nevirapine ( nvp , viramune , toc graphic ) 
 is a non - nucleoside reverse transcriptase inhibitor used for the treatment 
 of hiv-1 infections .
treatment with nvp is associated with a significant 
 incidence of idiosyncratic skin rashes and/or liver toxicity .
they are usually mild to moderate in nature ; however , 16% of nvp - induced 
 rashes are very severe , including stevens - johnson syndrome and toxic 
 epidermal necrolysis . in 2000
, the fda 
 placed a black box warning on nvp due to hepatotoxicity , which occurs 
 in 6% of patients and can be life threatening .
the incidence of elevated serum alanine transaminase ( alt ) in nvp - treated 
 patients , which is the first indication of liver injury , is between 
 8 and 18% and typically occurs within the first six weeks of treatment .
liver injury normally resolves when the drug 
 is stopped , but it can lead to fulminant liver failure and death
. 
 there also exists evidence for increased risk of liver injury in non - hiv 
 patients , which may be due to higher cd4 cell counts .
the mechanisms of idiosyncratic liver injury and 
 skin rashes are currently unknown , but most idiosyncratic drug reactions 
 appear to be mediated by reactive metabolites .
we developed an animal 
 model of nvp - induced skin rash in brown norway ( bn ) rats that is clearly 
 immune - mediated and has characteristics very similar to the rash in 
 humans ; however , the rats did not develop liver toxicity .
we postulated that the 12-hydroxylation pathway was involved in 
 the induction of the skin rash ; therefore , we replaced the hydrogens 
 on the methyl group with deuterium to slow down the rate of 12-hydroxylation 
 ( toc graphic ) .
we found that this analogue ( dnvp ) did not cause a 
 skin rash as predicted , but instead of higher blood levels because 
 one of the major metabolic pathways was inhibited , we found that the 
 blood levels of dnvp were actually much lower than those of nvp at 
 the same dose .
although the reason for 
 this was not immediately obvious , we ultimately concluded that , in 
 addition to oxygen rebound to form 12-oh - nvp , the intermediate free 
 radical in the p450-mediated oxidation could also lose a hydrogen 
 atom to form a reactive quinone methide ( toc graphic ) .
a glutathione 
 conjugate consistent with the quinone methide intermediate has been 
 reported ; however , it could also come from a sulfate 
 conjugate of the 12-oh - nvp . in this study
, we used an antiserum 
 against nvp to study the covalent binding of nvp , dnvp , and 12-oh - nvp 
 to hepatic proteins in mice , rats , and humans .
we also studied the 
 effects of chronic administration of nvp to various strains of mice 
 to determine if it causes liver injury .
in addition to c57bl/6 and 
 balb / c , we included the casitas b - lineage lymphoma - b ( cbl - b ) knockout 
 mouse ( cbl - b ) , which is bred on a c57bl/6 
 background . the cbl gene is a mammalian gene that encodes a variety 
 of proteins , specifically those involved in cell signaling and protein 
 ubiquitination .
lack of ubiquitination of nvp protein adducts could 
 lead to more persistent covalent binding and possibly toxicity .
this 
 also impairs immune tolerance ; therefore , if the liver injury is immune - mediated , 
 these animals should be at increased risk .
these animals also express 
 a mouse isoform of cyp3a4 ( cyp3a11 ) ; therefore , oxidative metabolism 
 of nvp should occur , and this has the 
 potential to lead to liver injury .
( ridgefield , ct ) . the majority of chemical reagents 
 ( 1-aminobenzotriazole ( abt ) , tris(hydroxymethyl)aminomethane base , 
 methanol , dmso , phosphate - buffered saline ( pbs , ph 7.4 ) , glycerol , 
 silica gel , etc . ) were obtained from sigma - aldrich ( oakville , on ) 
 unless otherwise noted in the methods .
sodium dodecyl sulfate and 
 tween-20 were obtained from bioshop ( burlington , on ) .
stock acrylamide / bis 
 solution ( 29:1 , 3.3% c ) , nonfat blotting grade milk powder , and nitrocellulose 
 membrane ( 0.2 m ) were purchased from bio - rad ( hercules , ca ) . 

amersham ecl plus western blotting detection system 
 was obtained from ge healthcare ( oakville , on ) .
horseradish peroxidise - conjugated 
 goat antirabbit igg ( h + l chains ) and monoclonal gapdh were purchased 
 from sigma - aldrich ( st . louis , mo ) .
expressed human cyp3a4 , rat cyp3a1 , 
 and rat cyp2c11 ( each with p450 reductase and cytochome b5 ) , 0.5 m potassium phosphate at ph 7.4 , and nadph regenerating 
 system solutions a and b were purchased from bd biosciences ( woburn , 
 ma ) .
alphaeasefc ( fluorchem 8800 ) , 
 manufactured by alpha innotech , now cell biosciences santa clara , 
 california , usa , was used to image blots .
integrated density values 
 were obtained using the spot denso function on the fluorchem 8800 
 imager .
synthesis of 
 dnvp was carried out using the method described by chen et al.h nmr ( cdcl3 ) :  
 0.310.41 ( m , 2h ) , 0.830.90 ( m , 2h ) , 3.603.64 
 ( m , 1h ) , 7.06 ( d , j = 4.8 hz , 1h ) , 7.19 ( dd , j = 4.8 , 7.5 hz , 1h ) , 8.01 ( dd , j = 2.1 , 6.6 hz , 1h ) , 8.08 
 ( d , j = 4.8 hz , 1h ) , 8.50 ( dd , j = 1.8 , 4.8 hz , 1h ) , 9.90 ( bs , 1h ) . 

