ism Minimizes Liver Damage and Improves Survival in Rats With Thioacetamide Induced Fulminant Hepatic Failure

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Hypothyroidism Minimizes Liver Damage and Improves Survivalin Rats With Thioacetamide Induced Fulminant Hepatic Failure

RAFAEL BRUCK,1 RAN OREN,1 HAIM SHIRIN,1 HUSSEIN AEED,1 MOSHE PAPA,1 ZIPORA MATAS,2 LILIANA ZAIDEL,3

YONA AVNI,1 AND ZAMIR HALPERN1

Recent data from animal studies suggest th at inducedhypothyroidism prevents the hyperdynamic circulation inportal vein ligated rats, liver cirrhosis in rats chronicallytreated with t hioacetamide (TAA), and immune-mediatedacute liver injury induced in mice by concanavalin A.Therefore, the aim of this present study is to determinewhether hypothyroidism would likewise prevent fulminanthepatic failure (FHF) in rats. FHF was induced by 3consecutive ip injections of TAA (400 mg/kg) at 24-hour

intervals. Hypothyroidism was induced in rats by eithermethimazole (MMI) or propylthiouracil (PTU) and surgicalthyroidectomy and was confirmed by elevated serum thy-roid stimulating hormone levels. Serum levels of liverenzymes, blood ammonia, and prothrombin time weresignificantly lower in all 3 groups of hypothyroid rats. Thestage of hepatic encephalopathy (HE) and the survival rateswere significantly improved in the hypothyroid rats (PF.01); the histologic examination of their livers showed lessnecrosis and inflammation (P F .01). In the hypothyroidrats, the serum levels of malondialdehyde 48 hours afterthioacetamide (TAA) administration were lower than incontrol rats (P F .01). Exogenous supplementation of hypothyroid rats with L-thyroxine started 48 hours before

TAA administration abrogated the protective effects of hypothyroidism. The serum levels of tumor necrosis factoralfa (TNF-), interleukin (IL) 2 and IL-6 after 24 hourswere slightly lower in the hypothyroid rats, but the adminis-tration of soluble receptor of TNF (10-1,000 g/rat) did notprevent the induction of fulminant liver failure by TAA.Oxygen extraction, studied in isolated perfused liver prepa-ration, was significantly lower in livers of hypothyroid rats(PF .01). These results suggest that induced hypothyroid-ism decreases the development of liver injury in a rat modelof FHF. Th e mechanism may involve d iminished oxidativecell injury caused by decreased oxygen utilization andhypometabolism associated with hypothyroidism. (HEPATOL-OGY 1998;27:1013-1020.)

Several lines of evidence suggest that thyroid status mayaffect the induction and clinical course of both animals andhumans with various liver diseases. Drugs used for thetreatment of portal h ypertension, such as beta adrenergicblocking agents, have also proved useful in controlling thecardiovascular manifestations of t hyrotoxicosis. Moreover,propylthiouracil (PTU), a commonly used drug for thetreatment of hyperthyroidism, was proposed for the manage-ment of patients with alcoholic liver disease.1 Data fromrecent studies suggest that induced hypothyroidism preventsthe development of liver injury in several animal models. In arat model of portal vein ligation, hypothyroidism causedamelioration of th e hyperdynamic circulation followed byreduction of the portal pressure.2 Hypothyroidism, inducedeither medically or surgically, p revented liver cirrhosis in ratschronically-treated with thioacetamide (TAA),3 and immune-mediated T cell-dependent acute liver injury in mice inducedby the lectin concanavalin A.4

Fulminant hepatic failure (FHF) is a rare but severecomplication of acute hepatitis. FHF is characterized bymassive hepatic necrosis and encephalopathy and carries avery high mortality. Viral hepatitis, drugs, and hepatotoxic

chemical-induced liver injury account for most cases of FHF.5,6 Although a wide variety of medical therapies, such asbenzodiazepine antagonists,7 L-dopa and branched chainamino acids,8 and prostaglandin E1,9 as well as extracorporealperfusion techniques,10 have been used for the managementof this ominous condition, very few therapies have beenevaluated in controlled clinical trials.11,12 The only treatmentof proven efficacy for those patients is emergency livertransplantation. 13,14

Recently, a rat model of TAA-induced FHF has beendescribed. Following 2 to 3 consecutive doses of TAA, ratsdevelop FHF characterized by massive liver necrosis, rapidneurologic deterioration, and death caused by severe encepha-lopathy and brain edema.15,16

In the present study, we demonstrate that hypothyroidisminduced either pharmacologically or by surgical thyroidec-tomy, inhibited the development of TAA-induced fulminantliver failure in rats.

