El

SYMa

b

c

d

e

a

A

R

A

A

K

A

I

L

N

1

ApaepSdaahft

1d

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 1 ( 2 0 1 1 ) 165–170

avai lab le at www.sc iencedi rec t .com

journa l homepage: www.e lsev ier .com/ locate /e tap

levated serum neopterin levels in acetaminophen-inducediver injury

eref Demirbasa, Erdinc Cakirb, Emin Ozgur Akgulb, Melik Seyrekc,∗, Tuncer Caycib,asemin Gulcan Kurtb, Bulent Uysald, Ibrahim Aydinb, Bulent Kurte, Halil Yamanb,ehmet Kemal Erbil b

Department of Internal Medicine, Gulhane Military Medical Academy, 06018 Etlik, Ankara, TurkeyDepartment of Medical Biochemistry, Gulhane Military Medical Academy, 06018 Etlik, Ankara, TurkeyDepartment of Medical Pharmacology, Gulhane Military Medical Academy, 06018 Etlik, Ankara, TurkeyDepartment of Physiology, Gulhane Military Medical Academy, 06018 Etlik, Ankara, TurkeyDepartment of Pathology, Gulhane Military Medical Academy, 06018 Etlik, Ankara, Turkey

r t i c l e i n f o

rticle history:

eceived 30 April 2010

ccepted 17 October 2010

vailable online 23 October 2010

a b s t r a c t

Neopterin is synthesized in macrophage/Kupffer cells by interferon-gamma and other

cytokines. This study aimed to evaluate the utility of using neopterin as a biomarker of

acetaminophen (APAP)-induced liver injury. Wistar rats, randomly divided into two groups

(APAP and normal), received APAP (1.0 g/kg) and distilled water, respectively, by gastric tube.

The APAP group had a higher degree of liver necrosis than the control group. The APAP

eywords:

cetaminophen

njury

iver

group also had significantly higher serum neopterin levels than the normal group. Serum

neopterin levels correlated with serum AST, ALT activities, and degree of necrosis. This study

demonstrates the preclinical utility of neopterin as a biomarker for the animal model of

APAP-induced liver injury. Further research studies are required to determine the preclinical

g neo

Elevations of ALT and AST indicate liver damage (Hamadeh

eopterin opportunities of usin

. Introduction

cetaminophen (4-hydroxyacetanilide, N-acetyl-p-amino-henol, paracetamol; APAP), a widely used over-the-counternalgesic and antipyretic drug, has pharmacologicalffects generally considered to be based on inhibition ofrostaglandin synthesis (Bessems and Vermeulen, 2001).ome authors consider APAP as one of the most reliablerugs, and even working pilots are allowed to use it (Sennd Akin, 2004). Although considered well tolerated at ther-peutic doses, an overdose of APAP produces centrilobular

epatic necrosis that can lead to fatal fulminant hepatic

ailure (Larson, 2007). (The liver represents an importantarget for the toxicity of drugs in terms of oxidative stress.) A

∗ Corresponding author. Tel.: +90 312 304777; fax: +90 312 3043300.E-mail address: melikseyrek@gmail.com (M. Seyrek).

382-6689/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.etap.2010.10.003

pterin as a marker of APAP-induced liver injury.

© 2010 Elsevier B.V. All rights reserved.

characteristic feature of APAP-induced hepatotoxicity is theaccumulation of macrophages in centrilobular regions of theliver (Laskin and Gardner, 1998), and medical professionalsincreasingly recognize APAP hepatotoxicity as a significantpublic health problem (Kaplowitz, 2004; Lee, 2007). APAP canalso induce renal failure and eventually cause death in severecases (Nelson, 1990). Liver injury by acetaminophen overdoseis caused by increased production of reactive oxygen species,antioxidant (glutathione) depletion, and cytokine upregula-tion (Dahlin et al., 1984; Ferret et al., 2001; James et al., 2003).

and Afshari, 2004). Necrosis of the liver usually results in hep-atocellular plasma membrane leakage of AST and ALT intothe bloodstream. However, although elevated levels of these

d p h

166 e n v i r o n m e n t a l t o x i c o l o g y a n

serum enzymes indicate hepatocellular damage, they serveas poor prognosticators for the severity of the liver injury oracute liver failure (Huang et al., 2008).

