rats and treatment with sb203580 : inbred strains of lec and wkah / hkm 
 ( wkah ) rats were obtained from the institute for animal reproduction inc . and the institute 
 for animal experimentation , school of medicine , hokkaido university , respectively . all research protocols were approved by the animal 
 research committee of rakuno gakuen university . the rats were housed at a temperature of 25 
  2c and exposed to a daily cycle of 14-hr light and 10-hr darkness . a solid diet ( mf - food , 
 oriental yeast co. , ltd . , tokyo , japan ) and water were provided ad libitum . twenty four male lec and 12 male wkah rats were used in the present study . although 
 long - evans agouti ( lea ) strain rats have been established as a sibling strain of lec rats , 
 lea rats show some genetic abnormalities such occurrence of diabetes mellitus ( n. kasai , 
 tohoku university , personal communications ) . in the present study , sb203580 ( 4-[4-fluorophenyl]-2-[4-methylsulfinyl]-5-[4-pyridyl]1h - imidazole ) , a p38 
 mitogen - activated protein kinase ( p38 mapk)-specific inhibitor , was purchased from wako 
 chemicals ( osaka , japan ) and was dissolved in dimethyl sulfoxide ( dmso ) . sb203580 inhibits 
 selectively an activation of p38 mapk , but not c - jun n - terminal kinase ( jnk ) and p48 mapk . injection at doses of 2.5 and 5 
 mg / kg body weight twice a week from 10 to 13 weeks of age . rna purification : rats at 4 and 13 weeks of age were anesthetized with 
 pentobarbital , and livers were flushed with ice - cold phosphate buffered saline ( pbs ) . total 
 rna was isolated from liver tissue using a polytron homogenizer ( quiagen , hilden , germany ) 
 and tri reagents ( sigma - aldrich , st . louis , mo , u.s.a . ) and purified using rnaeasy columns 
 ( quiagen ) . preparation of crna : total rna ( 3 g ) was used as the 
 starting material for cdna preparation . cdna synthesis and labeling of arna were performed 
 using an amino - allyl rna amplification kit ver.2 ( sigma - aldrichi ) and cy3 and cy5 
 mono - reactive dye pack ( amersham bioscience , uppsala , sweden ) according to the 
 manufacturers instructions . labeled arna was purified using microcon - ym30 ( millipore , 
 billerica , ma , u.s.a . ) . the quality of the labeling products was tested with nano drop 
 nd-1000 ( nanodrop technology , rockland , me , u.s.a . ) before hybridization to the 
 oligonucleotide array . array hybridization and scanning : crna was fragmented at 95c for 15 min 
 in a fragmentation buffer and hybridized to a panorama rat micro array ( sigma - aldrich ) in a 
 humid chamber ( sigma - aldrich ) at 60c for 16 hr . the probe array was washed with 4 x 
 standard saline citrate ( ssc ) containing 0.1% sodium dodecyl sulfate ( sds ) at 50c for 5 
 min , followed by washing with 2 x ssc containing 0.1% sds at 50c for 5 min and 2 x ssc at 
 50c for 5 min . the probe arrays were scanned by scanning service of sigma - aldrich . in the 
 comparison of two arrays ( lec rats at 4 vs 13 weeks or wkah rats at 4 vs 13 weeks ) , the 
 original output was signal log ratio to log base 2 , which was subsequently converted to 
 linear fold changes by lowess standardization . assay for jnk and p38 mapk : small pieces of freshly resected rat liver 
 were lysed in cell lysis buffer ( cell signaling , danvers , ma , u.s.a . ) using a sample 
 grinding kit ( ge healthcare bioscience , uppsala , sweden ) at 4c . protein concentrations of 
 the lysates were determined by the method of lowry et al . . measurements of activities of jnk and p38 mapk were 
 carried out using a jnk assay kit ( cell signaling ) and p38 map kinase kit ( cell signaling ) 
 according to the manufacturer s instructions . after sds polyacrylamide gel electrophoresis 
 and dry electrotransfer ( iblot dry blotting system , invitrogen , carlsbad , ca , u.s.a . ) to 
 nitrocellulose paper ( invitrogen ) , equivalent protein loading was confirmed by staining with 