MATERIALS AND METHODS

Materials

Animals. Male Wistar rats (range, 250-300 g), obtained fromTel-Aviv University Animal Breeding Center (Tel Aviv, Israel), werekept in the animal breeding house of the Wolfson Medical Centerand fed a Purina chow ad libitum. Animals were kept in a 12-hourlight-dark cycle at constant temperature and humidity.

Abbreviations: FHF, fulminant hepatic failure; HE, hepatic encephalopathy; TAA,

thioacetamide; TNF-, tumor necrosis factor alfa; IL, interleukin; MMI, methimazole;

PTU, propylthiouracil; s-TNF-R, soluble t umor necrosis factor receptor.

From the 1Departments of Gastroenterology, 2Biochemistry, and 3Pathology, The E.

Wolfson Medical Center, Holon, Israel.

Received February 6, 1997; accepted November 20, 1997.

Presented at t he Digestive Disease Week held in San Francisco , California, May 18-24,

1996, and was published in an abstract form: Gastroenterology 1966;110:1159A.

Address reprint requests to: Rafael Bruck, M.D., Department of Gastroenterology, The

E. Wolfson Medical Center, Holon 58100, Israel. Fax: 972-3-5035111.

Copyright 1998 by th e American Association for the Study of Liver Diseases.

0270-9139/98/2704-0017$3.00/0

1013



Methods

Induction of Hypothyroidism. Hypothyroidism was induced by theadministration of either methimazole (MMI) 0.04% (Taro, Herzlia,Israel) or propylthiouracil (PTU) 0.05% (Teva, Netanya, Israel) indrinking water for 3 weeks. Surgical thyroidectomy was performedwith the animals under chloral hydrate anesthesia (400 mg/kg) 3weeks before th e induction of FHF. To confirm that correction of hypothyroidism would reverse its protective effects on the liver, 3groups of hypothyroid rats (induced by PTU, MMI, or thyroidec-tomy) were supplemented with thyroxine (eltroxin, Glaxo, C.T.S.,Petah Tikva, Israel) 5 µg/day by gavage, started 24 hours before TAAand con tinued d uring th e 3 days of the stu dy. All rats had free accessto tap water durin g the week before the beginning of the stud y.

Induction of Fulminant Hepatic Failure. For induction of FHF, ratswere given ip injections of TAA (Sigma Chemical Co., St. Louis,MO), 400 mg/kg, three times with a 24-hour interval, as previouslydescribed.15,16 Control rats were treated with ip injections of NaCl0.9%. Supportive therapy by subcutaneous administration of 5%dextrose (25 mL/kg) and NaCl 0.9% with potassium (20 mEq/L)every 12 hours were administered to avoid weight loss, hypoglyce-mia, and renal failure, as p reviously described.17

Evaluation of Liver Injury. Four hours following the third TAAinjection, blood samples were drawn for analysis of aminotransfer-ase levels, serum glucose and bilirubin, prothrombin time, Interna-

tional Neutralization Ratio, and blood ammonia to evaluate th edegree of liver failure. Commercial enzyme-linked immunosorbantassay kits were u sed according to the manufacturer’s specificationsto determine th e serum levels of tumor necrosis factor alfa (TNF-),interleukin (IL)-2, and interleukin (IL)-6 (Genzyme Corp., Cam-bridge, MA). The concentrations of malondialdehyde were mea-sured as previously described.18,19

Effect of Soluble Receptor of TNF-. Recombinant human solubleTNF receptor (sTNF-R; Interpharm, Israel), was produced inchinese hamster ovaries cells and purified by immunoaffinitycolumn, using monoclonal antibodies to the sTNF-R1. Purity95%was verified by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis and size exclusion high-performance liquid chromatogra-phy. sTNF-R at TNF:sTNF-R molar ratio of 1:10 3, 1:104, and 1:105

(10, 100, and 1,000 µg/rat), was administered subcutaneously to

each rat 16 hou rs and 1 h our before each of the TAA treatments and24 h ours afterwards, as previously described.20

