| In t he p ost-genomic era, new families of technologies come available to provide a comprehensive analysis of many classes of cellular molecules, such as RNAs, DNAs, proteins, intermediary metabolites, etc. Termed as "-omics", these technologies have characteristics of high-through, large-scale and computer statistical analysis in methodology. Such "-omics" technologies that allow global analysis and multi-endpoint analysis are of particular importance in the field of toxicology. At the same time, chemical-related toxicity testing strategy has undergone little change, where traditional toxicity evaluating techniques remain mainstream still. To introduce "-omics" technologies into chemical safety evaluation is one of important tasks in pharmatoxicological studies.As standard antitubercular regimen currently, Isoniazid (INH) and Rifampicin (RFP) continue to be highly effective drugs in the chemoprophylaxis and treatment of tuberculosis. Unfortunately, concomitant administration of INH with RFP resulted in increased hepatotoxicity in patients, whose exact mechanism responsible for the hepatotoxicity remain poorly understood yet. Former studies considered hepatotoxicity mechanisms of INH, RFP and INH+RFP neither from the aspect of systematical biology, nor from global assessment of functional cellular alterations. In the present study, a platform based on metabonomics technique was established to explore of the effects, induced by the conjunctive treatment of INH and RFP, on rats' liver. In the present study, the primary hepatotoxic mechanism caused by co-admistration ofINH and RFP by using the metabonomics associated with proteomics and genomics techniques was studied; furthermore, the possibility of applying metabonomics, proteomics and genomics to toxicological study was explored.Wistars rats, randomly assigned into four groups, were administrated daily a single dose (po) of physiological saline, INH, RFP, or INH+RFP respectively for consecutive 7 days. The urine samples over 24 h of each rat were collected before the treatment and so did daily during 7-day treatments. 'H NMR spectra of these samples were acquired, and subjected to data preprocess and principal component analyses (PCA) and Simple independent modeling of class analogy (SIMCA) analysis. The platform, based on metabonomics technique, for chemical toxicity study was successfully established.Using the metabonomics technique accompanied by traditional toxicity evaluating techniques to explore liver toxicity, the dose-time-response relationship of INH and RFP alone and their combination were studied. Hepatotoxicity, evaluated by the traditional techniques, was evidently found in INH and INH+RFP treated rats with higher dosage and longer treatment, while minor hepatotoxicity was shown in RFP treated rats. Results also showed that the hepatotoxicity caused by INH+RFP was more severe than that of INH, and dose-time-response relationships were shown in both of the two treatments. The result of dose-related and time-related metabnomics changes was in accordance with t hat o f t raditional t oxicity e valuation. It w as w ell i n a greement w ith t he hepatic toxic injury differentia of differently treated rats with different doses that the metabonomics spectra of urine from these rats could also be distinguished easily from each other. Just after administration, the urine metabonomics trajectory biased from that of the controls or pre-administration. In addition, the longer treated period was, the further bias occurred, which means the severer toxic injury was. An animal model, through analyzing data about the dose-time-response relationship in liver toxicity induced by INH, RFP and INH+RFP, was set up for evaluating the hepatic toxic injury induced by INH+RFP.The established animal model was applied to study the primary hepatotoxic mechanism caused by co-admistration of INH and RFP. The contents of malondialdehyde (MDA) in liver homogenate, levels of hepatic thiols, activity of microsomal cytochrome P450 (CYP450) and CYP2E1 were examined, where hepatic CYP2E1 activity was measured by aniline hydroxylase (ANH). Oligonuleotide microarray was used to detect the alteration of the gene expression profile of rat liver; 2DE/M0LDI-T0F-MS was used to detect the alteration of the protein expression profile; the *H NMR spectroscopic and pattern recognition methods was used to detect alteration of the metabolite expression profile. Results showed: (1) The combination of INH and RFP treatments, compared with the control or the INH or RFP treatment alone, caused a significant reduction of hepatic thiols contents and mean body weight, and an increase in liver index, liver levels of MDA, microsomal CYP450 and ANH. (2) The alteration number of the gene expression, induced by co-administration