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Expression of liver-specific functions in rat hepatocytes following sublethal and lethal acetaminophen poisoning
Journal of Hepatology1996; 25: 183-190 Printedin Denmark All rights reserved Munksgaai-d
Copyright 0 European Association for the Studyof the Liver 1996
Copenhagen
JournalofHepatology ISSN0168.8278
Expression of liver-specific functions in rat hepatocytes following sublethal and lethal acetaminophen poisoning Niels Tygstrup, Soren Astrup Jensen, Bjorg Krog and Kim Dalhoff Departmentof Medicine A, Rigshospitalet, Copenhagen, Denmark
Aim: In order to study the short-term
effect of moderate and severe reduction of liver function by acetaminophen poisoning of different severity on gene expression for liver-specific functions, rats were given 3.75 and 7.5 g per kg body weight acetaminophen intragastrically. The lower dose is associated with low mortality; after the higher dose, most rats die at between 12 and 24 h. Methods: In the morning l’/,, 3,6,9, and 12 h after the injection, the rats were killed and RNA was extracted from liver tissue. By slot-blot hybridization mRNA steady-state levels were determined for enzymes involved in metabolic liver functions, i.e. ureagenesis, gluconeogenesis, and drug metabolism, for acute phase proteins, “house-keeping” proteins, and for proteins related to liver regeneration. Results were expressed as per cent of the level in similarly fasted, untreated rats of the same stock Results: After the smaller dose of acetaminophen, most of the examined mRNA levels were increasing during the experimental period, being two- to four-fold elevated in relation to control after 6 to
I
with acetaminophen is the most frequent cause of fulminant liver failure in many countries, including Denmark (1) and the UK (2). Whereas the chemical reactions by which metabolites of acetaminophen can modify hepatocellular proteins are fairly well known (3), the pathogenesis of the associated liver failure and why it may be fatal are not known. Measurement of liver functions following different degrees of acetaminophen intoxication is NTOXICATION
Received 21 September; revised 29 November: accepted 14 December 1995
Correspondence: Niels Tygstrup, M.D., Rigshospitalet 2152, DK 2100 Copenhagen, Denmark. Phone: +45 3545 2150 Fax: +45 3545 2913 e-mail: [email protected]
12 h. Rats receiving the lethal dose either showed no or a later and smaller increase, and in several cases a fall towards the end of the experiment. The greatest differences were seen for mRNA of arginase, P-fibrinogen, al-acid glycoprotein, a-tubulin, histone 3, TGFP, and cyclin d, i.e. proteins associated with acute phase response and liver cell replication and maintenance. Conclusions: It is concluded that reversible intoxication with acetaminophen induces an adaptive modulation of mRNA expression of liver functions and regeneration which is lacking after severe intoxication. This adaptation, with emphasis on acute phase response and regeneration, may be crucial for recovery after acetaminophen intoxication. If this also applies to the intoxication in man, estimates of the corresponding variables may be clues to the prognosis of acetaminophen-induced fulminant hepatic failure.
Key words: Acetaminophen;
Acute phase proteins; Gene expression; Hepatotoxicity; Liver function; Paracetamol.
not likely to answer the question if many functions are reduced. It may not be possible to discriminate between functions essential for survival and less essential functions. However, natural selection may be assumed to have favoured repair of functions essential for survival, transmitting this information in the genes. Accordingly, what may separate fatal from non-fatal liver damage is that essential liver functions which are inhibited by liver damage, causing liver failure, are preferentially repaired by production of new proteins, i.e. increased expression of the relevant genes. The purpose of the present work was to identify such proteins by -comparing changes in gene expression, as reflected by mRNA levels, for different types I83
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of function following sublethal and lethal acetaminophen intoxication in the rat. Increases in gene expression seen after sublethal intoxication, which are lacking or weaker following lethal intoxication, may identify functions which are essential for recovery. The models used were one from which 90% of the rats will recover, and one which kills more than 90% of the rats after 12 to 24 h (4). For this reason the experimental period was restricted to 12 h.