esi - ms : m / z ( % ) 270 ( mh , 100% ) .
the ratio of the peaks at m / z 267:268:269:270 as determined by mass spectrometry was 0:0.007:0.124:0.869 , 
 indicating only trace amounts of nvp .
the synthesis of the 
 immunogen is outlined in scheme 1 . the first 
 step in producing
the anti - nvp antiserum was to synthesize 12-oh - nvp 
 ( 2 ) and convert this to the benzylic chloride ( 12-cl - nvp , 3 ) .
the method to produce 12-oh - nvp followed the protocol 
 described previously with minor modifications . 

esi - ms ; m / z ( % ) 283 ( mh+ , 100% ) . 
 to convert 12-oh - nvp to 12-cl - nvp
( 200 mg ) in dry dichloromethane 
 ( 10 ml ) at 0 c was added n , n - diisopropylethylamine ( 0.14 ml ) followed by thionyl chloride ( 3 
 ml ) , and it was stirred under argon at room temperature for 3 h after 
 which the thionyl chloride was evaporated by rotary evaporation . the 
 reaction mixture
the ethyl acetate layer was washed with water ( 10 ml ) , dried 
 over anhydrous sodium sulfate , and concentrated to yield crude product , 
 which was purified with open column chromatography ( silica gel , pore 
 size 60  , 70230 mesh , column dimensions 30  200 
 mm ) eluted with 50% ethyl acetate / hexanes to yield 0.386 g of yellow 
 solid .
esi - ms ; m / z ( % ) 301 ( mh , 100% ) .
the 12-cl - nvp ( 1.78 g , 3.55 mmol ) was dissolved 
 in 18 ml of tetrahydrofuran and reacted with n - acetylcysteine 
 ( nac , 2.31 g , 14.18 mmol ) in 5 ml of triethylamine under argon reflux 
 for 2 h. the crude mixture was cooled to room temperature , acidified 
 to ph 34 by 1 n hcl and extracted with chcl3 .
the nevirapine - nac conjugate was 
 obtained as a pale yellow solid ( 4 ) .
formation of the 
 nevirapine - nac conjugate was confirmed by mass spectrometry esi - ms ; m / z ( % ) 428 ( mh , 100% ) .
activation of the carboxy groups 
 on nac of the synthesized 12-nac - nvp occurred as follows : to 61.4 
 mg 12-nac - nvp was added 108.5 mg of n - hydroxysuccinimide 
 and 103.9 mg of 1-ethyl-3-(3-dimethyllaminopropyl ) carbodiimide hydrochloride . 

the 
 entire mixture was sealed with a rubber stopper and stirred at 0 c 
 for 2 h under n2 .
methylene chloride ( 8 ml ) was added , 
 followed by washing with water ( 3  8 ml ) , and then the organic 
 layer was partially evaporated in vacuo to yield 
 a pale yellow solution ( 0.5 ml , 5 ) .
dmf ( 4 ml ) was added 
 followed by keyhole limpet hemocyanin ( klh , 8 mg ) , and the mixture 
 was stirred for 1 h at 4 c .
the reaction mixture was then concentrated 
 under a n2 stream , and 1 ml water was added .
centrifugal 
 filtration was performed to collect the protein solution , which was 
 then lyophilized . a final white powder ( 10.4 mg )
the same method was used 
 to prepare a conjugate with bovine serum albumin ( bsa ) maldi ms ; m / z 67,13968,569 .
the hapten density 
 of the bsa conjugate was approximately 4.5 molecules of nvp - nac / bsa 
 as determined by the increase in mass on mass spectrometry .
polyclonal 
 anti - nvp - nac - klh antibodies were raised in two individual 2 kg , male , 
 pathogen - free new zealand white rabbits ( charles river , quebec ) housed 
 in the animal care facility at the division of comparative medicine , 
 university of toronto .
each animal was immunized with the nvp - nac - klh 
 conjugate ( 1 mg antigen + 100 l of glycerol in 1.8 ml of phosphate 
 buffered saline emulsified with an equal volume of freund s 
 complete adjuvant ) subcutaneously at multiple sites .
injections with 
 500 g of nvp - nac - klh in freund s incomplete adjuvant 
 divided into six to eight subcutaneous sites were repeated 4 , 6 , 8 , 
 and 12 weeks after the initial immunization .
the animals were exsanguinated 
 while under pentobarbital anesthesia 10 days after the final immunization . 

the serum was heat - inactivated at 56 c for 30 min before being 
 stored at 80 c .
nvp - nac - bsa , bsa , or klh ( 100 l , 10 g / ml 
 in carbonate  bicarbonate coating buffer ) were coated into the 
 wells of a flat - bottom 96-well plate ( costar , cambridge , ma ) , and 
 the plate was incubated overnight at 4 c .
the plates were washed 
 with elisa wash buffer ( 50 mm tris(hydroxymethyl)aminomethane - buffered 
 saline , ph 8.0 , and 0.05% tween-20 ) three times and blocked by the 
 addition of 100 l of postcoat solution ( 50 mm tris - buffered 
 saline , ph 8.0 , and 1% bsa ) for 30 min at room temperature .
following 
 the blocking step , the wells were washed three times , and various 
 dilutions of the anti - nvp - nac - klh antiserum or preimmune serum were 
 added to the plates , which were then incubated at room temperature 
 for 2.5 h. the plates were subsequently washed three times with elisa 
 wash buffer , and horseradish peroxidase - conjugated goat antirabbit 
 igg ( diluted 1:5000 in postcoat solution ; 100 l ) was added 
 to each well .
the elisa plates were incubated at room temperature 
 for 2 h. plates were then washed three times with elisa wash buffer . 

enzyme substrate ( 3,3,5,5-tetramethylbenzidine peroxidase 
 substrate and peroxidase solution b , kirkegaard & perry laboratories ) 
 was mixed in equal volumes , and 100 l of the enzyme substrate 
 was added to each well .
sulfuric acid ( 2 m , 100 l ) was added 
 to each well to quench the reaction .
absorbance was measured with 
 the basic end point option of softmax pro 5 software , using the spectra 
 maxplus384 plate reader ( molecular devices technologies ) set at 450 
 nm .
male ( 200250 g ) or female bn rats 
 ( 150175 g ) were obtained from charles river ( montreal , quebec ) . 

