analysis using the  omics  ( transcriptomics , proteomics and metabolomics ) has become 
                 widespread in the post - genomics era , with one objective being the large - scale 
                 quantitative determination of mrnas , proteins and metabolites to establish gene 
                 function and ascertain the connectivity within the  omics  hierarchy . 1   metabolomics can be defined as an attempt to 
                 measure all the metabolites within a cell , tissue or organism with a genetic 
                 modification , in response to a physiological stimulus , or under specific conditions 
                 at a specified time . 2   metabolites are the 
                 end - products of a biological system and changes in metabolites can therefore be 
                 regarded as the final response of an organism to changes in gene expression . genome - wide expression of genes is amplified gradually through the hierarchy of the 
                 transcriptome , proteome and metabolome . changes in the concentrations of enzymes 
                 ( and the transcripts that encode them ) may have only small effects via metabolic 
                 pathways , but can have substantial effects on the concentration of metabolic 
                 intermediates . consequently , the metabolome may be more sensitive to perturbations 
                 compared with either the transcriptome or proteome . 3 
                
  the technologies that enable performance of metabolomics have recently increased in 
                     power . 4   transcriptomics and proteomics 
                 are based on target chemical analysis of biopolymers composed of 4 different 
                 nucleotides ( transcriptome ) or 20 amino acids ( proteome ) . in contrast , the metabolome 
                 has a large variety of chemical structures and properties , from ionic inorganic 
                 species to hydrophilic carbohydrates , volatile alcohols and ketones , organic amino 
                 and non - amino acids , hydrophobic lipids , and complex natural products . 5 6 
                  therefore , it is common to perform quantitative determination of one or a 
                 few metabolites related to a specific metabolic pathway after extensive sample 
                 preparation and separation from the sample matrix using chromatographic separation 
                 and sensitive detection , which is refered to as  target analysis  . the following two 
                 metabolomic approaches are common : i ) metabolic profiling to identify and quantify 
                 metabolites related through similar chemistries or metabolic pathways using 
                 chromatographic separation before detection with minimal metabolite isolation after 
                 sampling and ii ) metabolic fingerprinting for rapid global analysis of crude samples 
                 or extracts for classification or screening of samples without identification and 
                 quantification .   the technologies for metabolomics can also be classified into categories based on 
                 detection using mass spectrometry ( ms ) , nuclear magnetic resonance ( nmr ) 
                 spectroscopy , and fourier transform infrared ( ft - ir ) spectroscopy . the high 
                 sensitivity of ms makes it an important method for measuring metabolites in complex 
                 biosamples . gas chromatography ( gc ) and gc - ms are used for quantitative metabolic 
                 profiling , with gc first used over 20 years ago for disease diagnosis . 7   there has been rapid growth in metabolomic 
                 applications based on gc - ms for analysis of volatile and thermally stable polar and 
                 nonpolar metabolites , while developments in liquid chromatography ( lc)-ms and 
                 capillary electrophoresis ( ce)-ms have broadened the applicability of ms - based 
                 metabolomics . tandem ms and accurate mass ( time of flight ) methods are normally used 
                 to validate the identities of unknown metabolites . nmr and ft - ir spectroscopy are 
                 used for structural analysis of compounds , but have relatively low sensitivity 
                 compared with ms . nevertheless , nmr- and ft - ir - based metabolic profiling have been 
                 used successfully in many fields because both methods are highly quantitative and 
                 reproducible . the utility of metabolomics for toxic evaluation of compounds with nmr was 
                 comprehensively assessed by the consortium for metabonomic toxicology ( comet ) , which 
                 was founded by five major pharmaceutical companies and imperial college london , 
                     u.k . 8   the main objectives of comet were 
                 to assess and develop methodologies to generate a metabolomics database using nmr 