Hepatic Encephalopathy and Survival. The stage of hepatic encepha-lopathy (HE) and survival of the rats in the different treatmentgroups were determined 4 hours after the third TAA injection. Thestage of HE was determined by the following neurobeh avioral scale:stage 1, lethargy; stage 2, mild ataxia; stage 3, lack of spontaneousmovement and loss of righting reflex, but still responsive; and stage4, coma and lack of response to pain.21

For liver histopathology analysis, midsections of the left lobes of the liver were processed for light microscopy. This processing consistedof fixing the specimen in a 5% neutral formol solution, embeddingthe specimensin paraffin, slicingsections to 5-µm thick, and staining thesections with hematoxylin and eosin. The tissue slices were scannedand scored semiquantitatively by two expert pathologists who were

not aware of sample assignment to experimental groups. The degreeof inflammation and necrosis were expressed as the mean of 10different fields within each slide that had been classified on a scale of 0 to 3 (normal, 0; mild, 1; moderate, 2; and severe, 3).

Measurement of O2 Extraction in Isolated Perfused Rat Liver. Thesurgical procedures were essentially performed as described previ-ously.22 Briefly, the pancreaticoduodenal branch of the portal veinwas ligated and the bile duct, portal vein, and inferior vena cavawere cannulated under chloral hydrate anesthesia. The liver wastransferred into a heated perfusion chamber maintained at 37°C andperfused at a constant rate of 40 mL/min with Krebs-Ringer-Bicarbonate buffer containing 5.5 mmol/L glucose and gassed with95%O2 /5% CO2. With the use of a single pass system, O2 extractionby the rat liver was measured after 30 minutes of equilibration,expressed as a percentage and calculated as:

Extraction rate (%) O2 inflow O2 outflow

O2 inflow 100

Experimental Design. Five groups of rats were studied, as follows:control groups: 1) norm al controls: 3 NaCl 0.9% injections; 2) FHFcontrols: 3 TAA injections in 24-hour interval; 3) TAA sTNF-R(10-1,000 µg/rat); hypothyroid: 4) TAA MMI; 5) TAA PTU; 6)TAA surgical thyroidectomy; 7) TAA PTU eltroxin 5 µg/daystarted 24 hours before TAA; 8) TAA MMI eltroxin given as

described; and 9) TAA ST eltroxin.Statistical Analysis. The data are presented as the means SEM forliver enzymes. All other data are presented as means SD. Thesignificance of differences among different groups was determinedby ANOVA followed by a post-h oc test.

RESULTS

Induction of Hypothyroidism

The induction of hypothyroidism was confirmed by ele-vated serum levels of TSH. The administration of PTU andMMI, as well as surgical thyroidectomy, were each followedby a significant elevation of serum TSH (7.8 0.9, 7.6 0.8,and 6.9 0.9 µIU/mL, respectively) as compared with theuntreated control groups (0.31 0.05 and 0.32 0.04

µIU/mL, respectively).

Effect of TAA on Liver Enzymes, Prothrombin Time,

and Blood Ammonia

Rats were injected with 3 doses of TAA at 24-hour intervalsand bled 4 hours after the third injection; the serum levels of hepatic enzymes, bilirubin and glucose, as well as of prothrom-bin time an d blood ammonia were th en analyzed. Severe liverinjury, as manifested by elevation of serum aspartate amino-transferase, alanine aminotransferase, and lactate dehydroge-nase levels was observed 24 an d 52 hou rs after TAA adminis-tration ( Table 1 and Fig. 1). The serum levels of bilirubin andalkaline p hosphatase were not significantly elevated in re-

sponse to TAA administration in both the hypothyroid andcontrol groups (data not shown).Prothrombin time (and International Neutralization Ratio)

were markedly prolonged in the TAA-treated rats 24 and 52hours after TAA administration (53.5 5.0 and 82.4 8.0seconds, respectively) compared with pretreatment levels(18.2 3.8 seconds) (Fig. 2), although bleeding phenomenawere not observed in the TAA treated rats. Blood ammonialevels in the TAA treated group was elevated fivefold overcontrol untreated rats (8.5 2.1 vs. 1.7 0.2 µg/mL, P

.001) (Fig. 3).