Scholars still do not completely understand the exactmechanism of APAP-induced cell injury (Gujral et al., 2001).The initial event in APAP-induced hepatotoxicity, a toxic-metabolic injury, leads to hepatocyte death by necrosis andapoptosis. This results in secondary activation of the innateimmune response involving upregulation of inflammatorycytokines with activation of natural killer (NK) cells, NKT cells,and neutrophils (Liu et al., 2004, 2006). Liu et al. reported thatNK and NKT cells, major components of the liver’s innateimmune system, play a critical role in the severity and pro-gression of APAP-induced liver injury by secreting cytokineinterferon gamma (IFN-�), modulating chemokine production,and recruiting inflammatory cells into the liver (Liu et al.,2004).

Recent evidence suggests cytokines as possible candidatesto determine severity of liver toxicity (Blazka et al., 1995; Ishidaet al., 2004) and as role players in the regulation of toxicity(James et al., 2005). Many findings suggest cytokines, suchas tumor necrosis factor-� (TNF-�), interleukin (IL)-1� (Blazkaet al., 1995), and INF-� (Ishida et al., 2002), play in the patho-genesis of APAP-induced liver injury, while other cytokines,such as IL-6 (Masubuchi et al., 2003) and IL-10 (Bourdi et al.,2002) play protective roles in liver injury.

Neopterin is synthesized from guanosine triphosphate inmacrophages and monocytes by the activation of guanosinetriphosphate cyclohydrolase I (Muller et al., 1991). IFN-� andother cytokines greatly enhance the activity of the enzyme.Plasma or urine neopterin concentrations reflect the acti-vation of the macrophage/monocyte line cells (Ruokonenet al., 2002). Neopterin has become a useful tool in assess-ing the intensity of the cell-mediated immune response.Since neopterin excretion takes place before clinical symp-toms appear, scholars have accepted biochemical follow-upof neopterin levels as a strong indicator for the clinical sever-ity of some diseases (Huber et al., 1984; Muller et al., 1991;Turgan et al., 2001). In patients suffering from hematological,gynecological, lung, and gastrointestinal tumors, an elevatedneopterin level correlates with the disease stage and predicts apoor prognosis regarding relapse and overall survival (Sucheret al., 2010).

Clinical studies have examined the role of neopterin pre-dominantly as a biomarker of disease and injury in liver. Astudy by Yaman et al. (2005) revealed elevated urine neopterinlevels in patients with nonalcoholic steatohepatitis (NASH).

In this study, we aimed to investigate whether the con-centration of neopterin was elevated in serum samples in agroup with APAP-induced liver injury as compared to a controlgroup and to evaluate the utility of neopterin as a biomarkerof APAP-induced liver injury.

2. Materials and methods

2.1. Animals

This study used adult, male Wistar rats (Health SciencesInstitute, Gulhane Military Medical Academy, Ankara, Turkey),

a r m a c o l o g y 3 1 ( 2 0 1 1 ) 165–170

weighing 200–250 g each. We randomly assigned the rats to2 groups containing 10 rats each: normal and APAP groups.They had free access to food and water, were kept in an air-conditioned room at 21 ◦C, with a 12 h:12 h light:dark cycle, andwere handled humanely, in accordance with European UnionDirective 609/86 for the care and use of laboratory animals.The animals received no food for 12 h before APAP treatment.We performed the present study in the Research Center ofthe Gulhane Military Medical Academy, Ankara, Turkey, withthe approval of the Gulhane Military Medical Academy AnimalEthics Committee.