Materials and Methods Female Wistar rats, weighing about 200 g, were kept on a 12-h light/dark cycle and fed Alt.romin@ pellets ad lib. with free access to water. After fasting overnight, one group of 20 rats received acetaminophen 7.5 g/kg body weight (the lethal model) by gastric tube, and one group of 15 rats 3.75 g/kg body weight (the sublethal model), suspended in 0.2% tragacanth gum. Water was given ad lib., no food was offered. Three rats in each group were killed after 90 min, 3, 6, 9, and 12 h after administration. Nine rats from were killed at zero time after administration of the vehicle. After 6 h the rats receiving the high dose were slow and weak, and one died. The rats receiving the lower dose showed normal behaviour. The experiments were approved by the Danish Council for Supervision of Experimental Animals. About 200 mg of liver tissue was taken from the same site and stored in liquid nitrogen until total RNA was isolated using a Promega kit Z 5 110, based on the thiocyanate method, modified by ethanol precipitation as previously described (5). The samples were diluted to contain 10 l.tg RNA in 10 l.tl from OD values, treated with CH,-HgOH 0.02 M (final), and placed with loading buffer on a denaturing 1% agarose gel, subjected to 20 V overnight in a borate formaldehyde electrophoresis buffer. The gel was immersed in 0.1 M NH,acetate with 1.3 PM ethidium bromide for 30 min, and then in bidistilled H,O for 30 min to remove excess ethidium bromide, followed by inspection under UV light to check for RNA degradation and for equal loading of RNA in the lanes. Loading was adjusted when necessary and rechecked. The following cDNA probes were used: Carbamoylphosphate synthetase I (6), omithine transcarbamylase (7), argininosuccinate synthetase (8), argininosuccinate lyase (9), arginase (lo), glutamine synthetase (1 l), a2-macroglobulin (12), al -acid glycoprotein (13), haptoglobin ATCC? No. 63 138 (14), thiostatin (15), B-fibrinogen (16), albumin (17), CYPIIB l/2 (1 S), phosphoenolpyruvate carboxykinase (19), glyceraldehyde-3-phosphate dehydrogeI84
nase (20), galactokinase (21), p-actin (22), a-tubulin (23), histone 3 ATCC@’No. 63143 (24), transformation growth factor p ATCC’ No. 63197 (25), c-myc ATCC@ No. 41029 (26), cyclin-d (27), insulin-like growth factor II (28), and v-fos ATCC@ No. 41042 (29). Restriction enzymes were purchased from Promega and Boehringer. DNA fragments (inserts) were separated by agarose gel electrophoresis, excised from the gel under UV light, and DNA eluted on SpinBind@ DNA recovery system (FMC) as specified by the supplier. The probes were labelled using an Amersham multiprime kit RPN 1601 Z and isolated by a QIAquick nucleotide removal kit (Quiagen). The Hybond-NTM membrane was placed in a hybridization vessel (HybaidTM 150x35 mm), hybridization solution (formamide 22.5 M (Merck), Denhard solution 10x (from stock 50x, Sigma), Tris 0.05 M pH 7.4, NaCl 1 M, SDS l%, Na-pyrophosphate 0.1%. Salmon sperm DNA 0.5 mg/ml (Sigma) sonicated and immersed in boiling water for 15 min and then added to the solution) was added for prehybridization at 42 “C for 4 h. The solution was removed and fresh hybridization solution as above, but salmon sperm DNA reduced to half, was added preheated at 37 “C. The labelled probe was immersed in boiling water for 15 ruin, then added to the vessel, which was returned to the 42 “C incubator overnight. The filters were washed in SSC 2x (from stock 20x: 3 M NaCl, 0.3 M Na-citrate, pH 7.0) and SDS 0.1% at room temperature for 30 min, with the same solution at 65 “C for 30 min, and then twice with SSC lx and SDS 0.1% at 65 “C for 15 min. Specificity of the probes was ascertained by autoradiography of Northern blots, showing signals from hybridized mRNA at the expected position in relation to ribosomal RNA 18s and 28s. For determination of mRNA levels by slot blot analysis, a freshly prepared nylon membrane for each probe was incubated in bidistilled H,O for 10 min, then in SSC 10x for 10 min, and placed in a Schleicher & Schuell Minifold@. SSC 10x was loaded into the slots and vacuum applied until all slots were empty. To each slot SSC 10x50 p.1 was added, followed by 5 p.g RNA of a sample diluted to 50 l,tl with TE buffer (Tris-Cl pH 8.0 10 n&I, EDTA 1 n&I, SSC 20x), mixed with SSC 20x30 l.tl and formaldehyde 37% 20 l.tl, incubated for 15 min at 65 C, placed on ice, and diluted by SSC 10x300 pl before loading. After emptying the slots by vacuum, further SSC 10x400 l.t.1was added and vacuum applied. The filter was treated as above; only the amount of
Gene expression after acetaminophen poisoning
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Fig. 1. Changes in mRNA level for the urea cycle enzymes carbamoylphosphate synthetase (CPS), omithine transcarbamylase (OTC), argininosuccinate synthase (ASS), argininosuccinate lyase (ASL), arginase (ARG), and glutamine synthetase (GLS) in liver tissue following intragastric administration of acetaminophen 3.75 g/kg (A, N=3, sublethal model) and 7.5 g/kg (H, N=3, lethal model) in fasted rats at the periods indicated, given as per cent (meansEM) of the level in fasted, intact rats.