rats were housed in pairs in standard cages in a 12:12 h light / dark 
 cycle with access to water and agribrands powdered lab chow diet ( leis 
 pet distribution , inc .
wellesley , ontario ) ad libidum . following a 
 one week acclimatization period , rats were either maintained on control 
 chow or started on drug containing diet ( treatment groups ) .
the drug 
 was mixed thoroughly with powdered lab chow if it was to be administered 
 orally .
the amount of drug administered to animals was calculated 
 based on body weight of the rats and their daily food intake .
male balb / c 
 or c57bl/6 mice ( 68 weeks age ) were obtained from charles 
 river ( montreal , quebec ) .
cbl - b knockout 
 mice were bred in house from animals first developed by dr .
j. penninger 
 at the institute of molecular biotechnology of the austrian academy 
 of science , vienna , with his kind permission .
the average weight gain was approximately 0.75 g per week ( data 
 not shown ) .
animal experiments were approved by the university 
 of toronto animal care committee in accordance with guidelines of 
 the canadian council on animal care .
female bn rats were treated with nvp or dnvp at 150 mg / kg / day , or 
 12-oh - nvp at 159 mg / kg / day ( equimolar dose ) orally in standard rat 
 chow for either 8 , 10 , or 21 days .
dosages were based on previous 
 work showing the induction of rash at these levels .
injection lasted 21 days 
 with a dose of 75 mg / kg / day of either compound .
abt was dissolved 
 in water ( 20 mg / ml ) and administered via gavage at a dose of 50 mg / kg / day . 
 if abt was to be given to animals , the dose of nvp was 50 mg / kg / day 
 via gavage .
methylcellulose ( 0.5% ) was used to suspend nvp or metabolites 
 given to rats by gavage or s.c .
all mice were started on 
 nvp at 950 mg / kg / day in standard chow after preliminary studies showing 
 no apparent toxicity or mortality of mice at either 550 or 950 mg / kg / day .
livers were 
 homogenized in ice - cold 1.15% kcl using a polytron 2100 homogenizer 
 and centrifuged at 26,400 g for 10 min at 4 c . 

the supernatant was then centrifuged at 100,000 g for 
 50 min at 4 c .
the protein 
 concentration of the prepared microsomes was quantified using a bca 
 protein assay kit ( novagen , emd biosciences inc . ) .
nvp , 12-oh - nvp , or dnvp stock solutions 
 were prepared in methanol , and the final methanol concentration in 
 the reactions did not exceed 1% for any incubation .
the microsomal incubations consisted of 100 mm potassium 
 phosphate buffer ( ph 7.4 ) , an nadph - regenerating system ( solution 
 a final concentrations , 1.3 mm nadp , 3.3 mm glucose-6-phosphate , and 
 3.3 mm mgcl2 ; solution b final concentration , 0.4 units / ml 
 glucose-6-phosphate - dehydrogenase ) and microsomal homogenate ( final 
 protein concentration varying from 0.3 mg / ml to 15 mg / ml ) .
edta2na 
 ( 0.4 mm ) was added to rat cyp3a1 and 2c11 incubations , and water was 
 added to each incubation to reach a final volume of 400 l for 
 rat and mouse or 200 l for human 3a4 incubations .
incubations consisting of all reaction components 
 except the nadph - regenerating system or drug were preincubated for 
 5 min .
the nadph - regenerating system or drug was added to each of 
 the test and control tubes after the 5 min of preincubation .
reactions 
 were stopped by placing the sample vials on dry ice and storing at 
 80 c .
if microsomal incubations 
 were to be analyzed via lc / ms , 250 l of ice cold acetonitrile 
 was used to quench the reaction , and internal standard ( ethyl - nvp 
  a nvp derivative in which the cyclopropyl group has been 
 replaced with an ethyl group , 5.4 g / ml , 50 l ) was added 
 to each tube , contents were centrifuged , separated by solid phase 
 extraction ( strata solid phase extraction column c18-e , 100 mg , by 
 phenomenex ) , evaporated in vacuo at 50 c , and 
 reconstituted to 50 l prior to analysis .
samples were reconstituted to 50 l with mobile phase ( 16% 
 acetonitrile and 84% water with 2 mm ammonium acetate and 1% acetic 
 acid ) .
the separation was performed on an ultracarb c18 30  2.0 mm , 
 5 m column ( phenomenex ) under isocratic conditions with a mobile 
 phase consisting of 16% acetonitrile and 84% water with 2 mm ammonium 
 acetate and 1% acetic acid .
mass spectrometry was carried 
 out using a pe sciex api 3000 quadrupole system with an electrospray 
 ionizing source .
the ion pairs used for this analysis were 267.0/226.1 
 for nvp , 283.1/223.1 for 12-oh - nvp , 297.1/210.1 for 4-cooh - nvp , 283.1/161.0 
 for 2-oh - nvp , 283.1/214.0 for 3-oh - nvp , and 255.1/227.2 for ethyl - nvp 
 ( positive ionization mode ) .
standard curves prepared for 2-oh - nvp 
 ( 0.43102.9 g / ml ) , 3-oh - nvp ( 0.3686.8 g / ml ) , 
 12-oh - nvp ( 0.3891.0 g / ml ) , 4-cooh - nvp ( 0.2661.8 
 g / ml ) , and nvp ( 0.74176.9 g / ml ) had r values of > 0.99 .
livers were homogenized in working cell lysis buffer ( cell signaling 
 technologies , pickering , on ) containing 1 halt protease inhibitor 
 cocktail ( pierce , rockford , il ) with a polytron 2100 homogenizer and 
 centrifuged at 1000 g for 15 min , and the supernatant 
 was collected and again centrifuged at 10  000 g for 30 min .
the supernatant was mixed with pierce reducing sample 
 loading buffer in a 4:1 protein to buffer ratio and boiled for 5 min . 

