Thioacetamide-induced changes in hepatic hexokinase isoenzymes

Toxicology, 58 (1989) 21--31 Elsevier Scientific Publishers Ireland Limited.

Thioacetamide-induced changes in hepatic hexokinase isoenzymes Gillian M. L a w r e n c e a*, A. C h r i s t o p h e r H. Beesley a, M a r k A. Jepson b a n d Deryck G. W a l k e r c *School o f Life Sciences, Leicester Polytechnic, Scraptoft Campus, Leicester LE7 9SU, bBiochemistry Department, The Medical School, Newcastle upon Tyne University, Newcastle upon Tyne, NE2 4HH and ¢Biochemistry Department, Birmingham University, P.O.Box 363, Birmingham, B15 2TT (United Kingdom) (Received July 20th, 1988; accepted March 20th, 1989)

Summary In the 48 h following a single i.p. injection of 200 mg of thioacetamide/kg body wt there is a progressive rise in Iow-Km hexokinasc activity and a concomitant decrease in high-K glucokinase activity. Loss of glucokinase activity occurs as a consequence of its predominantly perivenous zonal distribution in normal liver since it is hcpatocytcs in this region of the acinus that arc selectively damaged by thioacetamide. Increased Iow-Km activity is mainly present in inflammatory infiltrates which invade perivenous and mid-zone regions in response to tissue injury. Low- and high-K hcxokinase activities return to control levels 10 days after thioacctamide treatment when the infiltration zones disappear and necrotic perivcnous hcpatocytes arc replaced by cells with normal morphology. Changes in low- and high-Km hexokinase activities induced by thioacctamide thus appear to primarily reflect alterations in cell population rather than changes in gene expression within existing hepatocytes.

Key words: Glucokinase; Hepatoxicity; Hexokinase; Liver; Thioacetamide

Introduction Four hexokinase isoenzymes contribute to the total glucose phosphorylating activity in normal adult rat liver [1]. The major, high-Km form, glucokinase (type IV hexokinase), constitutes about 88°/0 of the total activity, is entirely cytosolic and occurs only in parenchymal cells where it has a predominantly perivenous distribution [2,3]. Hepatocytes also contain small amounts of low-Km hexokinase activity (types I, I1 and III) [4]. The majority of this Iow-Km activity is, however, non-parenchymal in origin [5], occurring mainly in Kupffer cells and in bile *Address all correspondence to: Dr G.M. Lawrence, School of Life Sciences, Leicester Polytechnic, Scraptoft Campus, Leicester LE7 9SU, U.K. Abbreviations: ATP, adenosine 5'-triphosphatc; DEAE-cellulose, diethylaminoethyl-ccllulose; DTT, dithiothreitol; EDTA, ethylencdiaminetetraethylacetic acid; G6-PDH, glucose 6-phosphate dehydrogenase; KCI, potassium chloride; LCA, leucocyte common antigen; NAD', nicotinamide adenine dinucleotide; NADP °, nicotinamide adenine dinucleotide phosphate; 6-PGDH, 6-phosphogluconatc dehydrogenase; PK, pyruvate kinase; TAA, thioacetamide; TBS, Tris-buffered saline; Tris, Tris(hydroxymethyl)aminomcthane.

0300-483X/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

21

ducts, arteries and nerves in the portal triad [3]. Up to 70°7o of the Iow-K m activity is due to type I hexokinase and 10°70 of the activity is tightly associated with the outer mitochondrial membrane [3]. It has been suggested [6] that changes in hepatic hexokinase isoenzymes induced by thioacetamide (TAA) reflect the expression, by mature hepatocytes, of genes normally characteristic of undifferentiated cells in a process analogous to that occurring in neoplastic transformation [7,8]. These conclusions were, however, based mainly on biochemical measurements made on liver homogenates and little consideration was given to the effect of changes in cell population known to occur following acute TAA treatment [9]. In the present study we have used a combination of biochemical, histochemical and immunocytochemical techniques to follow the alterations in hepatic hexokinase activity induced by acute TAA treatment. Our results indicate that the changes in hexokinase isoenzymic composition induced by TAA can for the most part be explained in terms of cell population changes. Materials and methods