Fig. 2 Changes in mRNA level for acute phase proteins (a2-macroglobulin, al -acid glycoprotein, haptoglobin, thiostatin, P-fibrinogen, and serum albumin) in liver tissue following intragastric administration of acetaminophen 3.75 g/kg (A, N=3, sublethal model) and 7.5 g/kg (W, N=3, lethal model) in fasted rats at the periods indicated, given as per cent (meansEM) of the level in fasted, intact rats.
probe for hybridization was doubled. Autoradiography was made on an imaging plate BASIIITM under lead shield and the hybridization signal analyzed in a FUJIX bioimaging analyzer system BAS 2000TM (Fuji Photo Film Co). Results are expressed as per cent fSEM of the mean of nine control rats killed at zero time, i.e. when the other groups received acetaminophen. The coefficient of variation of this mean was on average 7%.
fold increase is seen for arginase (cfr. Fig. 6) in the sublethal model, compared with the later and slower rise in the lethal model. The mRNA level for the acute phase reactants (Fig. 2) showed minor differences for haptoglobin, thiostatin, and albumin; aZmacroglobulin was increased in the sublethal model alone, and the increase in al -glycoprotein and P-fibrinogen (cfr. Fig. 6) were delayed in the lethal model. The enzyme mRNA of the “liver-specific” functions (CUP IIB1/2, phosphoenolpyruvate carboxykinase, glyceraldehyde-3-phosphate dehydrogenase, and galactokinase) did not clearly separate the models (Fig. 3), but at the end of the experimental period they were all lower in the lethal model. The great variation in glyceraldehyde-3-phosphate dehydrogenase mRNA in the sublethal model is unexplained. The “house-keeping” protein mRNA (Fig. 4) was
Results Changes in mRNA levels for urea cycle enzymes following sublethal and lethal intoxication with acetaminophen are shown in Fig. 1. The level for carbamoylphosphate synthetase, argininosuccinate synthetase, and argininosuccinate lyase mRNA are essentially similar, for ornithine transcarbamylase and glutamine synthetase the level is falling in the lethal model. A 4-
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@-Actin
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Fig. 4. Changes in mRNA level for the “house-keeping” proteins j3-actin, a-tubulin, histone 3, and transformation growth factor p (TGFP) in liver tissue following intragastric administration of acetaminophen 3.75 g/kg (A, N=3, sublethal model) and 7.5 g/kg (I N=3, lethal model) in fasted rats at the periods indicated, given as per cent (meansEM) of the level in fasted, intact rats.
similar for p-actin except for a low value at the end of the experimental period in the lethal model, that of atubulin was three-fold increased after 6 h in the sublethal model and was falling slightly in the lethal model. Among the growth related proteins the difference in mRNA level was greatest for cyclin d which 186
Hours
12
HO”l3
Fig. 3. Changes in mRNA level for “liver-specific” function enzymes, the inducible drug metabolizing P450 (CYPIIBIR) and phosphoenolpyruvate carboxykinase (PEPCK}, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and galactokinase (Gal.kinase) in liver tissue following intragastric administration of acetaminophen 3.75 g/kg (A, N=3, sublethal model) and 7.5 g/kg (I N=3, lethal model) in fasted rats at the periods indicated, given as per cent (mean SEM) of the level in fasted, intact rats.
‘House-keep
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Fig. 5. Changes in mRNA level for the growth-related factors c-myc, v-fos, cyclin d, and insulin growth factor II (IGF II) in liver tissue following intragastric administration of acetaminophen 3.75 g/kg (A, N=3, sublethal model) and 7.5 g/kg (m, N=3, lethal model) in fasted rats at the periods indicated, given as per cent (meansEM} of the level in fasted, intact rats.
immediately increased four-fold in the sublethal model and gradually was reduced to half of the control value in the lethal model (Fig. 5). TGFP and histone 3 mRNA were increased two-fold at the end of the experiment in the sublethal model, in contrast no rise or a fall, respectively, in the lethal model. For the protooncogenes c-myc and v-fos, the difference in mRNA level increased during the experimental period, being higher in the sublethal model. For IGF II there was no change in the sublethal model and a slight fall in the lethal model.
Discussion Acetaminophen is a direct (Type I) hepatotoxin, i.e. intoxication is dose dependent and reproducible. In the rat liver cell damage occurs more rapidly than in man, but the pathogenesis appears to be the same. Hepatocellular damage is mainly due to acetaminophen oxidation by CYP450 isoenzymes (30) to the
Gene expression after acetaminophen poisoning
mRNA After Acetaminophen Arginase Lethal
Sublethal
al -acid glycoprotein Lethal
Sublethal
Poisoning p-fibrinogen Lethal
Sublethal
Oh
1.5 h
3h
6h
9h
12 h
Fig. 6. Autoradiograms of slot blots of mRNA for arginase (cfr Fig. 1) and for al -acid glycoprotein and P-fibrinogen (cfr: Fig. 2) at different time periods in relation to administration of a sublethal (3.75 g/kg) and a lethal (7.5 g/kg) dose of acetaminophen in three fasted rats at each point in time.