                 analysis of rodent urine and blood serum dosed with hepato- and nephrotoxins for 
                 preclinical toxicological screening of candidate drugs and to build an expert system 
                 for prediction of the site and mechanism of toxicity . this approach was used 
                 successfully to determine metabolic interspecies variation between rats and mice 
                 dosed with a toxin . 9   the goal of the second 
                 comet project ( comet 2 ) is to improve on the knowledge obtained in comet by detailed 
                 testing of hypotheses in metabolomics using nmr and lc - ms . 10 
  the discovery of biomarkers is a major objective in determining the toxicity of drug 
                 and identification of toxic biomarkers through a metabolomics approach is an 
                 emerging area . there are many reports of potential biomarkers in administration of a 
                 variety of drugs , and identification of toxicological biomarkers may contribute to 
                 assessment of the mechanisms of toxicity in drug development . fourier transform ion cyclotron resonance mass spectrometry ( ft - icr ms ) can be used 
                 to obtain an ultra - high resolution ( > 100,000 ) mass spectrum in about one second . separation of the metabolites is possible with this method alone , eliminating the 
                 need for time - consuming chromatography and derivatization . identification of 
                 putative metabolites , or the class to which they belong , can be achieved by 
                 determining the elemental composition of the metabolite based upon the ultra - high 
                 mass accuracy . 11   ,   12   the utility of this approach has been demonstrated in 
                 phenotyping of plants , in which the ripening process has been investigated in 
                 strawberries and transgenic mutant tobacco plants . 
                     13   in this review , we summarize our recent findings for metabolic 
                 fingerprints of rat urine using ft - icr ms . we used phospholipidosis ( pld ) , which is a well - examined drug - induced symptom , to 
                     evaluate the utility of ft - icr ms for toxicological metabolomics . 14   pld can be induced by cationic 
                     amphiphilic drugs ( cads ) 15   and is 
                     characterized by an abnormal intracellular accumulation of phospholipids and 
                     multilamellar bodies , as observed by electron microscopy . 16   amiodarone ( amd ) , perhexiline , fluoxetine and 
                     gentamicin induce pld in humans , 15   and 
                     pld in animal models has been observed with cads at doses far in excess of those 
                     used clinically . histopathological examinations using light and electron 
                     microscopy have traditionally been used to detect pld in drug - treated animals , 
                     but these methods are time - consuming and can not be performed in humans . urine samples from rats treated orally with amd at 300 mg / kg / day for 3 days were 
                     analyzed using an ft - icr ms instrument equipped with a 7.0-t actively shielded 
                     superconducting magnet ( ionspec , lake forest , ca , u.s.a . ) . higher intensity 
                     signals at m / z 192.06676 and 212.00239 and a lower intensity 
                     signal at m / z 178.05101 were observed in the amd - treated urine 
                     samples compared to the controls ( fig . 1 ) . 
                     a loading plot revealed that these metabolites contributed to discrimination 
                     among the samples . the ions at m / z 178.05101 , 192.06676 and 
                     212.00239 were identified as hippurate ( ha ) , phenylacetylglycine ( pag ) and 
                     indican ( idn ) , respectively . pag is the end product of phenylalanine metabolism 
                     in rodents 17   and has been reported as a 
                     biomarker using nmr 18   and lc - ms . 19   elevation of urinary pag after 
                     treatment with a cad such as amd that causes phospholipid accumulation may be 
                     related to changes in gut flora or the integrity of the intestine . 20   idn is derived from the action of 
                     intestinal bacteria containing tryptophanase , which converts tryptophan to 
                     indole . indole is then absorbed and conjugated in the liver to form idn , which 
                     was also shown to be a urinary pld biomarker on the loading plot . amd treatment 
                     has been linked to changes in the gut or the integrity of the intestine that 
                     result in increased absorption of the pag precursor , phenylacetate ( pa ) , leading 
                     to an elevated level of urinary pag . 20 
                     a decrease in urinary ha levels has been detected in urinary metabolic 
