glutathione s-transferases in neonatal liver disease · 7 clin, pathol 1992;45:679-683 glutathione...

7 Clin, Pathol 1992;45:679-683

Glutathione S-transferases in neonatal liverdisease

J Mathew, A R Cattan, A G Hall, J E Hines, R Nelson, E Eastham, A D Burt

AbstractAims: To investigate the distribution of aand it class glutathione S-transferases(GST) in normal fetal, neonatal, and adultliver; and to examine changes in GSTexpression in neonatal liver disease.Methods: a and X class GST wereimmunolocalised in sections of formalinfixed liver tissue obtained from humanfetuses (n =21), neonates (n = 8), youngchildren (n = 9) and adults (n = 10), andfrom neonates with extrahepatic biliaryatresia (n = 15) and neonatal hepatitis(n = 12). Monospecific rabbit polyclonalantibodies were used with a peroxidase-antiperoxidase method.Results: Expression of X GST was loc-alised predominantly within biliary epi-thelial cells of developing and mature bileducts of all sizes from 16 weeks' gestationuntil term and in neonatal and adult liver.Coexpression of a and a GST was seen inhepatocytes of developing fetal liver bet-ween 16 and 34 weeks' gestation. Althoughi GST was seen in occasional hepatocytesup to six months of life, this isoenzymewas not expressed by hepatocytes in adultliver. By contrast, a GST continued to beexpressed by hepatocytes in adult liver;this isoenzyme was also seen in someepithelial cells of large bile ducts in adultliver. No change was observed in thedistribution of a GST in either neonatalhepatitis or extrahepatic biliary atresia.However, aberrant expression of Xt GSTwas identified in hepatocytes of all but onecase of extrahepatic biliary atresia but inonly two cases of neonatal hepatitis.Conclusions: The phenotypic alterationsnoted in extrahepatic biliary atresia mayresult from the effect of cholate stasis.Evaluation of the pattern of X and a GSTdistribution by immunohistochemicalstaining may provide valuable informa-tion in distinguishing between these twoforms of neonatal liver disease.

Glutathione S-transferases (GST) are a familyof isoenzymes that have multifunctional bio-logical roles in the detoxification of xenobioticsand lipophilic ligands; they have overlappingsubstrate specificities and function as cytosolicbinding proteins.'`4 The cytosolic GST arehomo- and heterodimeric, chemically indu-cible isoenzymes3 4 which are the products ofthree distinct autosomal loci, GST1, GST2,

and GST3,5 ' and are classified as near neutral(p), basic (a), and acidic (7t) on the basis oftheir electrophoretic mobility.4GST are present in greatest concentration in

the liver where they comprise up to 10% ofcytosolic protein.3 Serum and plasmaconcentrations of GST change in a variety ofliver diseases, and have been shown to be amore sensitive index of acute hepatocellulardamage than conventionally used biochemicalmarkers, including aminotransferases and yglutamyl transferase.7-12 These include alco-holic cirrhosis,'0 autoimmune hepatitis," andparacetamol overdose.'2 Until recently, iden-tification and measurement of GST subtypeshave involved the use of starch gel electro-phoresis and chromatofocusing.4 The develop-ment of antibodies to GST-" has enabledinvestigators to study the distribution of thedifferent isoenzymes in normal and diseasedadult human liver. Immunohistochemicalstudies have shown that the distribution ofGST may be changed in alcoholic cirrhosis'6and malignant liver tumours. 7

In this study we examined the immunohisto-chemical pattern ofGST isoenzyme expressionin the developing fetal and neonatal liver andcompared this with that of the adult. We alsoinvestigated changes in the expression of theseisoenzymes in two common neonatal liverdiseases, neonatal hepatitis and extrahepaticbiliary atresia.8

MethodsFormalin fixed liver tissue from fetuses(n = 21; 16 weeks' gestation to term), neo-nates (n = 8), children (n = 9; 3 months to 5years of age) and adults (n = 10) were used asnormal controls to examine GST expression inthe developing liver. Biopsy specimens werealso obtained from 27 jaundiced babies aged 6to 8 weeks in which diagnosis of extrahepaticbiliary atresia (n = 15) and idiopathic neona-tal hepatitis (n = 12) was subsequently madeon clinical and histopathological grounds; inthe latter group no underlying infective ormetabolic disorders could be detected in anycase. Biopsy specimens were stained withconventional histochemical stains: haema-toxylin and eosin, picromallory, reticulin,Shikata's orcein, and diastase periodic acidSchiff and 3 pm sections were obtained forimmunohistochemical studies.Two primary antibodies were used in this

study: (i) rabbit polyclonal anti-ic GST, dilu-tion 1 in 400 in 20% normal swine serum

Division of Pathology,Department of ChildHealth, Department ofHaematology(Leukaemia ResearchFund Laboratory),University ofNewcastle upon Tyne,Royal VictoriaInfirmary, Newcastleupon Tyne NE1 7RUJ MathewA R CattanA G HallJ E HinesR NelsonE EasthamA D BurtCorrespondence to:Dr A D BurtAccepted for publication20 December 1991