highly reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) which is detoxified by glutathione (GSH) to form 3-(GSH-S-yl)acetaminophen. An
acetaminophen overdose saturates the nontoxic metabolic pathways, i.e. sulphation, glucuronidation, and detoxification of NAPQI by glutathion. 187
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The reactive NAPQI may oxidize and arylate cysteinyl thiol groups, forming adducts which inhibit the function of cellular proteins. Adduct formation has been demonstrated for a selenium-binding protein (31), for a microsomal subunit of glutamine synthetase (32), for certain nuclear proteins (33), and a large number of mostly unidentified proteins (34), including several apparently not associated with liver damage. Other mechanisms, such as oxidation of pyridine nucleotides and lipid peroxidation, may contribute to cell damage by an acetaminophen overdose (3). The dose-dependent effect of acetaminophen intoxication, i.e. what separates non-lethal from lethal intoxication, is presumably related to the extent of liver cell damage. Formation of the 3-(cysteine-syl)acetaminophen protein adduct (35) which precedes the appearance of necrosis, progresses from the perivenous zone where the CYP isoenzymes are most abundant (36), towards the periportal zone (37). Liver cell injury as estimated from the increase in serum alanine aminotransferase is most intense during the first 12 h (38). The extent of liver cell necrosis shows a dose dependent progression from the perivenous to the periportal zone (4). With this background, the reduced mRNA level for most of the proteins studied in the lethal model in relation to the sublethal model was expected. The data show, however, that the change in mRNA levels for different proteins differs markedly between the models. Increased mRNA values for a2-macroglobulin, glyceraldehyde-3-phosphate dehydrogenase, atubulin, histone 3, transformation growth factor p, cyclin d, c-myc, and v-fos, were seen in the sublethal model only. In the lethal model mRNA levels only rose for arginase, al-glycoprotein, and P-fibrinogen, and the rise occurred later and was smaller than in the sublethal model. The difference indicates that in the sublethal model the cellular machinery is still able to compensate for the damage by an increase in the mRNA level for these proteins. In the lethal model this is the case for only a few of them, possibly those most urgently needed for recovery. Whether this difference can account for the difference in outcome, i.e. recovery or death, will have to be tested under experimental conditions where selected functions, among them those identified in the present study, are selectively inhibited or replaced, respectively. The role of the liver-specific functions examined may be underestimated due to the short duration of the experiment. Towards the end of the experimental period all mRNA levels were decreasing in the lethal model. That of arginase was an exception, but in this 188
situation the important function of the enzyme may be the production of polyamines required for regeneration (39) rather than contributing to urea synthesis, since mRNA of the rate limiting carbamoylphosphate synthase did not change appreciably. Marked changes in the level of transcripts for glyceraldehyde-3-phosphate dehydrogenase and p-actin, particularly following sublethal poisoning, demonstrate that these “constitutionally” expressed proteins are not suitable as reference for RNA loading under these conditions. Our data are interpreted to demonstrate that acetaminophen intoxication will be lethal when increased expression of proteins associated with the acute phase response and replication of hepatocytes is no longer possible. This is supported by the finding that induction of these functions by pre-treatment with other toxins such as thioacetamide protects against lethal acetaminophen intoxication (38). Our interpretation is based on the concept of a gene-regulated response to liver damage, aimed at survival and triggered by insufficient functional liver mass. The expected response is an increase in transcription or enhanced processing of mRNA (e.g. of early-immediate genes (40)) of genes for proteins with vitally important functions, resulting in increased mRNA levels. The concept applies to partial hepatectomy, i.e. where the remaining cells are intact, but in the case of intoxication the situation may be confounded by partly damaged hepatocytes. Furthermore, the intoxications may reduce the stability of mRNA. However, the constant level of albumin mRNA which has a long half-life speaks against a general destabilization by the reactive metabolite. Comparing sublethal intoxication with two-thirds hepatectomy, and lethal intoxication with nine-tenths hepatectomy (41), the pattern of changes in mRNA levels was largely similar. However, after 90% hepatectomy an increase in mRNA levels was found for some liver-specific enzymatic functions and acute phase proteins which was absent in the lethal intoxication model. This may indicate that modulation of gene expression, as a response to insufficient liver function, is less efficient after intoxication than after hepatectomy, either because signals from the periphery are weaker or delayed, or because the transcription factors are among the proteins inactivated by the reactive metabolite. Increased mRNA levels may not be synonymous with increased function, and vice versu, but they may reveal a genetic regulated reaction which, a priori, should be regarded as advantageous. We therefore consider the study of changes in mRNA levels a use-
Gene expression a&r acetaminophen poisoning
ful screening method to identify liver functions, with high significance for survival following liver injury.
Acknowledgements The study was supported by the Benzon Foundation and the Danish Health Research Foundation Grant 12-0853. We wish to thank D.A. Shafritz for providing plasmids containing al-acid glycoprotein, TJfibrinogen, phosphoenolpyruvate carboxykinase, albumin, CYPIIB l/2, glyceraldehyde-3-phosphate dehydrogenase, p-actin, and a-tubulin, S.M.J. Morris for urea cycle enzyme cDNA sequences, W. Lamers for glutamine synthetase, J.J. O’Donnell for galactokinase, F.C. Nielsen for insulin growth factor-II, H. Bauman for thiostatin, and S. Reed for cyclin d. Probes for a2-macroglobulin, haptoglobin, histone 3, c-myc, TGFP, and v-fos were procured from ATCC.