2.2. General procedures

We administered a single dose of APAP (1.0 g/kg body weight)(Ordu Ilac Fabrikasi, Ankara, Turkey) suspended in hot dis-tilled water by gastric tube. The dose of APAP administeredin our animal model of hepatotoxicity in rats is based onother studies using this APAP model (Gardner et al., 1998).Rats in the normal group received distilled water by gastrictube. We sacrificed all surviving animals under light diethylether anesthesia, at a time point 48 h after APAP treatment,and sacrificed the control group at the same time point. Theabdomen was opened, and livers were removed and cleaned.Liver tissue samples were stored in 10% formalin solution forhistopathology analysis.

2.3. Estimation of liver injury

We collected blood samples from all animals via cardiac punc-ture, allowed the samples to clot, and removed the serumcentrifugation at 1000 × g for 10 min at room temperature. Allserum samples were sterile and haemolysis-free. They wereprocessed to determine the enzymatic activities of aspartateaminotransferase (AST), alanine aminotransferase (ALT), alka-line phosphatase (ALP), �-glutamyl transferase (�-GT), anddirect bilirubin, total bilurubin, urea, and creatinine concen-trations were measured with a spectrophotometric techniqueby the Olympus AU-2700 autoanalyzer using commercial kits(Olympus, Hamburg, Germany) and presented as U/L andmg/dL.

For liver histopathology analysis, we processed midsec-tions of the left lobes of the liver for light microscopy. Thespecimen processing consisted of: fixation in 4% bufferedneutral formalin solution for 24 h, embedding in paraffinwax, slicing sections at 5 �m thickness, and staining withhematoxylin–eosin (H&E). The liver tissue sections were inde-pendently examined and scored, while the examiner wasunaware of the group to which the specimen belonged.The degree of necrosis was expressed as the mean of 10high power fields (HPFs), chosen at random and classifiedon a scale of 0–3 (normal, 0; mild, 1; moderate, 2; andsevere, 3).

2.4. Measurement of neopterin levels

We measured serum neopterin concentrations with ahigh-performance liquid chromatography device (AgilentTechnologies 1200 Series System, Santa Clara, CA, USA)using a fluorescence detector, as discussed by Alrashed

p h a r

ea

2

WaaSalapc

3

3

TMi(rmt

3

Ttir

e n v i r o n m e n t a l t o x i c o l o g y a n d

t al. (2002). Neopterin concentrations were presenteds nmol/L.

.5. Statistical analysis

e used SPSS 11.0 (SPSS Inc., Chicago, IL, USA) statistical pack-ge to perform all statistical analyses and Mann–Whitney Und chi-square tests to test differences between the groups.pearman’s rho correlation coefficient was applied to evalu-te the association between serum neopterin, ALT, and ASTevels and degree of necrosis. The results were expresseds mean ± standard deviation (SD) and median (min–max). Arobability level of <0.05 was considered statistically signifi-ant.

. Results

.1. Neopterin levels

able 1 shows the results of the neopterin measurements.ean serum neopterin levels were significantly increased

n the APAP group rats when compared to normal ratsmean ± SD; 11.1 ± 5.5 vs. 2.9 ± 0.6 nmol/L, p = 0.002; Table 1,espectively). The mean serum neopterin levels were approxi-

ately four-fold higher in the APAP-induced rats as comparedo the normal rats (Table 1).

.2. Liver injury

able 1 shows all laboratory parameters of both groups. Inhe APAP-induced liver-injury rats, mean serum ALT activ-ties were significantly higher than in the normal group ofats (p = 0.002). Mean serum AST activities also significantly

Table 1 – Laboratory parameters and the degree of necrosis belo

Normal (n = 10)Mean ± SD

Median (min–max

Aspartate aminotransferase (U/L)146 ± 24

140 (113–178)

Alanine aminotransferase (U/L)74 ± 17

67 (57–98)

Alkaline phosphatase (U/L)374 ± 130

337 (227–547)

�-Glutamyl transferase (U/L)1.9 ± 0.42 (1–2)

Direct bilirubin (mg/dL)0.05 ± 0.01

0.05 (0.03–0.06)

Total bilurubin (mg/dL)0.16 ± 0.03

0.16 (0.12–0.20)