                     fingerprinting for amd - induced pld using nmr , 21 
                      consistent with the findings from ft - icr ms . the decrease in urinary ha 
                     has been proposed as a nonspecific marker of toxicity that reflects a complex 
                     combination of factors such as dietary intake and gut microbial metabolism . 22   ,   23 
                      these results show that urinary metabolic fingerprinting with ft - icr ms 
                     can be used to detect potent biomarkers for amd - induced pld and suggest that 
                     this method is applicable to toxicological assessment of compounds . the liver is the organ that is most exposed to drugs , with the concentration in 
                     the liver often being higher than the peak plasma concentration . the liver is 
                     also the major site for metabolism of xenobiotics that can lead to formation of 
                     active metabolites . thus , evaluation of liver toxicity is particularly important 
                     in development of new therapeutic compounds . here , we describe the urinary 
                     metabolic fingerprint of rat urine following treatment with two hepatotoxins , 
                     thioacetamide ( taa ) and -naphthylisothiocyanate ( anit ) , to investigate the 
                     relationship between histopathology and serum biochemistry and to search for 
                     possible biomarkers involved in hepatic toxicity . 24   ,   25 
  taa - induced acute hepatic injury in rats : rats were administered a single 
                     intraperitoneal injection of taa ( day 0 ) at a dose of 300 mg / kg body weight . alt 
                     and ast in serum both increased on day 1 , indicating that hepatocyte injury had 
                     occurred . these values decreased on day 3 and were almost at pre - dose levels on 
                     day 5 . histopathologically , taa - induced centrilobular necrosis of hepatocytes 
                     accompanied by a small amount of mononuclear cell infiltration was observed on 
                     day 1 ( fig . hepatocyte injury and 
                     infiltration of mononuclear cells became more prominent in the centrilobular 
                     area on day 3 ( fig . 2c ) , and the majority 
                     of infiltrating cells were reactive with anti - ed1 ( fig . hepatocyte injury and cell infiltration had almost 
                     completely disappeared on days 5 ( fig . 2f ) .   in negative ion spectra , the intensities of the ions at m / z 
                     178.05038 , 191.01967 , 212.00226 , 242.01320 and 258.99494 decreased on days 1 and 
                     3 compared to the pre - dose intensities ( fig . the intensity of the ion at 
                         m / z 401.20737 increased on day 1 , that at 
                         m / z 266.05390 increased on day 3 , and that at 
                         m / z 429.23882 decreased on day 1 compared to the pre - dose 
                     intensities ( fig . 4a ) . the pca scores plot 
                     showed that the negative ion compositions of taa - treated rat urine samples on 
                     days 1 and 3 differed from the pre - dose compositions , with the largest change 
                     seen on day 1 ( fig . alt and ast are well - known serum biochemical markers for 
                     hepatocyte injury and increased levels of these markers were seen on day 1 , with 
                     subsequent decreases on day 3 . the shift on the pca scores plot for the negative 
                     ions showed a similar pattern for changes of alt and ast . the positive ion 
                     compositions of the taa - treated rat urine samples on days 1 and 3 also differed 
                     from those in the pre - dose samples , but were similar to the pre - dose 
                     compositions on days 5 and 7 ( fig . histopathologically , hepatocyte injury accompanied by cell infiltration was seen 
                     on day 1 and these lesions were more prominent on day 3 . these results indicate 
                     that the changes in positive ion compositions might reflect the 
                     histopathological changes . the loading plot suggested that the negative ions at m / z 
                     178.05038 , 191.01967 , 192.06628 , 212.00226 , 220.14673 , 242.01320 , 258.99494 and 
                     303.06000 and the positive ions at m / z 266.05390 , 401.20737 and 
                     429.23882 were potential biomarkers for acute hepatic injury . the ions at 
                         m / z 178.05038 , 192.06628 , 212.00226 and 242.01320 were 
                     identified as ha , pag , idn and 3-methyldioxyindole sulfate , respectively . the 
                     alterations of ha and idn depended on administration of taa , which caused 