 amido black . the nitrocellulose paper was incubated with primary antibodies to 
 phosphorylated atf-2 rabbit igg for p38 mapk and to phosphorylrated c - jun rabbit igg for 
 jnk . the proteins were detected with an antibody to hrp - anti rabbit igg and lumiglo . chemical luminescence was analyzed using lumi vision pro ( aisin , kariya , japan ) . measurements of activities of alt and ast and concentrations of bilirubin in 
 sera : samples for measurements of aspartate aminotransferase ( ast ) and alanine 
 aminotrasferase ( alt ) activities and concentrations of bilirubin were frozen with liquid 
 nitrogen and stored at 80c until used . the activities of ast and alt and concentrations of 
 bilirubin in sera were determined using an automatic analyzer tba-120 fr ( toshiba , tokyo , 
 japan ) . statistical analysis : all data are expressed as means  sd . differences 
 between means were analyzed statistically by one - way anova followed by mann - whitney u test . changes in gene expression analyzed by oligonucleotide arrays : when the 
 levels of gene expression in the livers of lec rats at 13 weeks of age were compared to 
 those in the livers of rats at 4 weeks of age using oligonucleotide arrays covering 7,700 
 genes , 764 genes were up - regulated and 856 genes were down - regulated at 13 weeks more than 
 2-fold compared to at 4 weeks ( table 
 1table 1.numbers of up - regulated and down - regulated genes in the livers of lec and wkah 
 rats at 13 weeks of age compared to those at 4 weeks of ageratsno . of up - regulated genesno . of down - regulated genes>2-fold>3-fold>4-fold>2-fold>3-fold>4-foldlec764272129856294130wkah39079334499023numbers of genes that showed differences in expression levels by more than 2 , 3 and 4 fold in the livers of lec and wkah rats at 13 weeks of age from those at 4 weeks of 
 age . ) . in the case of livers of wkah rats , 390 genes were up - regulated , and 449 
 genes were down - regulated . although expression levels of many genes were different at 4 and 
 13 weeks of age in the livers of both lec and wkah rats , only 25 and 30 genes that were 
 up - regulated and down - regulated were overlapped in lec and wkah rats at 13 weeks of age 
 ( fig . 1.number of genes for which expression levels changed by more than 2-fold in livers of 
 lec and wkah rats at 13 weeks of age compared to the levels in livers of rats at 4 
 weeks of age . up and down represent up - regulated and down - regulated genes in 
 expression levels showing more than 2-fold differences in the livers of lec and wkah 
 rats at 13 weeks of age compared to the levels in livers of rats at 4 weeks of 
 age . ) . gene ontology was followed according to classification of manufactures s software 
 ( sigma - aldrich ) . 2.number of genes for which expression levels in some groups of gene ontology changed 
 by more than 2-fold in the livers of lec and wkah rats at 13 weeks of age compared to 
 the levels in the livers of the rats at 4 weeks of age . number in parenthesis is the 
 number of genes analyzed in each group of ontology . shows the number of genes that were up - regulated and down - regulated more than 2-fold 
 in some groups of gene ontology in the livers of lec and wkah rats at 13 weeks of age 
 compared to the levels in livers of rats at 4 weeks of age . many genes related to oxidative 
 stress , dna damage , apoptosis and inflammation with acute - phase reaction were up - regulated 
 and down - regulated in the livers of lec rats at 13 weeks of age ( fig . these results are in good agreement with results obtained by 
 klein et al . . expression levels 
 of 11 of 29 genes related to sapk changed more than 2-fold in the livers of lec rats at 13 
 weeks of age ( fig . however , there were no 
 sapk - related genes that showed changes in expression levels more than 2-fold in the livers 
 of wkah rats at 13 weeks of age compared to the levels in the livers of rats at 4 weeks of 