Urea (mg/dL)44 ± 4.4

43 (38–49)

Creatinine (mg/dL)0.60 ± 0.03

0.61 (0.54–0.65)

Neopterin (nmol/L)2.9 ± 0.6

2.8 (2.3–3.6)

The degree of necrosis0.0 ± 0.0

0.0 (0.0–0.0)

APAP: acetaminophen.

m a c o l o g y 3 1 ( 2 0 1 1 ) 165–170 167

increased in the APAP group as compared to the normal rats’values (p = 0.002). However, there was no statistically signifi-cant difference between the two groups in mean serum ALPand �-GT activities and, direct bilirubin, total bilurubin, andurea concentrations. However, mean serum creatinine lev-els in the APAP group were higher than in the normal group(p = 0.046).

Table 1 also shows the degree of liver necrosis. Meanscore of necrosis (median (min–max)) significantly increasedin APAP-induced rats [3.0 (1.0–3.0)] as compared to normal rats[0.0 (0.0–0.0)], p = 0.003. Fig. 1 provides sample views of thehistopathological examination of normal and APAP-inducedlivers.

The increased serum ALT, AST (indicative of necrosis) activ-ities in APAP-induced rats agreed with the histopathologicalliver injury. In this study, the degree of necrosis most stronglycorrelated with the activities of serum AST (r = 0.896, p = 0.006),with no correlation between this degree and serum ALT activi-ties (r = −0.120, p = 0.799). Furthermore, serum neopterin levelscorrelated with serum AST, ALT activities, and degree of necro-sis, respectively (r = 0.714, p = 0.071; r = 0.000, p = 1.000; r = 0.896,p = 0.006).

4. Discussion

Drug-induced liver injury, a major cause of human morbid-ity and mortality, also represents the single greatest cause fortermination of drug development and withdrawal of approveddrugs from the market (Kaplowitz, 2001). APAP, is a direct

hepatotoxin, and APAP intoxication is dose-dependent andreproducible (Grypioti et al., 2005). Analgesic APAP causesmassive hepatic necrosis when taken in overdose (Ekong et al.,2006).

ng to normal and APAP groups.

APAP (n = 10) pMean ± SD

) Median (min–max)

2125 ± 2234 0.0021153 (626–6780)

1272 ± 454 0.0021100 (824–2120)

368 ± 95 0.749403 (230–448)

1.6 ± 0.8 0.2751 (1–3)

0.07 ± 0.03 0.2410.06 (0.03–0.12)

0.16 ± 0.06 0.9480.15 (0.06–0.23)

45 ± 4.2 0.70044 (41–52)0.65 ± 0.04 0.046

0.66 (0.60–0.72)11.1 ± 5.5 0.002

13.9 (4.7–16.5)2.4 ± 0.8 0.003

3.0 (1.0–3.0)

168 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 1 ( 2 0 1 1 ) 165–170

Fig. 1 – Representative photographs showing histopathological changes in liver tissues. The normal group shows normalgrou

hepatic structure and sinusoids. The acetaminophen (APAP)

arrows) (H&E, CV = Central vein, Scale bars = 60 �m).

The present study demonstrates the preclinical utility ofneopterin as a biomarker for drug-induced liver injury in vivo,readily detected in the serum of animals treated with APAP.The application of this diagnostic biomarker offers a valu-able addition to other clinical tests and invasive biopsy alreadyused to detect drug-induced liver injury. However, the sensi-tivity and specificity of neopterin as a useful indicator of liverinjury remains unclear and requires further investigation onanimals and humans. This study aimed to evaluate neopterinboth as an indicator of liver injury for preclinical evaluation ofliver drug-safety and as a potential blood and urinary indicatorof hepatotoxicity.

We have previously demonstrated higher urine neopterinlevels in patients with NASH compared to a control group.The high neopterin concentrations in the early phase of dis-ease may trigger the development of irreversible liver damage(Yaman et al., 2005).