                     changes in the distribution of the bacterial flora . decrease of an ion at 
                         m / z 242 has been reported in urinary metabolic 
                     fingerprinting in anit - induced intrahepatic cholestasis using lc - ms , 26   and this ion may be due to 
                     3-methyldioxyindole sulfate . therefore , these findings suggest that alteration 
                     of urinary 3-methyldioxyindole sulfate may correlate with hepatotoxicity .   the positive ion at m / z 266.05390 has the empirical formula 
                         [ c9h13n3o4k ] ( calculated [ m+k ] 
                     = 266.05377 , m / z 0.00013 ) . the best candidate for this ion in 
                     the compound library was the potassium ion ( k ) adduct of deoxycytidine ( dcyt ) . ms / ms analysis of m / z 266.05390 in taa - treated rat urine gave 
                     rise to an ion at m / z 150.0 that resulted from loss of 
                     deoxyribose ( 116 ) , and the ms / ms spectrum of m / z 266.05390 in 
                     dcyt - spiked urine was identical to that for the taa - treated sample . these data 
                     are consistent with the hypothesis that m / z 266.05390 in the 
                     taa - treated urine was the k adduct of dcyt . phagocytosis of cell debris by 
                     macrophages leads to release of dcyt from these cells in vitro , 
                     and normal rat tissues including the liver and kidney have no dcyt deaminase 
                     activity for conversion of dcyt to deoxyuridine . 27   an increased number of macrophages were seen on days 1 and 3 in 
                     taa - injected rats , in parallel with development of hepatocyte injury , and 
                     therefore macrophage infiltration may be responsible for the occurrence of dcyt 
                     in the urine on days 1 and 3 . the majority of macrophages in the injured liver 
                     were reactive with ed1 . antigens recognized by ed1 occur on the membranes of 
                     cytoplasmic granules , and especially on phagolysosomes of macrophages , and the 
                     degree of ed1 expression depends on the phagocytic activity . 27   the increased number of ed1-reactive 
                     macrophages on days 1 and 3 reflects increased debris from injured hepatocytes . a significant amount of dcyt released from infiltrated macrophages was excreted 
                     in urine on day 3 , indicating a close relationship between hepatic macrophage 
                     infiltration and the appearance of dcyt in urine after taa administration . anit - induced intrahepatic cholestasis in rats : rats were administered a single 
                     oral injection of anit ( day 0 ) at a dose of 100 mg / kg body weight . alt and ast 
                     in serum increased on day 1 , and these increases were more pronounced on day 2 . total bilirubin ( tbil ) was markedly increased on day 2 , and a lesser increase in 
                     alkaline phosphatase ( alp ) was also observed . these levels then decreased on day 
                     4 . in the anit - treated rats , bile duct degeneration and necrosis accompanied by 
                     peribiliary infiltration and edema of neutrophils , and bile duct obstruction by 
                     degenerative epithelium were seen on day 1 . on day 2 , regenerative bile ducts 
                     with edema and inflammation around portal tracts , and bile duct proliferation 
                     were observed . degenerative vacuolation and single cell necrosis of hepatocytes 
                     were evident on days 1 and 2 . bile duct proliferation and increased mitosis of 
                     hepatocytes were observed on day 4 .   in negative ion spectra , the intensities of the ions at m / z 
                     178.05168 and m / z 192.06730 decreased at 048 h and 731 h , 
                     respectively , and those of the ions at m / z 512.26845 and 
                     514.28485 increased at 755 h compared to the pre - dose intensities . the pca 
                     scores plot showed that the biochemical compositions of the anit - treated rat 
                     urine samples at 724 h , 2431 h , 3148 h and 4855 h differed from the pre - dose 
                     composition , with the maximum difference at 2431 h. the compositions of the 
                     urine samples at 5572 h and 7296 h were almost the same as the pre - dose 
                     composition . increased levels of ast and alt ( serum markers for hepatocyte 
                     injury ) and alp and tbil ( markers for cholestasis ) were seen on day 2 . however , 
                     there were no serum biochemical findings at 2431 h , whereas the shift on the 
                     pca scores plot indicated differences in the urine composition at this time . 
