Metabolomics has focused on toxicological applicationsto (1) understand the mechanisms of toxicity, (2) identifynovel biomarkers of toxicity, and (3) provide in vivoassessment in animal models through simple and fastmethods to date. The toxicological effects of nonylphenol(NP) were evaluated after intraperitoneal injection of ratswith 0, 50, and 250 mg kg
-1 day
-1 NP for four consecutive days. In the nontargeted approach, different extractionconditions were introduced to investigate the effects of NPon rats through gas chromatography/mass spectrometry(GC/MS). The GC/MS data obtained were further analyzed with partial least-squares discriminant analysis tocompare toxicological effects between control and treatedgroups. The targeted approach was also used in combination with GC/MS to quantify endocrine hormones and toidentify possible biomarkers in rat urine under optimalextraction conditions. In addition, we considered themetabolic trajectory to examine the metabolite profiles andpatterns related to steroid metabolism in rats that weretreated with NP, considering both treatment amount andtime. The data suggest that tetrahydrocorticosterone and5
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-tetrahydrocorticosterone are possible urinary biomarkers of NP-induced toxicity. This metabolomic approachis a promising tool to assist with screening in toxicologicalstudies.