The administration of a toxic dose of APAP results in livertoxicity that becomes apparent after the first 20 h of treat-ment exposure (Nelson, 1990). In our experimental animalmodel of APAP-induced liver toxicity, we demonstrated acutehepatic injury by the increased activities of serum hepaticenzymes (AST, ALT) and the degree of liver necrosis in theAPAP-induced rats. Hepatic enzymes released into the bloodby damaged liver offer the most useful tools in detecting hep-atotoxicity (Sturgill and Lambert, 1997). In our study, 1.0 g/kgdose of APAP caused significant histological and histochemi-cal changes consistent with liver injury, including increases inserum ALT and AST activities. The comparison of the increaseof serum neopterin concentrations with ALT and AST activ-ities demonstrates a parallel in APAP-induced animals. Inaddition, elevated serum neopterin levels were matched withincreased serum ALT and AST activities. We have shown aclose association between serum neopterin and other func-tional markers of liver injury.

In this study, similar statistical correlation analyses

between serum neopterin levels and other serum diagnostictests and histopathology examined in the experimental modelrevealed neopterin release to be highly correlated with manycommonly evaluated blood indicators and the degree of necro-

p reveals necrosis (N) and leukocyte infiltration (black

sis for liver injury. Serum ALT and AST activities increasedin APAP-treated rats correlating highly with elevated serumlevels of neopterin.

Factor or factors responsible for the progression of APAP-induced liver injury have not been identified, although recentevidence suggests cytokines as possible mediators that deter-mine severity of the liver toxicity. Particularly, some authorshave reported that TNF-� and INF-� play pathogenic roles inanimal models of APAP-induced liver injury (Blazka et al., 1995;Ishida et al., 2002).

In various animal studies, various authors have demon-strated that macrophages/Kupffer cells in the liver increaseafter APAP treatment (Dambach et al., 2002; Laskin andGardner, 2003; Laskin et al., 1995). At the same time, inflam-matory cytokines such as TNF-� and IFN-� increase in theliver after an APAP challenge (Blazka et al., 1995, 1996; Ishidaet al., 2002), and Ishida et al. have shown that IFN-� knock-out mice were highly resistant to APAP-induced liver injury,suggesting an essential role of IFN-� in the progression ofAPAP-induced hepatotoxicity (Ishida et al., 2002). IFN-� couldstimulate macrophages/Kupffer cells. The Kupffer cells of theliver represent a likely cellular source of the increase in serumneopterin levels. We did not measure cytokine levels in ourstudy. However, we evaluated histological markers of inflam-mation and neopterin associated with cytokines such as INF-�.

Neopterin is produced by monocyte-derived macrophagesupon stimulation with IFN-� (Denz et al., 1990). An ele-vated neopterin level is a marker of monocyte/macrophageactivities. Furthermore, neopterin is a sensitive indicator ofthe extent of cellular immune activation in various diseases(Fuchs et al., 1992). Alterations in the biosynthesis and pro-duction of pteridines like neopterin have proven useful asprognostic markers for cancer patients (Murr et al., 2002;Reibnegger et al., 1991). Elevated neopterin concentrationshave been observed in patients with various cancers. In allcancer types investigated, high neopterin levels were signifi-

cantly associated with a poor prognosis (Sucher et al., 2010).

This report, to the best of our knowledge, represents thefirst that focuses on serum neopterin levels following APAP-induced liver injury. In this study, we determined an increased

p h a r

sptdo1aoricHTnAip

5

TaiacoFpAii

C

T

A

TA

r

A

B

B

B

e n v i r o n m e n t a l t o x i c o l o g y a n d

erum concentration of neopterin in APAP-induced animals,ossibly reflecting a cellular immune response that con-ributes to progression of the disease in APAP-induced liveramage. Since neopterin is a sensitive indicator of the extentf cellular immune activation in various diseases (Fuchs et al.,992), the elevated serum neopterin in the APAP group couldlso be responsible for liver damage in those rats. On thether hand, neopterin elevation may be one of the factorsesponsible for progression of APAP-induced hepatotoxicity torreversible hepatocellulary damage. Elevated neopterin con-entrations correlated with serum transaminase activities.igh serum ALT and AST levels indicate hepatocellular injury.herefore, elevated serum neopterin levels might be a diag-ostic marker to indicate liver injury/damage caused by APAP.t the same time, in rats with APAP-induced liver injury,

ncreased serum neopterin concentration may signify a poorrognosis concerning disease progression.