Inhibition of TAA-Induced Liver Injury by Hypothyroidism

In all 3 groups of hypoth yroid rats, the serum levels of liver

enzymes were significantly decreased;although in the thyroid-ectomized rats the liver enzymes were slightly higher than inthe animals with the medically induced hypothyroidism(Table 1 and Fig. 1). The prolonged prothrombin timeobserved in t he TAA-treated rats was almost fully corrected inall 3 groups of hypothyroid rats (Fig. 2, P .001). Bloodammonia, measured 4 hours after the third TAA injection,was threefold lower in the hypothyroid rats than in theeuthyroid, TAA-treated rats (Fig. 3, P .001).

Reversal of Hypothyroidism by Thyroxin Administration

In the 3 groups of rats where the hypothyroidism wascorrected by exogenous supplementation with th yroxin 5

1014 BRUCK ET AL. HEPATOLOGY April 1998



µg/day started 24 hours before the first injection of TAA, theliver enzymes and blood ammonia were not different than incontrol rats treated with TAA only (Table 1; Fig. 3). Theresults of this experiment support the hypothesis that theprotective effects of hypothyroidism on the insulted liverwere not caused by drug interactions but rather may beattributed to consequences of the h ypothyroid state.

Serum Cytokine LevelsThe serum levels of TNF-, IL-2, and IL-6 measured 2 and

6 hours after the first injection of TAA were undetectable.Proinflammatory cytokines were first measurable in theserum of the TAA-injected rats after 24 hours and weresignificantly lower in the hypothyroid TAA-treated groupcompared with rats treated with TAA alone (Table 2, P .05).At this time point, liver damage was already established asassessed by the elevated levels of liver enzymes (Table 1 andFig. 1). In addition, the levels of all 3 cytokines reached lowlevels in all groups, including the control rats. These findingsindicate that the release of those cytokines may be a second-ary event that occurred in response to the products of celllysis, after hep atic damage had already been established.

Effect of Soluble Receptor of T NF-

To further investigate the role of TNF- in TAA-indu cedFHF, the in vivo protective effects of recombinant prepara-tions of TNF sTNF-R were assessed in rats in response to TAAadministration. sTNF-R, at a molar range of 1:103, 1:104, and1:105 to TNF- (10, 100, an d 1,000 µ g/rat, respectively, basedon TNF- serum value measured in mice two hours afterconcanavalin A inoculation) , had no ben eficial effects on therelease of aminot ransferases (Table 1), blood amm onia levels(Fig. 3) or the survival of the TAA-treated rats. Thu s, th eadministration of sTNF-R, that prevents immune-mediated

hepatic damage in mice in response to concanavalin A23 andtoxic liver injury in rats induced by CCl4,20 had no protectiveeffect in t his model of TAA-indu ced FHF.

Serum and Hepatic Levels of Malondialdehyde

Fifty-two hours after the first TAA injection, the serummalondialdehyde levels in the rats treated with TAA onlyincreased from 0.83 0.09 to 3.06 0.6 nmol/mL comparedwith 1.86 0.4 and 1.77 0.5 nmol/mL in the MMI and thePTU treated rats, respectively, P .01 (data not shown).Likewise, in the TAA-treated hypothyroid rats hepatic MDAlevels were significantly lower th an in the euthyroid rats(17.5 3.5 and 16.7 4.2 nmol/g wet tissue in the MMI andPTU groups vs. 32 5.8 nmol/g wet tissue in the livers of ratstreated with TAA only, P .01), and the administration of sTNF-R had no effect on the increased hepatic levels of malondialdehyde in TAA treated rats.

HE

Four hours after the third injection, all rats in theTAA-treated group were in stage 3 to 4 HE.21 The level of HEin all groups of the hypothyroid rats was significantly lower(Table 3) .

Survival

Short-Term. To determine the effect ofind uced hypothyroid-ism on the survival of rats with TAA-induced FHF, con troland hypothyroid rats ( PTU, MMI, and surgical thyroidec-

FIG. 1. Effect of hypothyroidism on serum levels of liver enzymes (A)alanine aminotransferase and (B) aspartate aminotransferase 24 and 52 hoursafter th e first TAA injection. Mean SD (n12) in TAA alone; and TAAMMI and TAA PTU (n5) in the thyroidectomy group. *P .05; **P

.01 compared with TAA alone.