polyacrylamide gel electrophoresis was 
 performed using the protean-3 minigel system ( biorad , mississauga , 
 on ) .
gels were hand - cast ( 8% ) or bought from bio - rad canada ( 12% ) 
 and were run at 130 v. electrophoresis running buffer ( bio - rad ) consisted 
 of 25 mm tris base , 192 mm glycine , and 0.1% sodium dodecyl sulfate , 
 ph 8.3 .
transfer to nitrocellulose membrane ( 0.2 m , biorad ) 
 occurred at 0.13 ma for 90 min at 4 c using the same protean-3 
 minigel system ( biorad , mississauga , on ) .
tris - glycine transfer buffer 
 ( bio - rad ) consisted of 25 mm tris , 192 mm glycine , and 20% methanol 
 at ph 8.5 .
membranes were washed twice in tris - buffered saline tween-20 
 ( tbst ) wash solution for 5 min .
membranes were then rinsed with three changes 
 of tbst for 5 min each and incubated with a 1:100 or 1:500 dilution 
 of primary anti - nvp antiserum and 10% normal goat serum in tbst overnight 
 at 4 c . a 20 min wash ( three changes ) in tbst after overnight 
 blocking was followed by a 90 min incubation in secondary antisera 
 ( 1:2000 or 1:5000 dilution ) in tbst containing 10% goat serum .
all blots were 
 incubated with enhanced chemiluminescence stain for 5 min and analyzed 
 with a fluorchem8800 imager . to probe for the glyceraldehyde 3-phosphate 
 dehydrogenase ( gapdh ) loading control , membranes were stripped of 
 primary anti - nvp antiserum using pierce restore plus buffer ( pierce , 
 rockford , il ) for 15 to 20 min at room temperature followed by a 1 
 h blocking step .
membranes were then incubated in mouse monoclonal 
 anti - gapdh antisera ( 1:40,000 ) and processed as described above except 
 that the secondary antisera were goat antimouse horseradish peroxidase 
 antisera diluted 1:10,000 ( jackson immunoresearch , baltimore pike , 
 west grove , pa . ) .
the liver samples were fixed in 10% 
 formalin and the paraffin block , hematoxylin / eosin slides , or unstained 
 sections were prepared at the toronto hospital for sick children . 
 for immunohistochemical staining ,
nonspecific sites were blocked with 
 10% goat serum for 1 h. at this point , the anti - nvp antiserum was 
 diluted to 1:100 in 10% goat serum and applied to each section overnight . 
 following a washing step ,
slides were submerged in 0.3% hydrogen peroxide 
 in methanol for 10 min to block endogenous peroxidases followed by 
 a washing step .
igg - hrp conjugated 
 antisera ) was applied to the sections at a dilution of 1:3000 in 10% 
 goat serum .
sections were incubated with secondary antiserum for 2 
 h. after a final washing step , vector novared stain was added as a 
 substrate for the peroxidases following package protocol .
sections 
 were then counterstained with mayer s hematoxylin ( sigma ) , 
 dehydrated by sequential immersion in increasing concentrations of 
 ethanol , cleared in xylenes , and mounted using permount mounting medium 
 ( fisher , markham , on ) .
alanine 
 transaminase ( alt ) was assayed using the infinity alt ( glutamic pyruvate 
 transaminase ) liquid stable reagent kit by thermo scientific .
screening 
 for cytokines was performed using a luminex immunoassay mouse cytokine / chemokine 
 kit from millipore corporation ( milliplex map kit ) .
homogenized liver 
 tissue or serum samples prepared accroding to the kit 's specifications 
 were plated and analyzed following the manufacturer s instructions .
elisa analysis showed that the anti - nvp - nac - klh antiserum recognized 
 the nvp - nac - bsa conjugate or klh but not bsa alone ( figure 1a ) .
the binding of the antisera to the nvp - nac - bsa 
 conjugate was inhibited by preincubating the anti - nvp - nac - klh antiserum 
 with nvp or its metabolites ( figure 1b ) .
inhibition 
 was much less with 2-oh - nvp , 3-oh - nvp , and 4-cooh - nvp ( the metabolite 
 in which the methyl group has been oxidized to a carboxylic acid ) . 