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Mathew, Cattan, Hall, Hines, Nelson, Easthanm, Burt

(NSS) in TRIS-buffered saline (TBS); and (ii)rabbit polyclonal anti-a GST, dilution 1 in 500in NSS/TBS. Full details of the generation andspecificity of these antibodies are given else-where.'3 ' In brief, i GST was derived fromthe splenic tissue of a patient with chroniclymphocytic leukaemia. a GST was isolatedfrom human liver. These isoenzymes werepurified using affinity chromatography and fastprotein liquid chromatography using a MonoQ column (Pharmacia). Their substrate specifi-icities, isoelectric points, and molecularweights were shown to be similar to those

described for human placental it and hepatic aforms. Polyclonal antiserum to a and 7t GSTwas produced by a standard immunisationprotocol in male New Zealand white rabbits.Protein (150-200 ,ig) was introduced by intra-muscular injection into the hind quarter oneach occasion using Freund's complete adju-vant, followed by monthly injections usingFreund's incomplete adjuvant. Immunoblot-ting and ELISA were used to assess antibodytitre and specificity. Antisera to a and 7t GSTisoenzymes did not cross-react with otherforms of GST or other cytosolic proteins at

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Figure 1 Distribution ofglutathione S-transferases in (A, B) fetal (18-20 weeks' gestation) and (C-D) adult liver:(A) Xt GST immunoreactivity can be seen in hepatocytes with intense staining of cells within the ductal plate (arrow).(B) a-GST immunoreactivity is present in hepatocytes throughout the acinus with striking nuclear labelling. Bile ducts(B) are negative. (C) GST immunoreactivity is not seen in adult hepatocytes but striking staining is noted in bileduct epithelium. (D) Intense cytoplasmic and nuclear stainiong for a GST is seen in adult liver.

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Figure 2 Glutathione S-transferases in neonatal liver disease: (A) 7t GSTimmunoreactivity in neonatal hepatitis: a similar pattern to that seen in normal niand adult liver is present. (B) In extrahepatic biliary atresia, hepatocytes, includi,multinucleate cells, show expression of Jl GS7T (C) Xt GST immunoreactivity is seein hepatocytes, proliferating ductules, and bile ducts in a case of extrahepatic biliaratresia.

dilutions of 1 in 1000 (ELISA, immunting) and 1 in 100 (immunocytochemistA peroxidase-antiperoxidase techniqu

used with all of the primary antibDewaxed 3 ,um sections were: (i) treate40-5% hydrogen peroxide in methanol toendogenous peroxidase activity; (ii) was]TBS, pH = 7-6; (iii) incubated withNSS/TBS to block non-specific bindin]incubated with primary antibody overni4°C; (v) rinsed in TBS then incubatecswine anti-rabbit immunoglobulin (DakcDenmark), diluted 1 in 20 in NSS/TBS,minutes at room temperature; (vi) riniTBS and incubated with rabbit peroxantiperoxidase complex (Dakopatts,mark), diluted 1 in 50 in NSS/TBS fminutes at room temperature; (vii) waslTBS and incubated with 3,3'-diaminodine (DAB) in the presence of hydperoxide; and (viii) washed in tap

-' ResultsGST EXPRESSION IN FETAL, NEONATAL, AND ADULTLIVER

#. tlGSTAt sixteen weeks' gestation almost all hepto-cytes in all zones of the acinus showed cyto-plasmic and nuclear positivity (fig IA). Theintensity of immunoreactivity in hepatocytesdecreased towards term. After 34 weeks only

4 occasional hepatocytes expressed this isoen-zyme. In infants after 6 months and in adultliver there was no expression of this isoenzymeby hepatocytes (fig 1 C). The ductal platestructures at the porto-parenchymal interfacein the second trimester (from 16 weeks'gestation) (fig IA), as well as the developingand mature bile ducts of all sizes after thisperiod and in adult liver showed intensenuclear and cytoplasmic immunoreactivity (fig1C). Intra-sinusoidal haematopoietic cells didnot contain this isoenzyme.

a GST- Hepatocytic nuclear and cytoplasmic a GST

immunoreactivity was seen at 16 weeks' gesta-tion throughout the acinus (fig 1 B). Thispattern of staining was present throughoutfetal development and in adult liver (fig 1D).Between 16 and 24 weeks' gestation there wasmild nuclear and cytoplasmic positivity in

( some cells in intermediate sized bile ducts,although most bile ducts and ductal platestructures were negative. Later, only veryoccasional cells in larger bile ducts showedintense cytoplasmic, nuclear, and membranepositivity to term and in adults. The primitiveintrasinusoidal haematopoietic cells were neg-ative.