                     therefore , these results suggest that urinary metabolic fingerprinting with 
                     ft - icr ms is more sensitive than traditional serum biochemistry methods . the loading plot indicated that the ions at m / z 178.05168 , 
                     192.06730 , 195.05139 , 242.01323 , 512.26845 and 514.28485 were potential urinary 
                     biomarkers for intrahepatic cholestasis . the ions at m / z 
                     178.05168 , 192.06730 , 242.01323 , 512.26845 and 514.28485 were identified as ha , 
                     pag , 3-methyldioxyindole sulfate , taurocholic acid ( ta ) with one double bond , 
                     and ta , respectively . ta and ta with one double bond are both bile acids , and an 
                     increase in the levels of urinary bile acids is a well - known feature of 
                     intrahepatic cholestasis . 25 
                     histopathologically , cholestasis - induced bile ductar cell necrosis and 
                     subsequent membrane breakdown caused by anit results in elevation of urinary 
                     bile acids after 755 h. in previous urinary metabolic fingerprinting in 
                     anit - induced intrahepatic cholestasis , changes in pag have been found by 
                         lc - ms 26   and a decrease in urinary 
                     ha has been shown using nmr . 29   ,   30 
  the levels of 3-methyldioxyindole sulfate , a tryptophan metabolite , were lower at 
                     024 h and higher at 3155 h , but the relationship of this metabolite with 
                     hepatic toxicity is unclear . a decrease of the ion at m / z 242 
                     has been found in urinary metabolic fingerprinting in anit - induced intrahepatic 
                     cholestasis using lc - ms , 25   and a 
                     similar decrease was observed in rat urine following taa - induced hepatic injury . these results indicate that decrease in urinary 3-methyldioxyindole sulfate may 
                     be correlated with hepatotoxicity . further study is required to examine the 
                     relationship between the toxicity of anit with potential biomarkers including 
                     pag , ha and 3-methyldioxyindole sulfate . we used phospholipidosis ( pld ) , which is a well - examined drug - induced symptom , to 
                     evaluate the utility of ft - icr ms for toxicological metabolomics . 14   pld can be induced by cationic 
                     amphiphilic drugs ( cads ) 15   and is 
                     characterized by an abnormal intracellular accumulation of phospholipids and 
                     multilamellar bodies , as observed by electron microscopy . 16   amiodarone ( amd ) , perhexiline , fluoxetine and 
                     gentamicin induce pld in humans , 15   and 
                     pld in animal models has been observed with cads at doses far in excess of those 
                     used clinically . histopathological examinations using light and electron 
                     microscopy have traditionally been used to detect pld in drug - treated animals , 
                     but these methods are time - consuming and can not be performed in humans . urine samples from rats treated orally with amd at 300 mg / kg / day for 3 days were 
                     analyzed using an ft - icr ms instrument equipped with a 7.0-t actively shielded 
                     superconducting magnet ( ionspec , lake forest , ca , u.s.a . ) . higher intensity 
                     signals at m / z 192.06676 and 212.00239 and a lower intensity 
                     signal at m / z 178.05101 were observed in the amd - treated urine 
                     samples compared to the controls ( fig . 1 ) . 