Materials Phenazine methosulphate, Nitro Blue tetrazolium, diaminobenzidine tetrahydrochloride, glucose 6-phosphate dehydrogenase (from baker's yeast and from Leuconostoc mesenteroides), polyvinyl alcohol (type 11, water soluble, low molecular weight) and other fine chemicals (ATP, NADP *, NAD', DTT, glycylglycine and Tris) were obtained from Sigma (UK). Peroxidase-conjugated rabbit-anti mouse immunoglobulin and rabbit-anti sheep immunoglobulin were purchased from Dako (UK) and mouse monoclonal MRC OX-I (anti-leucocyte common antigen) from Serotec (UK). All other reagents were of AnalaR or of best available grade from Fisons (UK). Methods Treatment of animals. Female Wistar rats (180--220 g) from the colony in the Department of Biochemistry, Birmingham University were used throughout. Thioacetamide (5o7o (w/v) in water) was administered to 32 rats as a single i.p. injection of 200 mg/kg body wt. The animals (4 at each time point) were killed by cervical dislocation 12 h, 24 h, 36 h, 48 h, 3 days, 4 days, 6 days and 10 days after TAA treatment. Control rats (4) injected with corresponding volumes of water were killed by cervical dislocation 12 h, 36 h, 3 days and 10 days after injection. Tissue preparation for assays and activity gels. Rat livers were rapidly removed, weighed and homogenised in 5 times their weight of a buffer comprising 20 mM potassium phosphate (pH 7.0); 10 mM glucose; 1 mM EDTA; 0.5 mM DTT; 100 mM KCI; and 10070 (v/v) glycerol. After centrifuging the homogenate at 30 000 g for 30 min at 5°C and decanting the supernatant ( - T r i t o n ) , the precipitates were rehomogenised and centrifuged again under the same conditions. They were then homogenised in an identical buffer except for the addition of 0.5% Triton X-100. Supernatants ( + Triton) were obtained after centrifugation at 30 000 g for 30 rain at 5 °C. 22

Determination o f hexokinase activity. Hexokinase activity was determined in liver supernatants prepared in the absence and presence of Triton X-100 using yeast glucose 6-phosphate dehydrogenase (G6-PDH) in a coupled assay procedure described as 'the standard assay' by Lawrence and Trayer [10]. Total activity (high-K m + l o w - K ) and I o w - g m activity were distinguished by performing the assays in the presence of 100 mM or 0.5 mM glucose, respectively; high-K m activity being obtained by subtraction. Blanks contained no A T P and enzyme activity was expressed as tamoles N A D P H produced per min per g tissue wet weight at 30°C. Activities of samples prepared in the absence of Triton X-100 were divided by a factor of 2.0 in order to correct for the effect of tissue 6-phosphogluconate dehydrogenase (6-PGDH) activity on assayable hexokinase activity [11]. This correction factor was determined in experiments with livers containing similar amounts of endogenous 6 - P G D H activity which were obtained from control and TAA-treated rats. Hexokinase activity gels. Liver supernatants prepared in the presence and absence of Triton X-100 were subjected to electrophoresis in 1°70 agarose gels. Methods for the preparation, running, staining and scanning of these gels have been described in detail elsewhere [101. Gels were loaded with the same volume (15 tal) of each homogenate so that direct comparisons between isoenzymic profiles at each time point could be made. Histochemical

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Transverse and longitudinal pieces of fresh rat liver, 3--5 mm thick were snap frozen in liquid nitrogen-cooled isopentane. Serial sections (8 tam) were cut at - 1 5 ° C , transferred to washed, adhesive free slides and left to air dry at 20°C for at least 1 h. Sections to be stained for Iow-Km hexokinase activity or for leucocyte c o m m o n antigen were subsequently fixed in acetone for 15 min at 20°C and air-dried at 20°C for 30 min before use. Those to be stained for total hexokinase activity (low- plus high-K m) were fixed for 2 min in acetone at 20°C. Immunocytochemical staining for glucokinase was carried out on sections fixed in periodate-lysine-paraformaldehyde fixative prepared as described by Collings et al. [12]. Hexokinase activity staining. Hexokinase activity was demonstrated by the method of Lawrence et al. [3] except that baker's yeast G 6 - P D H was replaced by the enzyme purified from Leuconostoc mesenteroides and NADP" by NAD" [11]. Incubations in the presence of 100 mM and 0.5 mM glucose were used to distinguish between total and low-K m isoenzymic activity. Indirect immunoperoxidase staining. Staining was carried out using the indirect procedure described in detail by Lawrence et al. [13]. When staining for leucocyte c o m m o n antigen (LCA), mouse monoclonal MRC OX-I was diluted 1 in 1000 and peroxidase conjugated rabbit anti-mouse immunogiobulin was diluted 1 in 20 with 20°7o (v/v) rat serum in Tris-buffered saline (TBS). When staining for glucokinase, sheep anti-glucokinase was diluted 1 in 1000 and peroxidaseconjugated rabbit anti-sheep immunoglobulin was diluted 1 in 50 with 20°7o (v/v) rat serum in TBS. All primary antisera were also initially diluted with an equal volume of normal serum taken from the species used to raise the corresponding secondary antiserum. Staining controls involved the omission of primary a n d / o r secondary antisera and the replacement of the primary antiserum with normal 23

mouse serum or normal sheep serum as appropriate. Staining for glucokinase could be blocked by preincubation of the antiserum with purified glucokinase. Details of the procedures used to raise and characterise the sheep anti-glucokinase have been given elsewhere [14]. Results