eonatal

Tn here GST ISOENZYME EXPRESSION IN NEONATAL LIVERly DISEASE

Neonatal hepatitisIn all 12 cases of neonatal hepatitis immuno-localisation of a GST was identical with thatseen in normal neonatal and adult liver. In 10

oblot- cases immunoreactivity for X GST did nottry). show any variation from that expected inie was mature livers: bile ducts of all sizes were)odies. immunoreactive and hepatocytes were negatived with (fig 2A). However, in two cases some hep->block atocytes including giant cells showed moderatehed in cytoplasmic positivity. Histologically, no. 20% apparent differences in the degree of bili-g; (iv) rubinostasis could be seen between these casesight at and the remaining 10.i with)patts, Extraheptic biliary atresiafor 30 a GST isoenzyme did not show any variationsed in from that expressed in mature adult livers.cidase- There was intense nuclear and cytoplasmicDen- positivity in hepatocytes, although there was an

for 30 apparent decrease in the number of nuclei thathed in were positive. Proliferating ductules were neg-,benzi- ative, even in those cases of extrahepatic biliarylrogen atresia with severe bridging fibrosis.water, i GST showed a striking difference from the

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expected pattern of immunoreactivity. All bileducts showed intense cytoplasmic and nuclearpositivity and, in all but one case, hepatocytesshowed moderate cytoplasmic positivity withvariable mild to moderate nuclear positivity(figs 2B and C).

DiscussionWe have shown a consistent chronologicalvariation in the pattern of GST isoenzymeexpression in the normal developing liver. Thisobservation is in keeping with the findings ofHiley et al22 and Hayes et al.23 a and i GSTimmunoreactivity was intense in hepatocytesbetween 16 and 24 weeks of gestation afterwhich a GST was the predominant isoenzymeexpressed in these cells in neonates and adults.X GST was positive in a few scattered hep-atocytes until 6 months of life after which theywere negative.X GST was present in the biliaryepithelial cells of bile ducts of all sizes and at allstages of development from the early fetalperiod onwards. In early gestation, ductal platestructures were intensely stained with theantibody to this isoenzyme and later thedeveloping bile ducts acquired this pattern ofimmunoreactivity which persisted in matureadult bile ducts of all sizes. In material from 16and 20 week old fetuses mild to moderate aGST immunoreactivity was observed in inter-mediate sized bile ducts in a few cases but wasconsistently negative after this in all biliaryradicles except large bile ducts. In these,intense a GST expression was observed in onlysome biliary epithelial cells.Neonatal hepatitis and extrahepatic biliary

atresia are thought to belong to a group ofinfantile bile duct disorders referred to as"idiopathic obstructive cholangiopathy" whichrepresent a spectrum of diseases that haveoverlapping clinico-histomorphological fea-tures. 18 20 Although current concepts suggestthat neonatal hepatitis and extrahepatic biliaryatresia may therefore overlap, their man-agement and prognosis are different anddependent on early accurate histological diag-nosis.18 119 24 Early surgical intervention, before60 days of life, in the form of portoenterostomyshows an improvement in the morbidity andmortality that is associated with untreated orundiagnosed extrahepatic biliary atresia,24whereas operative intervention in neonatalhepatitis is contraindicated. Although distinc-tion between extrahepatic biliary atresia andneonatal hepatitis can usually be made on thebasis of changes in portal tract areas, this maybe difficult in biopsy specimens obtained atearly time points.We have found a remarkable difference in

GST immunohistochemical expression fromthe normal pattern in cases of extrahepaticbiliary atresia. In 93% of cases there wasalmost universal coexpression of a and XtGSTswithin hepatocytes; only one case of extra-hepatic biliary atresia failed to show Xt GSTexpression. In contrast, only two cases ofneonatal hepatitis (17%) showed coexpressionof these isoenzymes in hepatocytes. This strik-ing difference in GST isoenzyme expression

may thus provide a valuable immunohisto-chemical method of further differentiatingneonatal hepatitis and extrahepatic biliary atre-sia on histological grounds.

Phenotypic alteration in GST immunohisto-chemical expression has been described inseveral forms of adult liver disease includingalcoholic cirrhosis'" and primary biliary cir-rhosis.7 In these studies coexpression of a and7t class GST in hepatocytes, similar to thatobserved during the earlier gestational phase ofthe evolving liver, was found. The mechanismsresponsible for altered it GST expression inextrahepatic biliary atresia are probably similarto those in these adult liver diseases, possiblyreflecting ductular metaplastic changes inresponse to prolonged cholestasis.We speculate that differences in the concen-

trations of retained bile acids (cholate stasis)may be responsible for the differing patterns ofGST expression in extrahepatic biliary atresiaand neonatal hepatitis. Although this hypoth-esis remains to be tested, we conclude thatimmunohistochemical evaluation of a and 7tGST expression in percutaneous liver biopsyspecimens may provide useful information indifferentiating between neonatal hepatitis andextrahepatic biliary atresia.

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