                     a loading plot revealed that these metabolites contributed to discrimination 
                     among the samples . the ions at m / z 178.05101 , 192.06676 and 
                     212.00239 were identified as hippurate ( ha ) , phenylacetylglycine ( pag ) and 
                     indican ( idn ) , respectively . pag is the end product of phenylalanine metabolism 
                     in rodents 17   and has been reported as a 
                     biomarker using nmr 18   and lc - ms . 19   elevation of urinary pag after 
                     treatment with a cad such as amd that causes phospholipid accumulation may be 
                     related to changes in gut flora or the integrity of the intestine . 20   idn is derived from the action of 
                     intestinal bacteria containing tryptophanase , which converts tryptophan to 
                     indole . indole is then absorbed and conjugated in the liver to form idn , which 
                     was also shown to be a urinary pld biomarker on the loading plot . amd treatment 
                     has been linked to changes in the gut or the integrity of the intestine that 
                     result in increased absorption of the pag precursor , phenylacetate ( pa ) , leading 
                     to an elevated level of urinary pag . 20 
                     a decrease in urinary ha levels has been detected in urinary metabolic 
                     fingerprinting for amd - induced pld using nmr , 21 
                      consistent with the findings from ft - icr ms . the decrease in urinary ha 
                     has been proposed as a nonspecific marker of toxicity that reflects a complex 
                     combination of factors such as dietary intake and gut microbial metabolism . 22   ,   23 
                      these results show that urinary metabolic fingerprinting with ft - icr ms 
                     can be used to detect potent biomarkers for amd - induced pld and suggest that 
                     this method is applicable to toxicological assessment of compounds . the liver is the organ that is most exposed to drugs , with the concentration in 
                     the liver often being higher than the peak plasma concentration . the liver is 
                     also the major site for metabolism of xenobiotics that can lead to formation of 
                     active metabolites . thus , evaluation of liver toxicity is particularly important 
                     in development of new therapeutic compounds . here , we describe the urinary 
                     metabolic fingerprint of rat urine following treatment with two hepatotoxins , 
                     thioacetamide ( taa ) and -naphthylisothiocyanate ( anit ) , to investigate the 
                     relationship between histopathology and serum biochemistry and to search for 
                     possible biomarkers involved in hepatic toxicity . 24   ,   25 
  taa - induced acute hepatic injury in rats : rats were administered a single 
                     intraperitoneal injection of taa ( day 0 ) at a dose of 300 mg / kg body weight . alt 
                     and ast in serum both increased on day 1 , indicating that hepatocyte injury had 
                     occurred . these values decreased on day 3 and were almost at pre - dose levels on 
                     day 5 . histopathologically , taa - induced centrilobular necrosis of hepatocytes 
                     accompanied by a small amount of mononuclear cell infiltration was observed on 
                     day 1 ( fig . hepatocyte injury and 
                     infiltration of mononuclear cells became more prominent in the centrilobular 
                     area on day 3 ( fig . 2c ) , and the majority 
                     of infiltrating cells were reactive with anti - ed1 ( fig . 2f ) .   in negative ion spectra , the intensities of the ions at m / z 
                     178.05038 , 191.01967 , 212.00226 , 242.01320 and 258.99494 decreased on days 1 and 
                     3 compared to the pre - dose intensities ( fig . the intensity of the ion at 
                         m / z 401.20737 increased on day 1 , that at 
                         m / z 266.05390 increased on day 3 , and that at 
                         m / z 429.23882 decreased on day 1 compared to the pre - dose 
                     intensities ( fig . the pca scores plot 
                     showed that the negative ion compositions of taa - treated rat urine samples on 
                     days 1 and 3 differed from the pre - dose compositions , with the largest change 
                     seen on day 1 ( fig . 3b ) . by day 5 , the 
                     composition was close to that in pre - dose samples ( fig . alt and ast are well - known serum biochemical markers for 
                     hepatocyte injury and increased levels of these markers were seen on day 1 , with 
                     subsequent decreases on day 3 . the shift on the pca scores plot for the negative 
                     ions showed a similar pattern for changes of alt and ast . the positive ion 
                     compositions of the taa - treated rat urine samples on days 1 and 3 also differed 
                     from those in the pre - dose samples , but were similar to the pre - dose 
                     compositions on days 5 and 7 ( fig . histopathologically , hepatocyte injury accompanied by cell infiltration was seen 
                     on day 1 and these lesions were more prominent on day 3 . these results indicate 