Changes in hepatic hexokinase activity and isoenzymic composition induced by TAA During the 2--3-day period following a single i.p. injection of 200 mg of T A A / k g body weight there was a 4.2-fold increase in low-K m hexokinase activity (Fig. 1) whereas high-K m activity decreased to 1407o of control values. Approximately 25°70 of the elevated l o w - K activity was membrane-bound and was released on homogenisation in the presence of the detergent Triton X-100. Recovery began 3 days after treatment and 7 days later low- and high-K" activities had returned to control levels (Fig. 1). Changes in hexokinase isoenzymic composition induced by TAA treatment were monitored using agarose gel electrophoretograms stained for hexokinase activity. Chromoscan traces of these gels (Fig. 2) confirmed the progressive loss of h i g h - K glucokinase activity (IV) from the soluble fraction ( - T r i t o n ) during the first 2 days after TAA treatment and the gradual return to control levels over the next 8 days. They also demonstrated that the increased low-K m activities induced in the soluble ( - T r i t o n ) and particulate ( + Triton) fractions were mainly due to the type II and type III isoenzymes (II and III in Fig. 2). Slightly more type III hexokinase activity was present at each time point in the soluble fractions compared with the type II isoenzyme but in the particulate fractions the latter activity was increased to a much greater extent than the former.

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24

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Changes in the cellular distributions of hexokinase isoenzymes induced by TAA Histochemical staining indicated that increases in Iow-Km hexokinase activity were mainly due to its presence in inflammatory infiltrates. These initially appeared as foci within perivenous and mid-zone regions and gradually increased in number to form large infiltration zones which occupied up to 50°7o of the liver acinus 48 h after TAA treatment (Figs. 3.1 and 3.3). This interpretation was supported by immunocytochemical staining patterns for the white cell marker leucocyte common antigen (LCA) which mirrored those obtained with the histochemical technique (compare Figs. 3.1 and 3.3 with Figs. 3.2 and 3.4). Histochemical staining also indicated that progressive decreases in high-K m hexokinase activity correlated with the induction of perivenous hepatocyte necrosis by TAA. In sections stained for total hexokinase activity there was a gradual reduction in the number of positively stained hepatocytes in perivenous zones such that, 24 h after treatment, only a narrow rim around necrotic areas remained (Fig. 4.1). This pattern was confirmed in serial sections stained immunocytochemically for glucokinase (compare Figs. 4.1 and 4.2). 48 h after the administration of TAA, only a few positively staining hepatocytes remained. Decreases in low-K m hexokinase activity apparent 3--4 days after TAA treatment correlated with the disappearance of the inflammatory infiltrates. This was evident in decreased histochemical staining for low-K m hexokinase activity and reduced immunocytochemical staining for LCA. The rise in high-K m hexokinase activity accompanying this loss of low-K m activity was associated with the