 age . numbers of genes that showed differences in expression levels by more than 2 , 3 and 4 
 fold in the livers of lec and wkah rats at 13 weeks of age from those at 4 weeks of 
 age . number of genes for which expression levels changed by more than 2-fold in livers of 
 lec and wkah rats at 13 weeks of age compared to the levels in livers of rats at 4 
 weeks of age . up and down represent up - regulated and down - regulated genes in 
 expression levels showing more than 2-fold differences in the livers of lec and wkah 
 rats at 13 weeks of age compared to the levels in livers of rats at 4 weeks of 
 age . number of genes for which expression levels in some groups of gene ontology changed 
 by more than 2-fold in the livers of lec and wkah rats at 13 weeks of age compared to 
 the levels in the livers of the rats at 4 weeks of age . number in parenthesis is the 
 number of genes analyzed in each group of ontology . sapk activity in the liver of lec rats : to examine the activities of sapk 
 in the liver of lec rats at 13 weeks of age , the activities of p38 mapk and jnk were 
 measured . although the activities of p38 mapk in the livers of lec rats at 13 weeks of age 
 were 8.1  2.1-fold higher than those in rats at 4 weeks of age ( fig . 3afig . 3.relative activities of p38 mapk in the liver of lec rats analyzed by western 
 blotting . ( a ) relative activities of p38 mapk in the livers of lec rats not 
 administered sb203580 at 4 ( lane 1 ) and 13 weeks ( lane 2 ) of age . relative activity of 
 p38 mapk normalized to the band intensity of rats at 4 weeks of age is 1.0 . the 
 relative activities represent an average of 3 to 4 separate experiments with standard 
 deviations . the activities at 13 weeks of age were significantly higher than those at 
 4 weeks of age ( p<0.01 ) . ( b ) activities of p38 mapk in the livers 
 of lec rats not administered ( lane 1 ) and administered sb203580 at 2.5 ( lane 2 ) and 5 
 ( lane 3 ) mg / kg body weight at 13 weeks of age . ( c ) relative activity of p38 mapk 
 normalized to the band intensity of non - administered rats is 1.0 . the activities 
 represent an average of 3 to 4 separate experiments with standard deviations . * 
 represents statistically significant difference from non - administered control 
 ( p<0.05 ) . ) , there was no significant difference between the activities of jnk in the livers of 
 lec rats at 4 and 13 weeks of age ( data not shown ) . when lec rats were administered 
 sb203580 , a p38 mapk - specific inhibitor , by s.c . injection at doses of 2.5 
 and 5 mg / kg body weight twice a week from 10 to 13 weeks of age , p38 mapk activities in the 
 livers of lec rats at doses of 2.5 and 5 mg / kg were 0.45  0.12- and 0.54  0.15-fold lower , 
 respectively , than those in livers of rats not administered sb203580 ( fig . 3b and 3c ) . relative activities of p38 mapk in the liver of lec rats analyzed by western 
 blotting . ( a ) relative activities of p38 mapk in the livers of lec rats not 
 administered sb203580 at 4 ( lane 1 ) and 13 weeks ( lane 2 ) of age . relative activity of 
 p38 mapk normalized to the band intensity of rats at 4 weeks of age is 1.0 . the 
 relative activities represent an average of 3 to 4 separate experiments with standard 
 deviations . the activities at 13 weeks of age were significantly higher than those at 
 4 weeks of age ( p<0.01 ) . ( b ) activities of p38 mapk in the livers 
 of lec rats not administered ( lane 1 ) and administered sb203580 at 2.5 ( lane 2 ) and 5 
 ( lane 3 ) mg / kg body weight at 13 weeks of age . ( c ) relative activity of p38 mapk 
 normalized to the band intensity of non - administered rats is 1.0 . the activities 
 represent an average of 3 to 4 separate experiments with standard deviations . * 
 represents statistically significant difference from non - administered control 
 ( p<0.05 ) . effects of sb203580 on acute hepatic injury in lec rats : activities of ast 
