to pig adrenal toxicity, restrained the pig body’s aldosterone synthesis, caused the imbalance of salt and water metabolism. In further study, it had been confirmed that Carbadox and its metabolites could not reversible inhibit adrenal cortical methadone to the transformation of aldosterone, could reduce the secretion of aldosterone. Poisoning doses of Olaquindox to chicken, could produce damage on adrenocortical function, decreased the aldosterone concentration of the plasma, led to low serum sodium and high potassium, suppressed the liver enzymes microsomal system, decreased the content of cytochrome P-450enzyme, and impaired renal excretion. In the long-term animal feeding study also found that the highest dose of Mequindox could damage rat adrenal tissue, made the plasma cortical ketone and aldosterone down, as the steroid hormone imbalance and the release of salt and water disorder. They inhibited the expressions of cytochrome P450enzyme CYP11B1and aldosterone synthase CYP11B2. On the pig cells from the adrenal cortical organs, it was found that Mequindox could reduce adrenal aldosterone reduction. In earlier research, it was also found that the pig adrenal cortical cells treated with Quinocetone had toxic injury, and the expression of related enzyme inaldosterone synthesis and secretion was significantly decreased. Other studies showed that the potential toxicity was relevant to its deoxy-metabolites. Therefore, it had great significance to study the toxicity induced by metabolites of Quinoxalines. This research mainly used NCI-H295R cells as the adrenal cortical cells model, by adding the deoxy-metabolites of Quinoxalines, screened the main toxic metabolites; and we further studyed the toxicity mechanism of toxic metabolites through the expression regulation of aldosterone synthesis main enzymes.The results of this study showed that Quinoxalines’metabolites had time and dose-dependent cell damages on adrenal cells, through testing the aldosterone hormone level. It was determined that the main toxic metabolites of Quinoxalines were Nl-deoxy metabolites, its order of toxic size was Nl-deoxy QCT>Nl-deoxy MEQ> Nl-deoxy CYA, consistented with the toxicity order of the Quinoxalines. The toxic metabolites screening had certain relation with its side chain structure of patent drugs. It was also first found that the adrenal toxicity induced by Nl-deoxy QCT, Nl-deoxy MEQ and Nl-deoxy CYA were closely related with the oxidative stress, which could induced the cell of lipid peroxidationand the damage the body of the unbalance of oxidation and reduction system. However, the different drug influences on oxidative stress indicators were different. The oxidative damage induced by Nl-deoxy QCT, Nl-deoxy MEQ, and Nl-deoxy CYA had positively correlate with the toxicity inhibition of aldosterone hormones. Because of the inhibition of aldosterone synthesis and secretion caused by drugs, natural inhibitors of oxidant stress (Oligomeric Proantho Cyanidins) could effectively improve the synthesis and secretion of aldosterone, mitigate the toxic damage of the drugs on the adrenal cells and reduce the lipid peroxidation of the drugs on the cells, reduced the oxidative stress damage caused by drugs.Nl-deoxy QCT as the major toxic metabolite of Quinocetone could significantly inhibit the gene expression of transcription factor NURR1and ATF-1, as well as effective suppressed gene expression of NGFIB, then down-regulated the gene expression of aldosterone synthase CYP11B2. Nl-deoxy QCT inhibited transcription factors CREB and SF-1expression, thus inhibited CYP11B1protein and gene expression. Through different ability to the transcription factors, Nl-deoxy QCT determined the differment combining strength about transcription factors and the purpose gene promoter, restrained the expression of all transcription factors, decreased main enzymes CYP11B1, CYP11B2gene and protein expression level of aldosterone synthesis, inhibited CYP11B1and CYP11B2gene expression, which eventually reduced the aldosterone hormone secretion. After by adding inhibitor of oxidant stresss, OPC could effectively increase the expression of CREB, and promoted the gene expression of NURR1, NGFIB, ATF-1, so as to enhance the combination of transcription factors and promoter, promoted the transcription of CYP11B1and CYP11B2; OPC alleviated the toxicity of the drugs on the adrenal cells and raised the aldosterone hormone secretion levels.Nl-deoxy MEQ as the main toxic metabolite of Mequindox, could inhibit aldosterone hormone secretion of NCI-H295R cells. On the one hand, by inhibiting the expression of CREB and SF-1gene, Nl-deoxy MEQ significantly inhibited the expression of SF-1, then regulated the expression of CYP11B1gene and protein. On the other hand, Nl-deoxy MEQ significant inhibited the gene expression of transcription factors NGFIB, NURR1, ATF-1, leading to the various transcription factor could not fully binding to the promoter binding sites of CYP11B2gene, inhibited the expression of CYP11B2, further inhibited the expression of aldosterone synthase, eventually led to the inhibition of aldosterone synthesis and secretion. OPC raised the expression of transcription factors NURR1, ATF-1genes, had not significant effect on the transcription factor NGFIB, thus effectively promoted the combination of transcription factors and promoter binding sites to promote CYP11B2expression. Then, the inhibitor of oxidant stresss could induce the expression of SF-1, and had not significant role on CREB; it promoted the combination of the transcription factors and CYP11B1, and promoted the CYP11B1gene expression. Eventually, OPC alleviated the toxic caused by Nl-deoxy MEQ on NCI-H295R cells and promoted the synthesis secretion of aldosterone hormone.Nl-deoxy CYA as one of the Cyadox’main toxic metabolites, was less toxic than other Quinoxalines’metabolites. It had no toxicity damages on the adrenal cell at low doses, and Nl-deoxy CYA induced a decline in aldosterone hormone secretion of adrenal cell only under the high drug dose and long time conditions. On the one hand, by treated with Nl-deoxy CYA, the transcription factors CREB, SF-1gene levels were significantly down-regulated, which allowed the transcription factor with the CYP11B1binding site combined with disabilities, so as to inhibit the gene and protein expressions of CYP11B1. The function of Nl-deoxy CYA, gene expression of transcription factors NURR1, NGFIB, ATF-1were restrained, transcription factors couldn’t fully combineing to the gene promoter site of CYP11B2, thus it inhibited the transcription expression of CYP11B2. Therefore, through the inhibition of CYP11B1expression, as well as the suppressed of CYP11B2, Nl-deoxy CYA down-regulated aldosterone synthesis and secretion, caused a decline in aldosterone hormone secretion on adrenal cell. In a short time, by inducing the expression of transcription factors SF-1, CREB, OPC significantly raised the gene and protein expressions and the actitivation of CYP11B1reduced by Nl-deoxy CYA. After the incubation time of drug extended, the ability to regulate transcription factors and the purpose gene of OPC was decreased. It was found that OPC significantly raised the expression of transcription factors NGFIB, NURR1, strengthened the ability of combining transcription factors with the CYP11B2promoter site, further promoted CYP11B2expression, alleviated high doses of Nl-deoxy CYA toxic effects on adrenal cells.Nl-deoxy metabolites of Quinoxalines, by different inhibition of the regulatory capacity of different transcription factors, regulated the binding capacity of transcription factors and CYP11B1, CYP11B2. Thus Nl-deoxy metabolites restrained the purpose genes and proteins under the control of promoting aldosterone synthesis process, limited aldosterone synthesis, further caused aldosterone hormone, significantly reduced the toxic damage of adrenal cells. Through different induction of regulatory capacity, OPC could up-regulat expression of different the transcription factor and mainly enzyme of aldosterone synthesis, raised different level of aldosterone hormone, abated the toxic effects of drugs.