                     that the changes in positive ion compositions might reflect the 
                     histopathological changes . the loading plot suggested that the negative ions at m / z 
                     178.05038 , 191.01967 , 192.06628 , 212.00226 , 220.14673 , 242.01320 , 258.99494 and 
                     303.06000 and the positive ions at m / z 266.05390 , 401.20737 and 
                     429.23882 were potential biomarkers for acute hepatic injury . the ions at 
                         m / z 178.05038 , 192.06628 , 212.00226 and 242.01320 were 
                     identified as ha , pag , idn and 3-methyldioxyindole sulfate , respectively . the 
                     alterations of ha and idn depended on administration of taa , which caused 
                     changes in the distribution of the bacterial flora . decrease of an ion at 
                         m / z 242 has been reported in urinary metabolic 
                     fingerprinting in anit - induced intrahepatic cholestasis using lc - ms , 26   and this ion may be due to 
                     3-methyldioxyindole sulfate . therefore , these findings suggest that alteration 
                     of urinary 3-methyldioxyindole sulfate may correlate with hepatotoxicity .   the positive ion at m / z 266.05390 has the empirical formula 
                         [ c9h13n3o4k ] ( calculated [ m+k ] 
                     = 266.05377 , m / z 0.00013 ) . the best candidate for this ion in 
                     the compound library was the potassium ion ( k ) adduct of deoxycytidine ( dcyt ) . ms / ms analysis of m / z 266.05390 in taa - treated rat urine gave 
                     rise to an ion at m / z 150.0 that resulted from loss of 
                     deoxyribose ( 116 ) , and the ms / ms spectrum of m / z 266.05390 in 
                     dcyt - spiked urine was identical to that for the taa - treated sample . these data 
                     are consistent with the hypothesis that m / z 266.05390 in the 
                     taa - treated urine was the k adduct of dcyt . phagocytosis of cell debris by 
                     macrophages leads to release of dcyt from these cells in vitro , 
                     and normal rat tissues including the liver and kidney have no dcyt deaminase 
                     activity for conversion of dcyt to deoxyuridine . 27   an increased number of macrophages were seen on days 1 and 3 in 
                     taa - injected rats , in parallel with development of hepatocyte injury , and 
                     therefore macrophage infiltration may be responsible for the occurrence of dcyt 
                     in the urine on days 1 and 3 . antigens recognized by ed1 occur on the membranes of 
                     cytoplasmic granules , and especially on phagolysosomes of macrophages , and the 
                     degree of ed1 expression depends on the phagocytic activity . 27   the increased number of ed1-reactive 
                     macrophages on days 1 and 3 reflects increased debris from injured hepatocytes . a significant amount of dcyt released from infiltrated macrophages was excreted 
                     in urine on day 3 , indicating a close relationship between hepatic macrophage 
                     infiltration and the appearance of dcyt in urine after taa administration . anit - induced intrahepatic cholestasis in rats : rats were administered a single 
                     oral injection of anit ( day 0 ) at a dose of 100 mg / kg body weight . alt and ast 
                     in serum increased on day 1 , and these increases were more pronounced on day 2 . total bilirubin ( tbil ) was markedly increased on day 2 , and a lesser increase in 
                     alkaline phosphatase ( alp ) was also observed . these levels then decreased on day 
                     4 . in the anit - treated rats , bile duct degeneration and necrosis accompanied by 
                     peribiliary infiltration and edema of neutrophils , and bile duct obstruction by 
                     degenerative epithelium were seen on day 1 . on day 2 , regenerative bile ducts 
                     with edema and inflammation around portal tracts , and bile duct proliferation 
                     were observed . degenerative vacuolation and single cell necrosis of hepatocytes 
                     were evident on days 1 and 2 . bile duct proliferation and increased mitosis of 
                     hepatocytes were observed on day 4 .   in negative ion spectra , the intensities of the ions at m / z 
                     178.05168 and m / z 192.06730 decreased at 048 h and 731 h , 
                     respectively , and those of the ions at m / z 512.26845 and 
                     514.28485 increased at 755 h compared to the pre - dose intensities . the pca 
                     scores plot showed that the biochemical compositions of the anit - treated rat 
                     urine samples at 724 h , 2431 h , 3148 h and 4855 h differed from the pre - dose 
                     composition , with the maximum difference at 2431 h. the compositions of the 
                     urine samples at 5572 h and 7296 h were almost the same as the pre - dose 
                     composition . increased levels of ast and alt ( serum markers for hepatocyte 
                     injury ) and alp and tbil ( markers for cholestasis ) were seen on day 2 . however , 
                     there were no serum biochemical findings at 2431 h , whereas the shift on the 
                     pca scores plot indicated differences in the urine composition at this time . 