. Conclusions

his study demonstrates the preclinical utility of neopterins a biomarker for the animal model of APAP-induced livernjury. Preclinical models available to validation of neopterins a marker of liver injury would be helpful in further clini-al trials. Our study supports the need for further evaluationf neopterin as a biomarker of APAP-induced liver injury.urthermore, other studies are needed to determine thereclinical opportunities of using neopterin as a marker ofPAP-induced liver injury. In the future, it may be interest-

ng to investigate the role of neopterin in APAP-induced livernjury in properly designed trials.

onflict of interest statement

he authors declare that there are no conflicts of interest.

cknowledgements

he Research Center of Gulhane Military Medical Academy,nkara, Turkey, supported this study.

e f e r e n c e s

lrashed, M., Abougoush, M., Akgul, E.O., Erbil, M.K., Boyunaga,H., Almarafi, A., 2002. Detection method of serum and urineneopterin levels by high performance liquid chromatographyand clinical applications. Gulhane MJ 44, 273–277 (Article inTurkish).

essems, J.G., Vermeulen, N.P., 2001. Paracetamol(acetaminophen)-induced toxicity: molecular andbiochemical mechanisms, analogues and protectiveapproaches. Crit. Rev. Toxicol. 31, 55–138.

lazka, M.E., Elwell, M.R., Holladay, S.D., Wilson, R.E., Luster, M.I.,1996. Histopathology of acetaminophen-induced liver

changes: role of interleukin 1 alpha and tumor necrosis factoralpha. Toxicol. Pathol. 24, 181–189.

lazka, M.E., Wilmer, J.L., Holladay, S.D., Wilson, R.E., Luster, M.I.,1995. Role of proinflammatory cytokines in acetaminophenhepatotoxicity. Toxicol. Appl. Pharmacol. 133, 43–52.

m a c o l o g y 3 1 ( 2 0 1 1 ) 165–170 169

Bourdi, M., Masubuchi, Y., Reilly, T.P., Amouzadeh, H.R., Martin,J.L., George, J.W., Shah, A.G., Pohl, L.R., 2002. Protection againstacetaminophen-induced liver injury and lethality byinterleukin 10: role of inducible nitric oxide synthase.Hepatology 35, 289–298.

Dahlin, D.C., Miwa, G.T., Lu, A.Y., Nelson, S.D., 1984.N-acetyl-p-benzoquinone imine: a cytochromeP-450-mediated oxidation product of acetaminophen. Proc.Natl. Acad. Sci. U.S.A. 81, 1327–1331.

Dambach, D.M., Watson, L.M., Gray, K.R., Durham, S.K., Laskin,D.L., 2002. Role of CCR2 in macrophage migration into theliver during acetaminophen-induced hepatotoxicity in themouse. Hepatology 35, 1093–1103.

Denz, H., Fuchs, D., Huber, H., Nachbaur, D., Reibnegger, G.,Thaler, J., Werner, E.R., Wachter, H., 1990. Correlation betweenneopterin, interferon-gamma and haemoglobin in patientswith haematological disorders. Eur. J. Haematol. 44, 186–189.

Ekong, U., Zeng, S., Dun, H., Feirt, N., Guo, J., Ippagunta, N.,Guarrera, J.V., Lu, Y., Weinberg, A., Qu, W., Ramasamy, R.,Schmidt, A.M., Emond, J.C., 2006. Blockade of the receptor foradvanced glycation end products attenuatesacetaminophen-induced hepatotoxicity in mice. J.Gastroenterol. Hepatol. 21, 682–688.