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Copyright 0 European Association for the Studyof the Liver 1996
Copenhagen
JournalofHepatology ISSN0168.8278
Expression of liver-specific functions in rat hepatocytes following sublethal and lethal acetaminophen poisoning Niels Tygstrup, Soren Astrup Jensen, Bjorg Krog and Kim Dalhoff Departmentof Medicine A, Rigshospitalet, Copenhagen, Denmark
Aim: In order to study the short-term
effect of moderate and severe reduction of liver function by acetaminophen poisoning of different severity on gene expression for liver-specific functions, rats were given 3.75 and 7.5 g per kg body weight acetaminophen intragastrically. The lower dose is associated with low mortality; after the higher dose, most rats die at between 12 and 24 h. Methods: In the morning l’/,, 3,6,9, and 12 h after the injection, the rats were killed and RNA was extracted from liver tissue. By slot-blot hybridization mRNA steady-state levels were determined for enzymes involved in metabolic liver functions, i.e. ureagenesis, gluconeogenesis, and drug metabolism, for acute phase proteins, “house-keeping” proteins, and for proteins related to liver regeneration. Results were expressed as per cent of the level in similarly fasted, untreated rats of the same stock Results: After the smaller dose of acetaminophen, most of the examined mRNA levels were increasing during the experimental period, being two- to four-fold elevated in relation to control after 6 to
I
with acetaminophen is the most frequent cause of fulminant liver failure in many countries, including Denmark (1) and the UK (2). Whereas the chemical reactions by which metabolites of acetaminophen can modify hepatocellular proteins are fairly well known (3), the pathogenesis of the associated liver failure and why it may be fatal are not known. Measurement of liver functions following different degrees of acetaminophen intoxication is NTOXICATION
Received 21 September; revised 29 November: accepted 14 December 1995
Correspondence: Niels Tygstrup, M.D., Rigshospitalet 2152, DK 2100 Copenhagen, Denmark. Phone: +45 3545 2150 Fax: +45 3545 2913 e-mail: [email protected]
12 h. Rats receiving the lethal dose either showed no or a later and smaller increase, and in several cases a fall towards the end of the experiment. The greatest differences were seen for mRNA of arginase, P-fibrinogen, al-acid glycoprotein, a-tubulin, histone 3, TGFP, and cyclin d, i.e. proteins associated with acute phase response and liver cell replication and maintenance. Conclusions: It is concluded that reversible intoxication with acetaminophen induces an adaptive modulation of mRNA expression of liver functions and regeneration which is lacking after severe intoxication. This adaptation, with emphasis on acute phase response and regeneration, may be crucial for recovery after acetaminophen intoxication. If this also applies to the intoxication in man, estimates of the corresponding variables may be clues to the prognosis of acetaminophen-induced fulminant hepatic failure.
Key words: Acetaminophen;
Acute phase proteins; Gene expression; Hepatotoxicity; Liver function; Paracetamol.
not likely to answer the question if many functions are reduced. It may not be possible to discriminate between functions essential for survival and less essential functions. However, natural selection may be assumed to have favoured repair of functions essential for survival, transmitting this information in the genes. Accordingly, what may separate fatal from non-fatal liver damage is that essential liver functions which are inhibited by liver damage, causing liver failure, are preferentially repaired by production of new proteins, i.e. increased expression of the relevant genes. The purpose of the present work was to identify such proteins by -comparing changes in gene expression, as reflected by mRNA levels, for different types I83
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of function following sublethal and lethal acetaminophen intoxication in the rat. Increases in gene expression seen after sublethal intoxication, which are lacking or weaker following lethal intoxication, may identify functions which are essential for recovery. The models used were one from which 90% of the rats will recover, and one which kills more than 90% of the rats after 12 to 24 h (4). For this reason the experimental period was restricted to 12 h.