TABLE 1. Effect of Hypothyroidism on Seru m Levels of Liver Enzymes in

TAA-Induced FHF

n Hou r s

AST

(IU/L)

ALT

(IU/L)

LDH

(IU/L)

Normal 5 52 160 22 45 6 1476 182

TAA alone 6 24 3780 425 1540 220 15,305 2780

TAA alone 12 52 6176 307 3260 396 17,033 1833

TAAMMI 12 52 2008 270** 1669 166** 4868 699*

TAA PTU 12 52 1595 286** 1185 159** 4176 481**

TAA ST 5 52 3972 267** 2213 119* 9828 1280*

TAA PTU

Elt 3 52 5017 408 1503 174 13,137 1327

TAA ST Elt 3 52 5293 895 2560 306 13,310 1875

TAA sTNF-R

100 µg/rat 5 52 5150 830 2920 344 15,560 38401000 µ g/rat 5 52 7220 1310 2970 465 14,864 3584

NOTE. Means SEM. Hepatic enzymes determin ed after 3 ip injections of

400 mg/kg TAA in 24-hour in tervals, 52 hours after the first administrat ion.

TAA alone, liver enzymes were determined 24 hours after a single ip injection

of 400 mg/kg TAA. L-Thyroxin 5 µg/day, administered to correct hypothyroid-

ism, started 24 hours before the induction of FHF. Note that serum

aminotransferase levels in rats that were pretreated with s-TNF-R, and in

hypothyroid rats that their hypothyroidism was corrected by supplementa-

tion with exogenous t hyroxine, is not different from TAA-treated control

rats.

Abbreviations: ST, surgical thyroidectomy; Elt, L-thyroxin.

*P .001 compared to TAA.

**P .001 compared with TAAalone (52 h) .

HEPATOLOGY Vol. 27, No. 4, 1998 BRUCK ET AL. 1015



tomy) that received 3 injections of 400 mg/kg TAA, werefollowed after the last TAA dose. Sevety-two hours after thethird TAA injection only 24% 8.9% of 25 control rats (TAAonly) survived, whereas 100% of the h ypothyroid rats treated

with PTU and MMI and 80% of ST rats were alive (Table 4).The survival rate of rats treated with s-TNF-R (30% 14.1%)was not different than that of TAA treated euthyroid rats(Table 4) .

Long-Term. Hypothyroid (MMI and PTU) and 10 controlrats that received 2 doses of 300 mg/kg TAA, 15 were followedfor up to 10 days after the indu ction of FHF. By the end of the

follow-up period, only 20% died in the hypothyroid (onePTU and one MMI) rats. Liver histology in the surviving rats10 days after the induction of FHF was normal. In theTAA-treated euthyroid rats, 70% died during the 72 hoursafter the first TAA injection and the rest survived thefollow-up period. The survival rate in this control group isvery similar to the data of a previous study that characterizedthe TAA-induced FHF as a model of HE.15 In th is study, wheretwo doses of 300 mg/kg TAA were administered, 77% of thecontrol rats were dead before 72 h ours following the first TAAdose, while the rest of the rats survived.15

Liver Histopathology. Histopathologic examination of liverspecimens taken 24 and 52 hou rs after the first TAA injectionshowed less necrosis (P .01) and inflammation ( P .05) in

the livers of the hypothyroid rats compared to control ratstreated with TAA only (Table 5, Fig. 4A-4F). However,moderate inflammatory changes were present also in theTAA-treated h ypothyroid livers, in dicating lesser liver inju ryin those rats. These inflammatory changes may be consistentwith the moderate increase of hepatic enzymes and prothrom-bin time observed in the hypothyroid rats as well.

O2 Extraction by Isolated Perfused Rat Liver. O2 extraction byisolated perfused rat liver of the hypothyroid rats wasdecreased to 39 % 7% compared with 82% 12%in controleuthyroid rats (Fig. 5, P .001).

FIG. 2. Effect of hypothyroidism on (A) prothrombin time and (B)International Neutralization Ratio in TAA-induced fulminant liver failure in24 and 52 hours. The prolonged prothrombin time (and InternationalNeutralization Ratio) in the TAA-treated euthyroid rats was partiallycorrected in all 3 groups of hypothyroid rats. Mean SD (n8) in TAAalone; and TAAMMI and TAA PTU (n 5 ) in the thyroidectomy group.**P .01 compared with TAA alone.