 and alt and concentrations of total bilirubin in sera of lec rats at 13 weeks of age 
 significantly increased compared to those in rats at 4 weeks of age ( table 2table 2.activities of ast and alt , and concentrations of total bilirubin in sera of 
 ratsratsweeks of ageconcentrations of 203580 ( mg / kg)activitiesconcentrations of total bilirubin 
 ( mg / dl)ast ( u / l)alt ( u / l)lec4053  12121  150.27  0.07100149  20 * 155 32130573  129 * 548  154 * 6.4  3.7 * 132.5286  144295  1550.29  0.17135.0219  92363  1143.26  1.91wkah4044  1072  110.20  0.0813054  1566 150.15  0.09the activities of ast and alt , and concentrations of total bilirubin represent an 
 average of 3 to 4 separate experiments with standard deviations . * and ! represent 
 statistically significant differences from 4 and 10 weeks of age , respectively 
 ( p<0.01 ) . # and # # represent statistically significant 
 differences from the control not administered at 13 weeks of age 
 ( p<0.05 and p<0.01 , respectively ) . ) . there were no significant differences in activities of ast and alt and 
 concentrations of total bilirubin in sera of wkah rats at 4 and 13 weeks of age . activities 
 of ast and alt in sera of lec rats rapidly and significantly increased from 10 to 13 weeks 
 of age ( p<0.01 ) . these results showed that acute hepatic injury with 
 jaundice occurred in lec rats at 13 weeks of age . when lec rats were administered sb203580 , 
 activities of ast and alt and concentrations of bilirubin in sera significantly decreased 
 compared to those in rats not administered sb203580 ( table 2 ) . the activities of ast and alt , and concentrations of total bilirubin represent an 
 average of 3 to 4 separate experiments with standard deviations . * and ! represent 
 statistically significant differences from 4 and 10 weeks of age , respectively 
 ( p<0.01 ) . # and # # represent statistically significant 
 differences from the control not administered at 13 weeks of age 
 ( p<0.05 and p<0.01 , respectively ) . since hepatic copper contents and frequencies in dna strand breaks in hepatic cells in lec 
 rats were maximized at 13 weeks of age under our experimental conditions [ 7 , 8 ] , the levels of 
 gene expression in the livers of lec rats at 13 weeks of age were compared to those in the 
 livers of rats at 4 weeks of age . although expression levels of many genes differed by more 
 than 2-fold in lec and wkah rats at 4 and 13 weeks of age , only 25 and 30 genes that were 
 up - regulated and down - regulated were overlapped in the livers of lec and wkah rats . these 
 results suggested that copper accumulation in the liver of lec rats induced expression of 
 different genes from those in wkah rats . in this study , we showed that the activities of p38 mapk in the livers of lec rats at 13 
 weeks of age were 8.1  2.1-fold higher than those in rats at 4 weeks of age . to determine 
 whether the increase in activity of p38 mapk is related to the occurrence of acute hepatic 
 injury in lec rats , lec rats were administered sb203580 , a p38 mapk - specific inhibitor . activities of ast and alt and concentrations of total bilirubin in sera of lec rats 
 administered sb203580 significantly decreased compared to those in rats not administered 
 sb203580 . the p38 mapk activities in the livers of lec rats administered sb203580 were also 
 significantly lower than those in rats not administered sb203580 . activities of ast and alt 
 in sera of lec rats rapidly increased from 10 to 13 weeks of age ( table 2 ) . therefore , inhibition of activation of p38 mapk by 
 sb203580 resulted in attenuation of the progression of acute hepatic injury in lec rats . it is well known that sapk is activated by a variety of stressors such ros , ultraviolet and 
 ionizing radiation [ 1 , 13 , 14 ] . excessive copper induces the 
 production of large amounts of ros [ 11 , 26 ] . therefore , it is thought that ros produced by 