Materials and Methods Female Wistar rats, weighing about 200 g, were kept on a 12-h light/dark cycle and fed Alt.romin@ pellets ad lib. with free access to water. After fasting overnight, one group of 20 rats received acetaminophen 7.5 g/kg body weight (the lethal model) by gastric tube, and one group of 15 rats 3.75 g/kg body weight (the sublethal model), suspended in 0.2% tragacanth gum. Water was given ad lib., no food was offered. Three rats in each group were killed after 90 min, 3, 6, 9, and 12 h after administration. Nine rats from were killed at zero time after administration of the vehicle. After 6 h the rats receiving the high dose were slow and weak, and one died. The rats receiving the lower dose showed normal behaviour. The experiments were approved by the Danish Council for Supervision of Experimental Animals. About 200 mg of liver tissue was taken from the same site and stored in liquid nitrogen until total RNA was isolated using a Promega kit Z 5 110, based on the thiocyanate method, modified by ethanol precipitation as previously described (5). The samples were diluted to contain 10 l.tg RNA in 10 l.tl from OD values, treated with CH,-HgOH 0.02 M (final), and placed with loading buffer on a denaturing 1% agarose gel, subjected to 20 V overnight in a borate formaldehyde electrophoresis buffer. The gel was immersed in 0.1 M NH,acetate with 1.3 PM ethidium bromide for 30 min, and then in bidistilled H,O for 30 min to remove excess ethidium bromide, followed by inspection under UV light to check for RNA degradation and for equal loading of RNA in the lanes. Loading was adjusted when necessary and rechecked. The following cDNA probes were used: Carbamoylphosphate synthetase I (6), omithine transcarbamylase (7), argininosuccinate synthetase (8), argininosuccinate lyase (9), arginase (lo), glutamine synthetase (1 l), a2-macroglobulin (12), al -acid glycoprotein (13), haptoglobin ATCC? No. 63 138 (14), thiostatin (15), B-fibrinogen (16), albumin (17), CYPIIB l/2 (1 S), phosphoenolpyruvate carboxykinase (19), glyceraldehyde-3-phosphate dehydrogeI84
nase (20), galactokinase (21), p-actin (22), a-tubulin (23), histone 3 ATCC@’No. 63143 (24), transformation growth factor p ATCC’ No. 63197 (25), c-myc ATCC@ No. 41029 (26), cyclin-d (27), insulin-like growth factor II (28), and v-fos ATCC@ No. 41042 (29). Restriction enzymes were purchased from Promega and Boehringer. DNA fragments (inserts) were separated by agarose gel electrophoresis, excised from the gel under UV light, and DNA eluted on SpinBind@ DNA recovery system (FMC) as specified by the supplier. The probes were labelled using an Amersham multiprime kit RPN 1601 Z and isolated by a QIAquick nucleotide removal kit (Quiagen). The Hybond-NTM membrane was placed in a hybridization vessel (HybaidTM 150x35 mm), hybridization solution (formamide 22.5 M (Merck), Denhard solution 10x (from stock 50x, Sigma), Tris 0.05 M pH 7.4, NaCl 1 M, SDS l%, Na-pyrophosphate 0.1%. Salmon sperm DNA 0.5 mg/ml (Sigma) sonicated and immersed in boiling water for 15 min and then added to the solution) was added for prehybridization at 42 “C for 4 h. The solution was removed and fresh hybridization solution as above, but salmon sperm DNA reduced to half, was added preheated at 37 “C. The labelled probe was immersed in boiling water for 15 ruin, then added to the vessel, which was returned to the 42 “C incubator overnight. The filters were washed in SSC 2x (from stock 20x: 3 M NaCl, 0.3 M Na-citrate, pH 7.0) and SDS 0.1% at room temperature for 30 min, with the same solution at 65 “C for 30 min, and then twice with SSC lx and SDS 0.1% at 65 “C for 15 min. Specificity of the probes was ascertained by autoradiography of Northern blots, showing signals from hybridized mRNA at the expected position in relation to ribosomal RNA 18s and 28s. For determination of mRNA levels by slot blot analysis, a freshly prepared nylon membrane for each probe was incubated in bidistilled H,O for 10 min, then in SSC 10x for 10 min, and placed in a Schleicher & Schuell Minifold@. SSC 10x was loaded into the slots and vacuum applied until all slots were empty. To each slot SSC 10x50 p.1 was added, followed by 5 p.g RNA of a sample diluted to 50 l,tl with TE buffer (Tris-Cl pH 8.0 10 n&I, EDTA 1 n&I, SSC 20x), mixed with SSC 20x30 l.tl and formaldehyde 37% 20 l.tl, incubated for 15 min at 65 C, placed on ice, and diluted by SSC 10x300 pl before loading. After emptying the slots by vacuum, further SSC 10x400 l.t.1was added and vacuum applied. The filter was treated as above; only the amount of
Gene expression after acetaminophen poisoning
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Fig. 1. Changes in mRNA level for the urea cycle enzymes carbamoylphosphate synthetase (CPS), omithine transcarbamylase (OTC), argininosuccinate synthase (ASS), argininosuccinate lyase (ASL), arginase (ARG), and glutamine synthetase (GLS) in liver tissue following intragastric administration of acetaminophen 3.75 g/kg (A, N=3, sublethal model) and 7.5 g/kg (H, N=3, lethal model) in fasted rats at the periods indicated, given as per cent (meansEM) of the level in fasted, intact rats.