FIG. 3. Effect of hypothyroidism on blood ammonia in TAA-inducedfulminant liver failure. Blood ammonia was significantly lower in all 3 groupsof hypothyroid TAA-treated rats. Note that in rats that were pretreated withs-TNF-R, and in hypothyroid rats that were supplemented with L-thyroxinbefore TAA admin istration (TAA ELT), to correct hypothyroidism, thehigh blood ammonia is not different from TAA-treated control rats. Mean SD (n8). **P .001 compared with TAA alone.

TABLE 3. Effect of Hypothyroidism on HE in TAA-Induced FHF

Group Grade of Encephalopathy

TAA alone 3-4

TAA PTU Elt 3-4

TAA ST Elt 3-4

TAA sTNF-R 3-4

TAA PTU 1-2

TAAMMI 1-2

TAA ST 1-2

NOTE. L-thyroxin 5 µg/day, administered to correct hypothyroidism,

started 24 h before the induction of FHF. HE was evaluated in all treatment

groups 4 hours following the 3 rd injection of TAA. n 12 in TAA alone;

TAA MMI and TAA PTU; n 5 in TAA ST group, TAA sTNF-R

(100 or 1,000 µg/rat); and n 3 in TAA PTU Elt, and TAA ST Elt.

Abbreviations: ST, surgical thyroidectomy; Elt, L-thyroxin.

TABLE 2. Effect of Hypothyroidism on Cytokine Release

in TAA-Induced FHF

Hours

After TAA

TNF- (pg/mL) IL-2 (pg/mL) IL-6 (pg/mL)

Cont MMI Cont MMI Cont MMI

2 0 0 0 0 0 0

6 0 0 0 0 0 0

24 2 0.4 0.4 0.1* 6.8 1.1* 0.4 0.1* 10 1.6 2 0.4*

48 0 0 0 0 0 0

NOTE. Mean SD (n 4). TAA 400 mg/kg injected ip. Cytokine levels in

normal untreated rats were undetectable in all time points (not shown in the

table).

*P .01 compared with control.




DISCUSSION

The present study was undertaken to examine whetherhypothyroidism that prevents liver damage in several animalmodels could also be protective in a model of FHF induced byTAA. This model was characterized previously by clinical,biochemical, and histologic methods, and it proved to be a

reliable and satisfactory model of FHF and HE.15,16,24

Hypothy-roidism, regardless of the mode of induction, essentiallyinhibited the development of FHF in this rat model. Theominou s man ifestations of FHF, in cluding severe coagulopa-thy, high grade HE and high mortality rate, were prevented.Consistent with t hese findin gs, liver histology in all groups of hypothyroid rats showed significantly less hepatic necrosis,although substantial infiltration of liver tissue with inflamma-tory cells was still observed (Table 5, Fig. 4), which isconsistent with moderate elevations of serum aminotransfer-ase levels in the hypothyroid groups.

TAA is a potent hepatotoxin in rats that acts via thehepatocyte mono-oxigenase cytochrome system. The activemetabolites responsible for hepatotoxicity of TAA are those

derived from TAA S-oxide, the product of oxidation of TAAby the flavin adenine dinucleotide-monooxigenase system.25

Free radicals are generated by th is oxidative pathway, causinglipid peroxidation and hepatocyte damage.26

To exclude th e possibility of dru g interaction between TAAand the anti-thyroid drugs used in the study, hypothyroidismwas induced also by surgical thyroidectomy. The results inthis group of rats also showed improvement in liver functionwhich is similar to those of the rats with drug-inducedhypoth yroidism, suggesting that th e hypothyroid status itself,and not drug interaction, inhibited the development of FHFin TAA-treated hypothyroid rats. However, the correction of liver function in the group of rats undergoing thyroidectomy

is somewhat less impressive than in the rats with thedrug-induced hypothyroidism. Therefore, our studies do notentirely exclude that the beneficial effect of hypothyroidismon the insulted liver could be augmented by other actionsinduced by the anti-thyroid drugs, such as suppression of themicrosomal flavin adenine dinucleotide-containing mo-nooxigenases in the liver by MMI.25 Other effects of anti-thyroid drugs, such as alteration of hepatic glutathione