of INH and RFP, was the largest; that induced by INH was medium; and that induced by RFP was the smallest. The up-regulated gene numbers were larger than the down-regulated gene numbers in the three microarrays. There were 55 different expression genes regulated in rat by INH+RFP, among which 19 genes were also regulated by INH and 3 genes were also regulated by RFP. All these different expression genes of the three microarrays were functionally related to metabolism, and the changes of CYP450 related gene were obvious. Different expression genes, regulated by INH+RFP, were functionally related to CYP450, liver function and antioxidation. The hepatotoxic mechanism caused by co-administration of INH and RFP can be better understood from the oligonuleotide microarrays. (3) The computer assisted image analysis showed there were 11 different expression proteins in the two-dimensional electrophoresis (2-DE) gels. The alteration of the protein expression, induced by co-administration of INH and RFP, changed most obviously, that induced by INH changed moderately, and that induced by RFP changed less. There were 8 different proteins measured by theMALDI-TOF-MS and analyzed by computer database, among which antioxidation related proteins changed obviously. It was shown by proteomics analysis that the drug toxicity related proteins could become biomarker candidates. (4) It was shown by metabonomics analysis that the urine metabonomic trajectories of rats treated with INH or RFP alone or in combination were obviously separated from those of control rats, but they were not separated from each other before treatment. The different metabonomic trajectories of different treated rats suggested that there were different mechanisms. Increased concentration of urinary taurine and glucose, together with decreased concentrations of urinary citrate and 2-oxoglutarate (2-OG), was the changes in the *H NMR spectra of urine from rats treated with RFP. Besides, increased concentration of urinary 2-aminoadipate (2-AA) was also revealed by the changes in the 'H NMR spectra of urine from rats treated with INH. In addition, increased concentration of urinary creatine and decreased concentrations of urinary succinate were also revealed by changes in the 'H NMR spectra of urine from rats treated with INH+RFP. Latent hepatotoxic mechanisms were hinted by analyzing the formation and transform of these metabolites. These findings suggest that the increase of hepatic toxicity induced by co-administration of INH with RFP is related to the injury of mitochondrial fundction, reductions energy metabolism in TCA cycle and perturbations in the metabolism of glucose and lipid. It was shown by metabonomics analysis that the drug toxicity related proteins could become biomarker candidates.Based on the results of this study, following conclusions could be made: (1) The platform of metabonomics technique for chemical toxicity study was successfully e stablished. It w as also p roved t hat t he m etabonomics s pectra o f urine could be distinguished readily by different toxic mechanism. It was also proved that metabonomics technique could be applied to toxicological study. (2) The metabonomics spectra of urine from rats treated with INH, RFP and INH+RFP of different doses could be distinguished from each other. The metabonomics technique could distinguish the onset and development oftoxicity, which could help tracking and identifying biomarkers. Although it was correlated with traditional measurements for evaluating toxicity, metabonomics method was more sensitive than traditional measurements. (3) The possible hepatotoxic mechanism caused by co-administration of INH and RPP was expounded by the three profiles of genes, proteins and metabolites. When the active metabolites of INH and RFP were induced by the increased activity of hepatic microsome enzyme, the balance between oxidation and antioxidation within organisms were wrecked, which resulted in oxidation stress. Then, oxidation stress induced oxidation injuries of membrane lipid peroxidation and exhaustion of antioxidation. Moreover, exhaustion of antioxidation aggravated the toxicity of hepatic cells. And the hepatotoxicity caused by co-admistration of INH and RFP is also related to the injury of mitochondrial fundction, reductions energy metabolism in TC A cycle and perturbations in the metabolism of glucose and lipid. (4) The chemical toxicity screen system based on gene, protein and metabolite expression profile was established. Drug toxicity related genes, proteins and metabolites were also found. (5) Genomics, proteomics and metabonomics can be recognized as an ideal and widely-used technique for exploring and evaluating the toxicity of drugs/food/chemical. |
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