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replacement of necrotic cells by parenchymal cells possessing normal morphological characteristics. These cells showed positive histochemical staining for high-K m hexokinase activity (Fig. 4.3) and positive immunocytochemical staining for glucokinase (Fig. 4.4). Discussion The present study suggests that changes in hepatic low- and high-K m hexokinase activities induced by acute TAA treatment largely reflect alterations in cell population within toxin-damaged liver rather than changes in hepatocyte gene expression as has previously been suggested [6]. Thus, decreases in h i g h - K (glucokinase) activity appear to be a consequence of the selectively perivenous necrosis induced by TAA [9,151 while increases in low-K m activity seem to be mainly due to its presence in inflammatory cell infiltrates which invade the liver in response to tissue injury. The concept of 'metabolic zonation' of liver parenchyma in which certain metabolic pathways predominate in specific zones of the acinus has gained widespread acceptance in recent years (for a review see [16]). Of particular relevance is the predominance of the glycolytic pathway in perivenous hepatocytes. The preferential localisation of glucokinase in these cells has been demonstrated biochemically in microdissection studies [2] as well as histochemically and immunocytochemically [3,13]. It is, therefore, perhaps not surprising to find that selective damage to perivenous hepatocytes by TAA results in the loss of glycolytic enzymes such as glucokinase. The isoenzymic composition of the increased low-K m hexokinase activity seen in the soluble fractions from TAA-treated livers, bears a remarkable resemblance to that previously reported for supernatants prepared in the absence of Triton X100 using livers from rats exposed to CCI 4 [171. Here, 48 h after a single oral dose of 20070 C C I 4 in liquid paraffin (0.5 ml/100 g body wt), DEAE cellulose elution profiles indicated 3.2-fold, 8.8-fold and 6.8-fold increases in hepatic type I, 1I and III hexokinase activities respectively. These and the present hexokinase isoenzyme profiles contrast sharply with those obtained in previous studies on foetal and regenerating liver and on hepatomas [17] in which soluble fractions of foetal liver were shown to contain low levels of type 1 and type II hexokinase activity while supernatants prepared from ascites tumour ceils demonstrated a massive peak of type I1 hexokinase activity and a much smaller pcak corresponding to the type III isoenzyme. On this evidence alone, therefore, it would appear unlikely that the changes seen in TAA-livers could be solely due to changes in gene expression analogous to those seen in neoplastic transformation I7,8]. Monocytes, lymphocytes and granulocytes are known to contain relatively high levels of low-K m hexokinase activity [18,19] but little is known about its isoenzymic composition and subcellular distribution. Soluble fractions prepared from human leucocytes have been shown to contain either type 1 or type 1 and type III hexokinase depending on their site of origin [18,20] but particulate activity was not investigated. Similarly, it has been shown [211 that lymphocytes stimulated

28

with Bordetella pertussis vaccine show 14-fold increases in mitochondrially bound activity but the isoenzymic composition of the particulate activity was not determined. Changes in hexokinase isoenzymic composition and subcellular distribution similar to those seen in TAA-damaged livers in the present study thus might well be expected to occur if massive inflammatory cell infiltration takes place. The presence of these infiltrates in toxin-damaged liver could also explain previously reported increases in glucose 6-phosphate dehydrogenase (G6-PDH) activity [6,22--24]. High levels of G6-PDH activity have been demonstrated histochemically in neutrophils and macrophages present in infiltrates seen in experimentally-induced choleostasis [251 and in TAA-induced liver injury (unpublished observations). Similarly, the presence of T and B lymphocytes within the infiltration zones of TAA-treated liver [26] may explain changes in pyruvate kinase (PK) isoenzymes previously reported to be a general characteristic of toxic liver injury [6,22,231. Both types of cell contain high levels of type K PK rather than the type L isoenzyme which is normally present in adult hepatocytes [18]. Increases in type K PK might thus be expected when inflammatory responses occur, while decreases in type L PK may again reflect a generalised loss of hepatic glycolytic enzymes which occurs as a result of the zone-specific cellular damage induced by TAA and CC14. Increases in low-K m hexokinase activity associated with an enhancement of non-parenchymal cell number have also been reported to occur after treatment with methapyrilene, a hepatotoxin which predominantly affects periportal hepatocytes [27]. In the absence of histochemical evidence, it was impossible in this study to say whether the increase in non-parenchymal cell number was due to intrahepatic proliferation or to infiltration by non-hepatic cells. It was, however, clear that the changes detected were not due to hepatocyte dedifferentiation since no increased activity was seen in parenchymal cell preparations. It is therefore evident that, when dealing with toxins which induce marked changes in cell population within a tissue, interpretations based purely on biochemical studies which take no account of these alterations may be grossly misleading. This type of problem should be investigated either by biochemical studies on separated cell types or by using a combined biochemical and histochemical approach.

Acknowledgements The authors are grateful to Dr J.B. Matthews from the Department Pathology at the Birmingham Dental School for arranging the departmental tissue processing and photomicrographic facilities. A.C.H. is supported by a Cancer Research Campaign Grant and M.A. Jepson recipient of an MRC studentship when this work was carried out.

of Oral use of Beesley was the

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3 4 5 6

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19 20 21 22 23

24

25

80

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carbohydrate melabolism in rat liver parenchyma after experimentally induced choleostasis. Virchows Arch. (Cell Pathol.)., 52 (1987) 501. 26 G.M. Lawrence and J.B. Matthews, Immunocytochemical characterisation of the inflammatory cells infiltrating liver during acute toxic injury. Abstract in the 10th European Drug Metabolism Workshop, Guildford (1986). 27 W. Fischer, S.R. Wagle and N.R. Katz, Altered distribution of hcxokinase and glucokinase between parenchymal and nonparenchymal cells of rat liver after methapyrilene intoxication. Biochem. Biophys. Res. Commun., 115 (1983) 1090.

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