content or kinetics, should also be considered. It has beendemonstrated in a recent study in rats that although TAAadministration had no effect on the total hepatic glutathionecontent, it changed the oxidative status of glutathione,inducing a significant increase in glutathione disulfide levels,and a glutathione-dependent mechanism has been suggestedas responsible for the protection of S-adenosyl-L-methionineagainst TAA hepatotoxicity.27 Nevertheless, in a recent studyfrom our lab, the continuous administration of the glutathi-one donor N -acetylcysteine, before and during the 48 hoursof TAA administration h ad no beneficial effects on eith er liverfunction tests or survival of rats with TAA-induced hepaticfailure.28 Because acute administration of PTU in rats canincrease portal blood flow, independent of its effect on

thyroid function,29 the inhibition of FHF by MMI and in thethyroidectomized rats likewise excluded the possibility thatthe beneficial effect of hypothyroidism in this model wascaused by a direct effect of PTU on th e liver.

The mechanism(s) responsible for the inhibition of fulmi-nant hepatitis in rats by hypothyroidism are not clear.Immunologic factors shou ld be considered, as studies u singthe TAA model have shown strong features of inflammationand cellular infiltration in the pericentral areas of livers fromTAA-treated rats,24 and in a rat model of chronic TAAadministration, immune cells are involved in the induction of liver cirrhosis by TAA.30 Furthermore, hypothyroidism pre-vents liver injury in a model of immun e-mediated concanava-lin A-indu ced acute h epatitis in mice which is associated withsignificantly reduced serum levels of TNF- in the hypothy-roid mice.4 Several lines of evidence suggest that the thyroidstatus may have immunomodulatory effects: decreased thy-roid function is associated with redu ced CD4 T lymphocytesactivation, increased number and activation of CD8 cellsand decreased soluble IL-2 receptors.31 In rats and mice,MMI-induced hypothyroidism suppressed the expression of TNF gene in peritoneal macrophages32,33 and reduced alveo-lar macrophage production under the stimulation of lipopoly-saccharide.34 In a recent stu dy, th e administration of solublereceptor of TNF that neutralizes circulating serum TNF-,prevented acute liver injury in rats which was induced by thehepatotoxin CCl4.20 Nevertheless, the cytokine response in

TAA-induced FHF was n ot ch aracterized in p revious studies,and, therefore, the role of TNF- and other proinflammatorycytokines as mediators of liver injury was not determined.

To address this issue, we measured the serum levels of TNF-, IL-2, and IL-6 for 2, 6, 24, and 48 hours followingTAA administration in hypothyroid and normal rats. Thelower serum levels of TNF- and the other cytokines in thehypothyroid compared with the euthyroid rats suggest thatthe suppression of cytokine release might have a role in theprevention of FHF by hypothyroidism. However, the increasein the serum levels of TNF- occurred late (TNF- levelswere measurable not earlier than 24 hours after TAA injec-tion) and reached low levels of only 2 pg/mL in control rats,250-fold lower than the serum TNF concentrations observed

TABLE 5. Effect of Hypothyroidism on Liver Histology

in TAA-Induced FHF

In flam mat ion ( 0-3 ) Necr os is ( 0-3 )

TAA alone 2.4 0.4 2.4 0.5

TAAMMI 1.6 0.3* 0.3 0.1**

TAA PTU 1.4 0.3* 0.4 0.1**

TAA sTNF-R

100 µg/rat 2.3 0.5 2.0 0.4

1,000 µg/rat 2.5 0.6 2.2 0.5

NOTE. Mean SD (n 5). Rats were sacrificed 52 hours after the first

TAA injection.

*P .05 compared with TAAalone.

**P .01.

TABLE 4. Effect of Hypothyroidism on Survival in TAA-Induced FHF

No.

Rats

TAAOnly

(%)

TAA PTU

(%)

TAA MMI

(%)

TAA ST

(%)

TAA sTNF-R

(%)

5 20 100 100 80 20

5 20 100 100 40

5 20 100 100

5 20 100 100

5 40 100 100 80

Mean 24 100* 100* 80 30

SD 8.9 0 0 0 14.1

NOTE. Survival was recorded in all treatment groups up to 72 hours

following the 3rd injection of TAA.

Abbreviations: ST, surgical thyroidectomy.

*P .01.