Fig. 2 Changes in mRNA level for acute phase proteins (a2-macroglobulin, al -acid glycoprotein, haptoglobin, thiostatin, P-fibrinogen, and serum albumin) in liver tissue following intragastric administration of acetaminophen 3.75 g/kg (A, N=3, sublethal model) and 7.5 g/kg (W, N=3, lethal model) in fasted rats at the periods indicated, given as per cent (meansEM) of the level in fasted, intact rats.
probe for hybridization was doubled. Autoradiography was made on an imaging plate BASIIITM under lead shield and the hybridization signal analyzed in a FUJIX bioimaging analyzer system BAS 2000TM (Fuji Photo Film Co). Results are expressed as per cent fSEM of the mean of nine control rats killed at zero time, i.e. when the other groups received acetaminophen. The coefficient of variation of this mean was on average 7%.
fold increase is seen for arginase (cfr. Fig. 6) in the sublethal model, compared with the later and slower rise in the lethal model. The mRNA level for the acute phase reactants (Fig. 2) showed minor differences for haptoglobin, thiostatin, and albumin; aZmacroglobulin was increased in the sublethal model alone, and the increase in al -glycoprotein and P-fibrinogen (cfr. Fig. 6) were delayed in the lethal model. The enzyme mRNA of the “liver-specific” functions (CUP IIB1/2, phosphoenolpyruvate carboxykinase, glyceraldehyde-3-phosphate dehydrogenase, and galactokinase) did not clearly separate the models (Fig. 3), but at the end of the experimental period they were all lower in the lethal model. The great variation in glyceraldehyde-3-phosphate dehydrogenase mRNA in the sublethal model is unexplained. The “house-keeping” protein mRNA (Fig. 4) was
Results Changes in mRNA levels for urea cycle enzymes following sublethal and lethal intoxication with acetaminophen are shown in Fig. 1. The level for carbamoylphosphate synthetase, argininosuccinate synthetase, and argininosuccinate lyase mRNA are essentially similar, for ornithine transcarbamylase and glutamine synthetase the level is falling in the lethal model. A 4-
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related
proteins
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0
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@-Actin
0
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Fig. 4. Changes in mRNA level for the “house-keeping” proteins j3-actin, a-tubulin, histone 3, and transformation growth factor p (TGFP) in liver tissue following intragastric administration of acetaminophen 3.75 g/kg (A, N=3, sublethal model) and 7.5 g/kg (I N=3, lethal model) in fasted rats at the periods indicated, given as per cent (meansEM) of the level in fasted, intact rats.
similar for p-actin except for a low value at the end of the experimental period in the lethal model, that of atubulin was three-fold increased after 6 h in the sublethal model and was falling slightly in the lethal model. Among the growth related proteins the difference in mRNA level was greatest for cyclin d which 186
Hours
12
HO”l3
Fig. 3. Changes in mRNA level for “liver-specific” function enzymes, the inducible drug metabolizing P450 (CYPIIBIR) and phosphoenolpyruvate carboxykinase (PEPCK}, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and galactokinase (Gal.kinase) in liver tissue following intragastric administration of acetaminophen 3.75 g/kg (A, N=3, sublethal model) and 7.5 g/kg (I N=3, lethal model) in fasted rats at the periods indicated, given as per cent (mean SEM) of the level in fasted, intact rats.
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Fig. 5. Changes in mRNA level for the growth-related factors c-myc, v-fos, cyclin d, and insulin growth factor II (IGF II) in liver tissue following intragastric administration of acetaminophen 3.75 g/kg (A, N=3, sublethal model) and 7.5 g/kg (m, N=3, lethal model) in fasted rats at the periods indicated, given as per cent (meansEM} of the level in fasted, intact rats.
immediately increased four-fold in the sublethal model and gradually was reduced to half of the control value in the lethal model (Fig. 5). TGFP and histone 3 mRNA were increased two-fold at the end of the experiment in the sublethal model, in contrast no rise or a fall, respectively, in the lethal model. For the protooncogenes c-myc and v-fos, the difference in mRNA level increased during the experimental period, being higher in the sublethal model. For IGF II there was no change in the sublethal model and a slight fall in the lethal model.
Discussion Acetaminophen is a direct (Type I) hepatotoxin, i.e. intoxication is dose dependent and reproducible. In the rat liver cell damage occurs more rapidly than in man, but the pathogenesis appears to be the same. Hepatocellular damage is mainly due to acetaminophen oxidation by CYP450 isoenzymes (30) to the
Gene expression after acetaminophen poisoning
mRNA After Acetaminophen Arginase Lethal
Sublethal
al -acid glycoprotein Lethal
Sublethal
Poisoning p-fibrinogen Lethal
Sublethal
Oh
1.5 h
3h
6h
9h
12 h
Fig. 6. Autoradiograms of slot blots of mRNA for arginase (cfr Fig. 1) and for al -acid glycoprotein and P-fibrinogen (cfr: Fig. 2) at different time periods in relation to administration of a sublethal (3.75 g/kg) and a lethal (7.5 g/kg) dose of acetaminophen in three fasted rats at each point in time.