elisa 
 analysis showing ( a ) binding of the anti - nvp - nac - klh antiserum to 
 the nvp - nac - bsa conjugate , klh , or bsa , and ( b ) the effect of preincubation 
 of the antiserum with nvp or its metabolites on the binding of the 
 antisera to the nvp - nac - bsa conjugate .
when microsomes produced from male bn rats 
 were incubated with nvp , 12-oh - nvp , or dnvp , the greatest covalent 
 binding observed was with nvp , and the stongest band was at 55 
 kda ( figure 2a ) , which corresponds to the mass 
 of the male dominant p450 2c11/3a1 isoforms .
incubation of mouse liver microsomes with nvp produced a band of 
 slightly higher mass , 57 kda ( figure 2b ) , corresponding to the mass of the dominant murine p450 3a11 .
significant covalent binding of 12-oh - nvp was 
 not observed with rat microsomes , and dnvp produced a much fainter 
 band at 55 kda than nvp in both rodent species tested . in 
 vivo experiments with either species
displayed a wide range 
 of covalently modified bands that were much more intense than from in vitro experiments .
the covalent binding of dnvp to both 
 rat and mouse hepatic microsomes was also much less than that of nvp 
 by almost 5-fold as determined by densitometry ( data not shown ) .
the 
 amount of binding did not increase significantly beyond 15 min ( figure 2c ) .
( a ) comparison of covalent binding of 12-oh - nvp ( lanes 
 2 and 5 ) and dnvp ( lane 3 , 6 ) with that of nvp ( lanes 4 and 7 ) after 
 a 30 or 60 min incubation with male bn rat microsomes ( 1 mg / ml protein ) 
 at a drug concentration of 1 mm . for comparison ,
covalent binding 
 to hepatic proteins is shown after 8 days of treatment of female rats 
 with 12-oh - nvp ( 159 mg / kg / day , lane 9 ) or nvp ( 150 mg / kg / day , lane 
 10 ) .
protein loading was 15 g for lanes 17 and 20 g 
 for lanes 810 .
( b ) comparison of covalent binding of 12-oh - nvp 
 ( lanes 2 , 5 ) and dnvp ( lanes 3 and 6 ) with that of nvp ( lanes 4 and 
 7 ) at a concentration of 1 mm after a 30 or 60 min incubation with 
 microsomes ( 1 mg / ml protein ) from male c57bl/6 mice .
for comparison , 
 covalent binding to hepatic proteins is shown after 6 weeks of treatment 
 of c57bl/6 mice with nvp at a dose of 950 mg / kg / day in food .
protein 
 loading was 13 g for lanes 17 and 20 g for lanes 
 89 .
( c ) comparison of covalent binding of nvp to hepatic microsomes 
 from male c57bl/6 mice ( lanes 24 ) or male bn rats ( lanes 68 ) 
 after a 15 , 30 , or 60 min incubation at a drug concentration of 1 
 mm and microsome concentration of 1 mg / ml protein .
the primary antiserum dilution was 1:500 , 
 and that of the secondary antisera was 1:5000 .
incubation of nvp with expressed rat cyp2c11 ( figure 3a ) or cyp3a1 supersomes ( figure 3b ) , 
 the dominant forms of p450 in male rats led to covalent binding 
 with major bands produced at 50 kda and 52 kda , respectively . 
 in the absence of nvp as indicated in the figures , there is a small 
 artifact band . binding to 2c11 and 3a4 was strongest at 30 min ; a 
 decrease in the intensity of the p450 band was observed from 30 to 
 120 min .
covalent binding of nvp to expressed male rat cyp2c11 ( a ) or cyp3a1 
 ( b ) in vitro .
protein concentration for each incubation 
 was 0.8 mg / ml with 0.5 mm of drug . for immunoblots ,
protein loading 
 was 9 g and 7.5 g per lane for a and b , respectively . 

( + ) indicates incubations containing nvp , while (  ) indicates 
 incubations lacking nvp .
proteins were resolved on 12% gels with 1:100 
 dilution of primary antiserum followed by 1:2000 dilution of secondary 
 antisera .
comparison of covalent binding of 12-oh - nvp ( lanes 2 and 
 5 ) or dnvp ( lanes 3 and 6 ) with that of nvp ( lanes 4 and 7 ) to human 
 cyp3a4 with a drug concentration of 1 mm and protein concentration 
 in each incubation of 1 mg / ml ( c ) .
dilutions of antisera were 1:500 for the primary 
 antiserum and 1:5000 for the secondary antiserum .
the incubation of expressed human 3a4 displayed 
 the greatest binding with nvp ( figure 3c ) versus 
 12-oh - nvp or dnvp .
however , 12-oh - nvp did bind to human cyp3a4 more 
 than expected , although less than nvp , and there was much less binding 
 of dnvp .
the nvp - modified band had a mass of 57 kda , which 
 is the mass of cyp3a4 .
female bn rats 
 were treated with nvp or 12-oh - nvp for a period of 8 days at doses 
 of 150 mg / kg / day or 159 mg / kg / day , respectively ( figure 4 ) .
the pattern of covalent binding was different for nvp and 
 12-oh - nvp ; this difference was most prominent for the lower molecular 
 mass proteins ( 30 60 kda ) .
nvp - treated female bn rats exhibited 
 greater covalent binding than 12-oh - nvp - treated rats at an equimolar 
 dose , but there was a prominent artifact band in the 12-oh - nvp blot 
 at about 60 kda .
preincubation of the anti - nvp serum with nvp blocked 
 almost all of the binding ( figure 4b ) .
( a ) covalent 
 binding to hepatic proteins from female bn rats fed nvp ( 150 mg / kg ) 
 or 12-oh - nvp ( 159 mg / kg ) for 8 days .
a 1:500 dilution of 
 primary antiserum was followed by 1:5000 dilution of secondary antisera . 