FIG. 4. Effect ofh ypothyroidism on liver histology in TAA-induced acute hepatic failure. Rats were sacrificed and livers fixed 24 and 52 hours after the firstTAA injection. (A and B) Liver section from a r at treated only with TAA, showing diffuse centrilobular necrosis and severe inflammatory reaction. ( C and D)TAA and hypothyroidism induced by PTU. Note that no significant h epatic necrosis is present. (E and F) TAA and hypothyroidism induced by MMI. Althoughsome portal and pericentral inflammatory changes are present, no substantial liver necrosis is observed. (G and H) TAA and hypothyroidism induced bythyroidectomy. Inflammatory infiltration is more intense [h], and small areas of hepatic necrosis can be observed around the central vein [g], however, noextensive necrosis is present (compared t o TAA alone, A and B). (Hematoxylin and eosin; original magnification80.)




in mice with immune-mediated liver injury induced byconcanavalin A.35 Likewise, the serum levels of IL-2 and IL-6measured 24 hours after TAA administration were low aswell, whereas severe liver d amage has already been confirmed(Table 2). Moreover, the administration of high doses of soluble TNF receptor, which neutralizes circulating serumTNF-, failed to prevent TAA-induced FHF in rats (Table 1,3, and 5; Fig. 3). Therefore, it seems that the suppression of TNF- and other proinflammatory cytokines do not play akey role in the p revention of severe liver damage by hypothy-roidism.

In this model of FHF, cell necrosis probably has thefollowing two major components: 1) metabolism of TAA togenerate reactive radicals leading to oxidative cell damage;

and 2) the secondary inflammatory response to the produ ctsof cell lysis. Hypothyroidism may, therefore, protect the liverby inhibiting the generation of free-oxygen radicals, causingcell necrosis via oxidation of cellular proteins, DNA, andlipids.26 To determine whether hypothyroidism decreaseslipid peroxidation, we measured the serum and hepatic levelsof malondialdehyde in response to TAA.24 In control euthy-roid rats, a marked increase in serum malondialdehydeconcentrations was observed 52 hours after the initiation of TAA treatment, indicating lipid peroxidation.36 In contrast, inthe hypothyroid rats there was only slight increase in theserum levels of malondialdehyde. Similar results were ob-tained from measurement of hepatic malondialdehyde concen-trations, suggesting that the increased serum malondialde-

hyde levels indicate hepatocyte oxidative damage, and notlipid peroxidation, of extrahepatic tissues. These findingssuggest that the hypothyroid state is protective in FHF andmay minimize oxidative damage to the hepatocytes in AA-treated rats. In a rat model of chronic TAA ingestion, livercirrhosis was completely prevented by hypothyroidism.3

Similar to TAA-induced FHF, in this chronic model h epaticdamage also results from toxic oxygen species causingchronic liver injury and leads to the development of fibrosis,probably through the products of lipid peroxidation.30,37,38 Inhyperthyroid rats, generalized hypermetabolism and in-creased hepatocyte oxygen demand lead to an accelerateddevelopment of TAA-induced liver cirrhosis and po rtal hyper-

tension.3 Thus, under conditions of hyperthyroidism, theliver is particularly susceptible to injury.39-41 It has beenshown in a previous study that hypothyroidism which isinduced by PTU, as well as by surgical thyroidectomy,protected rat livers from galactosamine-induced necrosis.This preventive effect has been ascribed to cellular hypome-tabolism, although the exact mechanism of ‘‘hepatic protec-tion’’was n ot elucidated.39 Therefore, it appears th at hypome-

tabolism and decreased hepatocyte oxygen demand, associatedwith hypoth yroidism, may be protective for the insulted liver.This is supported by the decreased oxygen extraction in theisolated perfused liver of hypothyroid rats observed in ourstudies and also by the prevention of liver damage byhypothyroidism in several experimental models based onmechanisms different than toxic liver injury, i.e., immune-mediated hepatitis4 or mechanically-induced liver damagesuch as portal vein and bile-duct ligation.2,3 Altogether, thefindings of our studies suggest that decreased thyroid func-tion may be beneficial for the insulted liver, regardless of themechanisms involved in t he initiation of liver injury.

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FIG. 5. Effect of hypothyroidism on oxygen extraction in isolatedperfused rat liver. Oxygen extraction by the liver was significantly reduced inhypothyroid (methimazole-treated) rats compared with control liver fromeuthyroid rats (82% 12% vs. 39 7%). Mean SD (n5); **P .001.




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ism Minimizes Liver Damage and Improves Survival in Rats With Thioacetamide Induced Fulminant Hepatic Failure

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