                     therefore , these results suggest that urinary metabolic fingerprinting with 
                     ft - icr ms is more sensitive than traditional serum biochemistry methods . the loading plot indicated that the ions at m / z 178.05168 , 
                     192.06730 , 195.05139 , 242.01323 , 512.26845 and 514.28485 were potential urinary 
                     biomarkers for intrahepatic cholestasis . the ions at m / z 
                     178.05168 , 192.06730 , 242.01323 , 512.26845 and 514.28485 were identified as ha , 
                     pag , 3-methyldioxyindole sulfate , taurocholic acid ( ta ) with one double bond , 
                     and ta , respectively . ta and ta with one double bond are both bile acids , and an 
                     increase in the levels of urinary bile acids is a well - known feature of 
                     intrahepatic cholestasis . 25 
                     histopathologically , cholestasis - induced bile ductar cell necrosis and 
                     subsequent membrane breakdown caused by anit results in elevation of urinary 
                     bile acids after 755 h. in previous urinary metabolic fingerprinting in 
                     anit - induced intrahepatic cholestasis , changes in pag have been found by 
                         lc - ms 26   and a decrease in urinary 
                     ha has been shown using nmr . 29   ,   30 
  the levels of 3-methyldioxyindole sulfate , a tryptophan metabolite , were lower at 
                     024 h and higher at 3155 h , but the relationship of this metabolite with 
                     hepatic toxicity is unclear . a decrease of the ion at m / z 242 
                     has been found in urinary metabolic fingerprinting in anit - induced intrahepatic 
                     cholestasis using lc - ms , 25   and a 
                     similar decrease was observed in rat urine following taa - induced hepatic injury . these results indicate that decrease in urinary 3-methyldioxyindole sulfate may 
                     be correlated with hepatotoxicity . further study is required to examine the 
                     relationship between the toxicity of anit with potential biomarkers including 
                     pag , ha and 3-methyldioxyindole sulfate . a number of metabolomics studies using nmr and/or ms have been performed to detect 
                 metabolic alterations induced by administration of compounds to assess their 
                 toxicity . the results shown above indicate that urinary metabolic fingerprinting 
                 with ft - icr ms can be used to detect metabolic changes and to identify potential 
                 biomarkers induced by compounds with different mechanisms of toxicity . therefore , 
                 metabolomics approaches including metabolic fingerprinting have the potential to 
                 reveal onset of toxicity at an early stage of drug development . to reach this goal , improvements in the technology for metabolomics are required to allow detection of 
                 the whole metabolome with a high degree of accuracy . improved informatics 
                 approaches , including database construction , are also needed for identification of 
                 biomarkers , with follow - up studies to validate biomarker concentrations in biofluid 
                 and metabolic pathways in vivo .
detection of the toxicity of a candidate compound at an early stage of drug 
                     development is an emerging area of interest . 
 it is difficult to determine all of 
                     the effects of metabolism of a compound using traditional approaches such as 
                     histopathology and serum biochemistry . 
 the goal of a metabolomics approach is to 
                     determine all metabolites in a living system , with the potential to detect and 
                     identify biomarkers involved in toxicity onset . here , 
 we summarize the metabolic 
                     fingerprints for detection and identification of metabolic changes and 
                     biomarkers related to drug - induced toxicity using fourier transform ion 
                     cyclotron resonance mass spectrometry ( ft - icr ms ) .