highly reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) which is detoxified by glutathione (GSH) to form 3-(GSH-S-yl)acetaminophen. An
acetaminophen overdose saturates the nontoxic metabolic pathways, i.e. sulphation, glucuronidation, and detoxification of NAPQI by glutathion. 187
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The reactive NAPQI may oxidize and arylate cysteinyl thiol groups, forming adducts which inhibit the function of cellular proteins. Adduct formation has been demonstrated for a selenium-binding protein (31), for a microsomal subunit of glutamine synthetase (32), for certain nuclear proteins (33), and a large number of mostly unidentified proteins (34), including several apparently not associated with liver damage. Other mechanisms, such as oxidation of pyridine nucleotides and lipid peroxidation, may contribute to cell damage by an acetaminophen overdose (3). The dose-dependent effect of acetaminophen intoxication, i.e. what separates non-lethal from lethal intoxication, is presumably related to the extent of liver cell damage. Formation of the 3-(cysteine-syl)acetaminophen protein adduct (35) which precedes the appearance of necrosis, progresses from the perivenous zone where the CYP isoenzymes are most abundant (36), towards the periportal zone (37). Liver cell injury as estimated from the increase in serum alanine aminotransferase is most intense during the first 12 h (38). The extent of liver cell necrosis shows a dose dependent progression from the perivenous to the periportal zone (4). With this background, the reduced mRNA level for most of the proteins studied in the lethal model in relation to the sublethal model was expected. The data show, however, that the change in mRNA levels for different proteins differs markedly between the models. Increased mRNA values for a2-macroglobulin, glyceraldehyde-3-phosphate dehydrogenase, atubulin, histone 3, transformation growth factor p, cyclin d, c-myc, and v-fos, were seen in the sublethal model only. In the lethal model mRNA levels only rose for arginase, al-glycoprotein, and P-fibrinogen, and the rise occurred later and was smaller than in the sublethal model. The difference indicates that in the sublethal model the cellular machinery is still able to compensate for the damage by an increase in the mRNA level for these proteins. In the lethal model this is the case for only a few of them, possibly those most urgently needed for recovery. Whether this difference can account for the difference in outcome, i.e. recovery or death, will have to be tested under experimental conditions where selected functions, among them those identified in the present study, are selectively inhibited or replaced, respectively. The role of the liver-specific functions examined may be underestimated due to the short duration of the experiment. Towards the end of the experimental period all mRNA levels were decreasing in the lethal model. That of arginase was an exception, but in this 188
situation the important function of the enzyme may be the production of polyamines required for regeneration (39) rather than contributing to urea synthesis, since mRNA of the rate limiting carbamoylphosphate synthase did not change appreciably. Marked changes in the level of transcripts for glyceraldehyde-3-phosphate dehydrogenase and p-actin, particularly following sublethal poisoning, demonstrate that these “constitutionally” expressed proteins are not suitable as reference for RNA loading under these conditions. Our data are interpreted to demonstrate that acetaminophen intoxication will be lethal when increased expression of proteins associated with the acute phase response and replication of hepatocytes is no longer possible. This is supported by the finding that induction of these functions by pre-treatment with other toxins such as thioacetamide protects against lethal acetaminophen intoxication (38). Our interpretation is based on the concept of a gene-regulated response to liver damage, aimed at survival and triggered by insufficient functional liver mass. The expected response is an increase in transcription or enhanced processing of mRNA (e.g. of early-immediate genes (40)) of genes for proteins with vitally important functions, resulting in increased mRNA levels. The concept applies to partial hepatectomy, i.e. where the remaining cells are intact, but in the case of intoxication the situation may be confounded by partly damaged hepatocytes. Furthermore, the intoxications may reduce the stability of mRNA. However, the constant level of albumin mRNA which has a long half-life speaks against a general destabilization by the reactive metabolite. Comparing sublethal intoxication with two-thirds hepatectomy, and lethal intoxication with nine-tenths hepatectomy (41), the pattern of changes in mRNA levels was largely similar. However, after 90% hepatectomy an increase in mRNA levels was found for some liver-specific enzymatic functions and acute phase proteins which was absent in the lethal intoxication model. This may indicate that modulation of gene expression, as a response to insufficient liver function, is less efficient after intoxication than after hepatectomy, either because signals from the periphery are weaker or delayed, or because the transcription factors are among the proteins inactivated by the reactive metabolite. Increased mRNA levels may not be synonymous with increased function, and vice versu, but they may reveal a genetic regulated reaction which, a priori, should be regarded as advantageous. We therefore consider the study of changes in mRNA levels a use-
Gene expression a&r acetaminophen poisoning
ful screening method to identify liver functions, with high significance for survival following liver injury.
Acknowledgements The study was supported by the Benzon Foundation and the Danish Health Research Foundation Grant 12-0853. We wish to thank D.A. Shafritz for providing plasmids containing al-acid glycoprotein, TJfibrinogen, phosphoenolpyruvate carboxykinase, albumin, CYPIIB l/2, glyceraldehyde-3-phosphate dehydrogenase, p-actin, and a-tubulin, S.M.J. Morris for urea cycle enzyme cDNA sequences, W. Lamers for glutamine synthetase, J.J. O’Donnell for galactokinase, F.C. Nielsen for insulin growth factor-II, H. Bauman for thiostatin, and S. Reed for cyclin d. Probes for a2-macroglobulin, haptoglobin, histone 3, c-myc, TGFP, and v-fos were procured from ATCC.
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