( b ) incubation of the anti - nvp serum with 2 mm nvp for 2 h at 37 c 
 blocked most of the binding ( left side of panel ) to samples from livers 
 of 12-oh or nvp treated rats .
samples for both panels a and b were 
 prepared , run , blocked , incubated with secondary antibody , and imaged 
 at the same time , and protein loading was 10 g / well of protein 
 per lane .
hepatic covalent binding 
 of nvp and dnvp was greatest in the centrilobular area ( figure 5 ) .
the pattern of binding was dramatically different 
 in rats treated with a combination of nvp and the p450 inhibitor aminobenzotriazole ; 
 specifically , treatment with abt blocked binding in the centrilobular 
 area and shifted it to the periportal area .
co - treatment with abt 
 also changed the pattern of binding by western blot , although there 
 was still significant binding ( data not shown ) .
clearance of nvp depends 
 on oxidative metabolism , and so even if p450 is inhibited , it causes 
 an increase in blood levels , but ultimately nvp is oxidized .
immunohistochemistry 
 of liver sections from female bn rats ; blank control , nvp treatment 
 ( 150 mg / kg / day  7 days in food ) , dnvp treatment ( 150 mg / kg / day 
  7 days in food ) , abt treatment ( 50 mg / kg / day  28 days 
 by gavage ) , or nvp ( 150 mg / kg / day ) + abt ( 50 mg / kg / day )  28 
 days by gavage .
slides were incubated with 1:100 dilution of primary 
 antisera and 1:2000 dilution of the secondary antisera .
the slides 
 were counterstained with mayer s hematoxylin ; magnification 
 20. the carboxylic acid ( 4-cooh - nvp ) of nvp was detected in the incubation 
 of 12-oh - nvp with nadph and hepatic microsomes from both male and 
 female bn rats ( figure 6 ) .
4-cooh - nvp concentrations from incubations 
 of 12-oh - nvp with microsomes from male ( n = 3 ) and 
 female ( n = 1 ) bn rats .
there was no change in plasma alt in bn rats treated 
 with nvp ( data not shown ) .
various strains of mice were treated with 
 nvp to determine if it causes liver damage , covalent binding , and/or 
 histological changes .
male balb / c mice treated with nvp had no increase 
 in alt ( data not shown ) , while there was an increase in alt in male 
 c57bl/6 mice at 3 weeks followed by normalization of alt levels ( figure 7a ) .
immunoblots revealed no significant differences 
 between the pattern and degree of binding in these two strains ( figure 7b ) .
alt levels in both male and female cbl - b mice increased at week 2 , with a somewhat 
 greater increase in male mice ( figure 8a ) than 
 female mice ( figure 8c ) .
the animals with the 
 largest alt increase displayed areas of gross hepatic necrosis evident 
 as areas of white on the surface of the liver upon sacrifice at 2 
 weeks .
immunoblot analysis showed the presence of a wide range of 
 modified hepatic protein in both male ( figure 8b ) and female ( figure 8d ) mice .
animals with 
 gross necrosis appeared to have slightly more binding of nvp to lower 
 molecular mass proteins ( figure 8b , d ) .
( a ) changes 
 in alt in male c57bl/6 mice treated with nvp ( 950 mg / kg / day ) for 4 
 weeks .
values are based on the mean of triplicate readings per time 
 point per animal  sd , n = 5 treated mice or n = 4 control mice .
( b ) corresponding covalent binding 
 of nvp at the same dose in male balb / c ( n = 2 ) or 
 c57bl/6 ( n = 3 ) mouse livers after 6 weeks of treatment . 

( a ) plasma alt levels in male cbl - b mice fed nvp orally for 14 days ( 950 mg / kg / day ) .
values are based 
 on the mean of triplicate readings per time point per animal  
 sd , n = 5 treated mice or n = 4 
 control mice .
( b ) covalent binding of nvp in the livers of the same 
 cbl - b mice .
( c ) plasma alt levels in nvp - treated 
 ( 950 mg / kg / day ) female cbl - b mice , n = 4 treated or n = 4 control mice .
values 
 are based on the mean of triplicate readings per time point per animal 
  sd , n = 5 treated mice or n = 4 control mice
( d ) covalent binding of nvp in the livers of 
 the same mice .
a 1:500 dilution of primary 
 antisera followed by 1:5000 dilution of secondary antisera was used .
luminex analysis for a broad range of cytokines 
 performed on serum of mice from the 2 week study on days 1 , 7 , and 
 14 of nvp treatment revealed an increase in interferon - gamma ( ifn- ) 
 in plasma samples of male mice on day 7 ( figure 9b ) , both in animals that developed significant necrosis and those 
 that did not , but the level was highest in an animal that did develop 
 necrosis .
il-6 was also increased at day 7 versus day 14 of nvp treatment 
 in plasma of male mice ( figure 9a ) . by day 
 14 of nvp treatment ,
the cytokine levels had decreased to or close 
 to the baseline ( data not shown ) for the majority of animals .
serum il-6 
 ( a ) or ifn- ( b ) from control and nvp - treated cbl - b mice at day 7 of nvp treatment .
changes in cytokines were less clear for serum 
 samples from female cbl - b   mice , and no 
 inferences could be made ( data not shown ) .
no significant changes 
 in cytokines were observed for gm - csf , il-10 , 1 l-12(p70 ) , il-13 , 
 il-17 , 1 l-1 , il-2 , il-4 , il-5 , il-7 , il-9 , mcp-1 , or tnf-. liver histology of cbl - b mice sacrificed after 2 weeks of nvp treatment 
 at the time of maximal alt elevation is shown in figure 10 .
the presence of gross necrosis , which was visible on the 
 surface of the liver as white areas was observed in 4 of 7 treated 
 animals .
two of 8 nvp - treated female mice with minor 
 liver necrosis had alt values of 286 and 80
histology in female 
 mice did not demonstrate as much injury as in males ( data not shown ) . 

histology of the livers of affected males showed the presence of focal 
 subcapsular areas of massive liver necrosis ( figure 10b , c ) sharply demarcated from the adjacent viable liver .
necrotic 
 areas were surrounded by and infiltrated by mononuclear cells , macrophages , 
 and neutrophils .
this pattern of liver necrosis suggests an ischemic 
 injury , but no evidence of thrombi or vasculitis was observed .
multifocal 
 necro - inflammatory hepatitis with neutrophil - rich inflammatory response 
 was observed in the absence gross necrotic lesions in male cbl - b mouse livers .
lower doses of nvp were also 
 tested with cbl - b mice , 
 but no injury was seen ( data not shown ) .
in contrast , hepatic histology 
 of c57bl/6 mice treated with nvp and sacrificed at 4 weeks displayed 
 hepatocyte death on the edge of the lobe in one animal , as well as 
 small focal areas of necrosis ( figure 11b ) . 