Ferret, P.J., Hammoud, R., Tulliez, M., Tran, A., Trebeden, H.,Jaffray, P., Malassagne, B., Calmus, Y., Weill, B., Batteux, F.,2001. Detoxification of reactive oxygen species by anonpeptidyl mimic of superoxide dismutase curesacetaminophen-induced acute liver failure in the mouse.Hepatology 33, 1173–1180.

Fuchs, D., Weiss, G., Reibnegger, G., Wachter, H., 1992. The role ofneopterin as a monitor of cellular immune activation intransplantation, inflammatory, infectious, and malignantdiseases. Crit. Rev. Clin. Lab. Sci. 29, 307–341.

Gardner, C.R., Heck, D.E., Yang, C.S., Thomas, P.E., Zhang, X.J.,DeGeorge, G.L., Laskin, J.D., Laskin, D.L., 1998. Role of nitricoxide in acetaminophen-induced hepatotoxicity in the rat.Hepatology 27, 748–754.

Grypioti, A.D., Theocharis, S.E., Papadimas, G.K., Demopoulos,C.A., Papadopoulou-Daifoti, Z., Basayiannis, A.C., Mykoniatis,M.G., 2005. Platelet-activating factor (PAF) involvement inacetaminophen-induced liver toxicity and regeneration. Arch.Toxicol. 79, 466–474.

Gujral, J.S., Bucci, T.J., Farhood, A., Jaeschke, H., 2001. Mechanismof cell death during warm hepatic ischemia-reperfusion inrats: apoptosis or necrosis? Hepatology 33, 397–405.

Hamadeh, H.K., Afshari, C.A. (Eds.), 2004. Toxicogenomics:Principles and Applications. Wiley-Liss, Hoboken, NJ.

Huang, L., Heinloth, A.N., Zeng, Z.B., Paules, R.S., Bushel, P.R.,2008. Genes related to apoptosis predict necrosis of the liveras a phenotype observed in rats exposed to a compendium ofhepatotoxicants. BMC Genomics 9, 288.

Huber, C., Batchelor, J.R., Fuchs, D., Hausen, A., Lang, A.,Niederwieser, D., Reibnegger, G., Swetly, P., Troppmair, J.,Wachter, H., 1984. Immune response-associated production ofneopterin. Release from macrophages primarily under controlof interferon-gamma. J. Exp. Med. 160, 310–316.

Ishida, Y., Kondo, T., Ohshima, T., Fujiwara, H., Iwakura, Y.,Mukaida, N., 2002. A pivotal involvement of IFN-gamma in thepathogenesis of acetaminophen-induced acute liver injury.FASEB J. 16, 1227–1236.

Ishida, Y., Kondo, T., Tsuneyama, K., Lu, P., Takayasu, T., Mukaida,N., 2004. The pathogenic roles of tumor necrosis factorreceptor p55 in acetaminophen-induced liver injury in mice. J.Leukoc. Biol. 75, 59–67.

James, L.P., Kurten, R.C., Lamps, L.W., McCullough, S., Hinson, J.A.,

2005. Tumour necrosis factor receptor 1 and hepatocyteregeneration in acetaminophen toxicity: a kinetic study ofproliferating cell nuclear antigen and cytokine expression.Basic Clin. Pharmacol. Toxicol. 97, 8–14.

d p h

HPLC-fluorometric method. Clin. Biochem. 34, 271–275.

170 e n v i r o n m e n t a l t o x i c o l o g y a n

James, L.P., Lamps, L.W., McCullough, S., Hinson, J.A., 2003.Interleukin 6 and hepatocyte regeneration in acetaminophentoxicity in the mouse. Biochem. Biophys. Res. Commun. 309,857–863.

Kaplowitz, N., 2001. Drug-induced liver disorders: implicationsfor drug development and regulation. Drug Saf. 24, 483–490.

Kaplowitz, N., 2004. Acetaminophen hepatoxicity: what do weknow, what don’t we know, and what do we do next?Hepatology 40, 23–26.