 accumulated copper activated p38 mapk in the liver of lec rats . activation of p38 mapk 
 regulates a variety of cellular processes such inflammation , cell cycle arrest and apoptosis 
 in a cell type - specific manner [ 4 , 29 , 31 ] . indeed , 
 expression levels of many genes that regulate apoptosis and inflammatory response changed in 
 the livers of lec rats at 13 weeks of age compared to those at 4 weeks of age . since it has 
 been reported that activation of p38 mapk induces apoptosis [ 20 , 31 ] , sb203580 might inhibit the 
 induction of apoptosis of hepatic cells in lec rats . the study concerning a role of 
 apoptosis in occurrence of acute hepatic injury in lec rats is now in progress . furthermore , 
 activation of p38 mapk increases expression of inflammatory cytokines including tnf- . since fong et al .   suggested that tnf- plays a role in pathogenesis of 
 copper - induced acute hepatitis in lec rats , inhibition of p38 mapk activation might cause 
 decrease in expression of inflammatory cytokines . although administration of lec rats with sb203580 lowered activation of p38 mapk , the 
 activities of p38 mapk at 13 weeks of age were about 4-fold higher than those at 4 weeks of 
 age . therefore , occurrence of hepatic injury might not be completely inhibited under the 
 conditions in this study . there was no significant difference in activities of alt and ast 
 in the sera of lec rats administered sb203580 between 2.5 and 5 mg / kg . administration of rats with high 
 concentration of sb203580 might inhibit other protein kinase such phosphatidylinositol 
 3-kinase / protein kinase b . it has been reported 
 that sb203580 also directly inhibits thromboxane synthase , cyclooxygenase-1 and 
 cyclooxygenase-2 . furthermore , it has been 
 reported that sb203580 activates extracellular - regulated protein kinase and jnk in primary 
 human hepatocyte . our preliminary results showed 
 that the protective effects by sb203580 at 10 mg / kg body weight on acute hepatic injury 
 reduced in lec rats compared to rats administered at 2.5 mg / kg body weight ( data not shown ) . therefore , activation or inhibition of other protein kinases might affect the occurrence of 
 hepatic injury in lec rats administered sb203580 at high concentrations . in the present study , it was shown that the increase in activities of p38 mapk was related 
 to the occurrence of acute hepatic injury in lec rats . an inhibitor of p38 mapk may be a 
 therapeutic agent for attenuation of acute hepatic injury in patients with wilson s 
 disease .
abstractthe long - evans cinnamon ( lec ) rat , an animal model of human wilson s disease , 
 spontaneously develops fulminant hepatitis associated with severe jaundice at about 4 
 months of age . in this study , we examined the changes in gene expression during 
 progression of acute hepatic injury . when levels of gene expression in the liver of lec 
 rats at 13 weeks of age were compared to those in rats at 4 weeks of age using 
 oligonucleotide arrays , 1,620 genes out of 7,700 genes analyzed showed more than 2-fold 
 differences . 
 expression levels of 11 of 29 genes related to stress - activating protein 
 kinase ( sapk ) changed by more than 2-fold in the liver of lec rats , but none of the 
 sapk - related genes showed changes in expression levels in the liver of control rats . 
 activity of p38 mapk in the liver of lec rats at 13 weeks of age was about 8.1-fold higher 
 than that in rats at 4 weeks of age . when lec rats were administered sb203580 , a p38 
 mapk - specific inhibitor , by s.c . 
 injection twice a week from 10 to 13 
 weeks of age , activities of p38 mapk in the liver , activities of ast and alt and 
 concentrations of bilirubin in sera of rats administered sb203580 significantly decreased 
 compared to those in rats not administered . 
 these results showed that the increase in 
 activities of p38 mapk was related to the occurrence of acute hepatic injury in lec 
 rats .