induction of smooth endoplasmic reticulum ( figure 11c ) , presumably including p450 induction , was present in the 
 histology of all mice strains tested but was most prominent for cbl - b male mice .
this marked induction may have 
 led to greater reactive metabolite formation contributing to the greater 
 toxicity in this strain , and this appeared to be the case although 
 the difference is subtle ( figure 12 ) .
h&e staining 
 of livers from cbl - b mice treated with 
 nvp for 2 weeks .
( a ) untreated control liver with normal alt ; ( b ) 
 the liver from a nvp - treated mouse with gross necrosis and an alt 
 of 271 u / l , and the ( c ) liver from another nvp - treated mouse with 
 gross necrosis and alt of 313 u / l .
areas of massive hepatocyte necrosis 
 surrounded by viable hepatocytes are shown in b and c. h&e staining of livers from male c57bl/6 mice treated 
 with nvp for 3 weeks .
( a ) untreated control liver with a normal alt ; 
 ( b ) the liver from a nvp - treated mouse with very mild necrosis ( appearing 
 as the thin band around the capsule ) and alt of 94 u / l ; and ( c ) the 
 liver from another nvp - treated mouse with an alt of 75 u / l .
changes 
 to the liver parenchyma due to enlargement of hepatocytes in the periacinar 
 regions and extensive expansion of the endoplasmic reticulum are also 
 present in both b and c. comparison of covalent binding of nvp to hepatic proteins 
 in mice and rats .
nvp was fed to rats in a time course manner from 
 1 to 8 days at 150 mg / kg orally in food .
mice were given 950 mg / kg / day 
 for 2 weeks or 10 weeks .
a 1:500 dilution 
 of primary antisera followed by a 1:5000 dilution of secondary antisera 
 was used .
female bn rats treated with nvp for 1 , 2 , 
 4 , or 8 days were sacrificed , and covalent binding was determined 
 ( figure 12 ) . in comparison with cbl - b knockout mice at 2 or 10 weeks of treatment or male c57bl/6 mice 
 at 2 weeks of treatment , rats had significantly greater binding from 
 day 4 onward . in all animals ,
the presence of a modified p450 band 
 at 55 kda was prominent and represents the largest modified 
 band in each lane .
while modified proteins in rats range from 20 to 
 100 kda , it appeared that lower molecular weight proteins were modified 
 in mice ( up to 70 kda ) .
treatment of cbl - b mice with nvp for 2 weeks led to greater binding than that at 10 
 weeks , and c57bl/6 mice displayed the least binding of the species 
 tested .
an anti - nvp antiserum was produced and used 
 to demonstrate that nvp covalently binds to hepatic proteins , both 
 in vitro and in vivo .
binding occurred 
 directly to p450 as demonstrated by covalent binding to expressed 
 p450s , both rat and human .
we have shown that the skin rash requires 
 oxidation of nvp to 12-oh - nvp , and most 
 recently , we have shown that covalent binding of the benzylic sulfate 
 of this metabolite formed by sulfotransferase in the skin is responsible 
 for the rash ( manuscript in preparation ) .
in contrast , the majority 
 of binding in the liver must involve direct oxidation by p450 as evidenced 
 by the marked shift in the pattern of binding from the centrilobular 
 region to the portal region caused by the p450 inhibitor abt , as shown 
 in figure 5 .
there is also less covalent binding 
 of 12-oh - nvp than nvp in the liver .
furthermore , substitution of the 
 methyl hydrogens with deuterium ( dnvp ) led to a marked decrease in 
 covalent binding .
given that oxidation of the methyl group is involved 
 in the covalent binding , but it does not involve 12-oh - nvp , these 
 data provide strong evidence that the chemical species responsible 
 for the covalent binding in the liver is a quinone methide formed 
 by the loss of a hydrogen atom from the p450-generated free radical 
 ( toc graphic ) .
others have found evidence for an epoxide reactive 
 metabolite , but these data suggest that 
 it is less important with respect to covalent binding than the quinone 
 methide .
some covalent binding of 12-oh - nvp was detected in 
 the in vitro experiments where phase ii pathways 
 such as sulfation would not occur , and the pattern of binding was 
 somewhat different from that of nvp .
this suggests that oxidation 
 of 12-oh - nvp can lead to a reactive metabolite , although the binding 
 is less than that for nvp .
this could be due to oxidation of the benzylic 
 alcohol to an aldehyde or oxidation of some other part of the molecule . 

oxidation of 12-oh - nvp by rat hepatic microsomes led to the carboxylic 
 acid ( toc graphic ) , but the intermediate aldehyde was not observed 
 ( figure 6 ) .
this suggests that 12-oh - nvp is 
 oxidized all the way to the carboxylic acid by p450 without release 
 of the intermediate aldehyde ; there is precedent for this .
it is conceivable that some of the aldehyde could 
 become covalently bound to p450 and be responsible for the observed 
 covalent binding ; however , the pattern of binding was broader than 
 that of nvp ; specifically , most of the binding was to proteins with 
 masses different from p450 .
therefore , the aldehyde seems unlikely 
 to be responsible for a significant amount of the covalent binding 
 of 12-oh - nvp .
to re - emphasize , the data strongly implicate the quinone 
 methide as being the major species responsible for covalent binding 
 of nvp in the liver .
although we had previously observed some 
 strange inclusion bodies in the livers of rats treated with nvp , we 
 did not observe an increase in alt even though there was a significant 
 degree of covalent binding .
this suggests that covalent binding may 
 be necessary but not sufficient to produce liver injury ; this is consistent 
 with an immune mechanism .
we attempted to develop an animal model 
 of nvp - induced liver toxicity in mice .
mice metabolize nvp much faster 
 than rats , and even higher doses did not produce easily detectable 
 blood levels of nvp or outward signs of toxicity . however , even with 
 higher doses and more rapid metabolism , the amount of covalent binding 
 in mice was less than that in bn rats .
treatment of c57bl/6 mice with 
 nvp led to a small increase in alt in some animals that resolved despite 
 continued treatment .
this is the pattern of adaptation frequently 
 observed in humans treated with a drug that can cause more severe 
 idiosyncratic liver injury .
liver histology in these mice revealed 
 moderate inflammatory nodules and areas of mild focal necrosis ( figure 11b , c ) .
although the covalent binding in balb / c mice 
 was similar to that in c57bl/6 mice , no increase in alt was observed 
 in balb / c mice .
cbl - b mice lack e3 ubiquitin ligase , which leads 
 to impaired immune tolerance ; however , the animals are phenotypically 
 normal .
this deficiency could also lead to increased covalent binding 
 if ubiquitin ligase is required for clearance of modified proteins , 
 and this appeared to be the case ( figure 12 ) .
we found that there was a much greater increase in alt in some 
 of the cbl - b mice than in the c57bl/6 
 mice , but the alt also returned to normal despite continued treatment 
 with the drug .
histology performed at the time of peak alt ( 14 days ) 
 showed areas of complete necrosis with a local inflammatory response . 