Larson, A.M., 2007. Acetaminophen hepatotoxicity. Clin. LiverDis. 11, 525–548, vi.

Laskin, D.L., Gardner, C.R., 1998. The role of nonparenchymalcells and inflammatory macrophages in hepatotoxicity. In:Plaa, G.L., Hewitt, W.R. (Eds.), Toxicology of the Liver. Raven,New York, USA, pp. 297–320.

Laskin, D.L., Gardner, C.R., 2003. Nonparenchymal cells,inflammatory macrophages, and hepatotoxicity. In:Kaplowitz, N., DeLeve, L.D. (Eds.), Drug-induced LiverDiseases. Marcel Dekker, New York, pp. 183–212.

Laskin, D.L., Gardner, C.R., Price, V.F., Jollow, D.J., 1995. Modulationof macrophage functioning abrogates the acutehepatotoxicity of acetaminophen. Hepatology 21, 1045–1050.

Lee, W.M., 2007. Acetaminophen toxicity: changing perceptionson a social/medical issue. Hepatology 46, 966–970.

Liu, Z.X., Govindarajan, S., Kaplowitz, N., 2004. Innate immunesystem plays a critical role in determining the progressionand severity of acetaminophen hepatotoxicity.Gastroenterology 127, 1760–1774.

Liu, Z.X., Han, D., Gunawan, B., Kaplowitz, N., 2006. Neutrophildepletion protects against murine acetaminophenhepatotoxicity. Hepatology 43, 1220–1230.

Masubuchi, Y., Bourdi, M., Reilly, T.P., Graf, M.L., George, J.W., Pohl,L.R., 2003. Role of interleukin-6 in hepatic heat shock protein

a r m a c o l o g y 3 1 ( 2 0 1 1 ) 165–170

expression and protection against acetaminophen-inducedliver disease. Biochem. Biophys. Res. Commun. 304, 207–212.

Muller, M.M., Curtius, H.C., Herold, M., Huber, C.H., 1991.Neopterin in clinical practice. Clin. Chim. Acta 201, 1–16.

Murr, C., Widner, B., Wirleitner, B., Fuchs, D., 2002. Neopterin as amarker for immune system activation. Curr. Drug Metab. 3,175–187.

Nelson, S.D., 1990. Molecular mechanisms of the hepatotoxicitycaused by acetaminophen. Semin. Liver Dis. 10, 267–278.

Reibnegger, G., Fuchs, D., Fuith, L.C., Hausen, A., Werner, E.R.,Werner-Felmayer, G., Wachter, H., 1991. Neopterin as a markerfor activated cell-mediated immunity: application inmalignant disease. Cancer Detect. Prev. 15, 483–490.

Ruokonen, E., Ilkka, L., Niskanen, M., Takala, J., 2002.Procalcitonin and neopterin as indicators of infection incritically ill patients. Acta Anaesthesiol. Scand. 46, 398–404.

Sen, A., Akin, A., 2004. Drug use attitude of Turkish armed forcespilots. TAF Prev. Med. Bull. 3, 213–220.

Sturgill, M.G., Lambert, G.H., 1997. Xenobiotic-inducedhepatotoxicity: mechanisms of liver injury and methods ofmonitoring hepatic function. Clin. Chem. 43, 1512–1526.

Sucher, R., Schroecksnadel, K., Weiss, G., Margreiter, R., Fuchs, D.,Brandacher, G., 2010. Neopterin, a prognostic marker inhuman malignancies. Cancer Lett. 287, 13–22.

Turgan, N., Habif, S., Parildar, Z., Ozmen, D., Mutaf, I., Erdener, D.,Bayindir, O., 2001. Association between homocysteine andneopterin in healthy subjects measured by a simple

Yaman, H., Cakir, E., Ozcan, O., Yesilova, Z., Ozcan, A., Akgul, E.O.,Erbil, M.K., Bagci, S., Bilgi, C., Dagalp, K., 2005. Elevated urineneopterin levels in nonalcoholic steatohepatitis. Clin.Biochem. 38, 187–190.