these appeared to represent ischemic lesions because cells close to 
 the liver capsule were spared presumably because they could benefit 
 from diffusion through the liver capsule .
luminex analysis of cytokines performed 
 on serum samples from cbl - b mice sacrificed 
 at the time of alt peak displayed a significant increase in serum 
 ifn- and il-6 in some of the animals ( figure 9a , b ) .
this increase was most prominent on day 7 rather than 
 day 1 or 14 in the majority of mice , and it occurred before the alt 
 increase at day 14 .
an elevation in cytokines or immune factors that 
 occurs earlier than increases in other toxicity markers ( i.e. , alt ) 
 is consistent with an immune response . at the study end point of 14 
 days ,
ifn- in liver samples of male cbl - b mice was also elevated to 100 pg / ml for two mice ( data not 
 shown ) with gross necrosis compared with 39 pg / ml for control mice . 

( 130 pg / ml ) on day 
 14 also had markedly elevated plasma ifn- ( 866 pg / ml ) on day 
 7 of treatment .
this cytokine is considered a pro - hepatotoxic mediator 
 leading to inflammation and tissue injury through activation of macrophages 
 and natural killer cells .
that found that incubation 
 of nvp with t - cells from a patient with nvp - induced skin rash led 
 to the production of ifn- by t cells .
reviews regarding the difficulties with production of animal 
 models of idiosyncratic drug reactions are available elsewhere , but 
 the major obstacle appears to be the development of immune tolerance .
this is consistent with the delayed onset of 
 liver injury and resolution despite continued treatment observed in 
 these mice .
we suspect that the liver injury in humans is immune - mediated 
 and that the reason that most humans and rats do not develop liver 
 injury is that the dominant response is immune tolerance .
it is known 
 that the dominant immune response in the liver is tolerance and that it is presumably why liver transplantations 
 are relatively easy compared to transplantation of , for example , skin . 

co - treatment of cbl - b mice with polyinosinic / polycytidylic 
 acid , imiquimod , and even -irradiation to deplete circulating 
 regulatory t - cells was used in an attempt to break the immune tolerance 
 and induce sustained liver damage .
all of these attempts were unsuccessful 
 in both male and female mice ( data not shown ) .
a clear picture 
 regarding the specific types of proteins covalently modified by hepatotoxic 
 drugs and the outcome of liver injury does not exist .
therefore , even 
 though mice and rats display a relatively similar pattern of nvp - induced 
 covalent binding , other individual or species - specific factors must 
 play a role in the development of liver injury . in support of this , 
 a recent clinical study demonstrated that patients who carried the 
 hla - drb*01 allele were at increased risk of developing nvp - induced 
 liver toxicity ( the alleles associated with the risk of skin rash 
 were different ) , but there was no association with the cyp2b6 genotype , 
 which is polymorphic and is one of the p450s involved in the metabolism 
 of nvp . in conclusion , we have clearly demonstrated 
 that nvp covalently binds to hepatic proteins in mice , rats , and humans . 

the major chemical species responsible for this covalent binding is 
 a quinone methide metabolite .
we have shown a mild delayed - onset liver 
 injury in c57bl/6 mice that may be the basis for an animal model if 
 a method can be found to increase liver injury . more significant injury 
 was observed in cbl - b mice , but the histology 
 suggests that the mechanism may be different .
 nevirapine ( nvp ) treatment is associated with a significant 
 incidence of liver injury . 
 we developed an anti - nvp antiserum to determine 
 the presence and pattern of covalent binding of nvp to mouse , rat , 
 and human hepatic tissues . 
 covalent binding to hepatic microsomes 
 from male c57bl/6 mice and male brown norway rats was detected on 
 western blots ; the major protein had a mass of 55 kda . 
 incubation 
 of nvp with rat cyp3a1 and 2c11 or human cyp3a4 also led to covalent 
 binding . 
 treatment of female brown norway rats or c57bl/6 mice with 
 nvp led to extensive covalent binding to a wide range of proteins . 
 
 co - treatment with 1-aminobenzotriazole dramatically changed the pattern 
 of binding . 
 the covalent binding of 12-hydroxy - nvp , the pathway that 
 leads to a skin rash , was much less than that of nvp , both in vitro and in vivo . 
 an analogue of nvp 
 in which the methyl hydrogens were replaced by deuterium also produced 
 less covalent binding than nvp . 
 these data provide strong evidence 
 that covalent binding of nvp in the liver is due to a quinone methide 
 formed by oxidation of the methyl group . 
 attempts were made to develop 
 an animal model of nvp - induced liver injury in mice . 
 there was a small 
 increase in alt in some nvp - treated male c57bl/6 mice at 3 weeks that 
 resolved despite continued treatment . 
 male cbl - b/ mice dosed with nvp had an increase in alt of > 200 u / l , which 
 also resolved despite continued treatment . 
 liver histology in these 
 animals showed focal areas of complete necrosis , while most of the 
 liver appeared normal . 
 this is a different pattern from the histology 
 of nvp - induced liver injury in humans . 
 this is the first study to 
 report hepatic covalent binding of nvp and also liver injury in mice . 
 
 it is likely that the quinone methide metabolite is responsible for 
 nvp - induced liver injury . 
Introduction
Materials and Methods
Results
Discussion