Molecular and Cellular Biochemistry 268: 33–43, 2005.c�2005 Kluwer Academic Publishers. Printed in the Netherlands.

Serine dehydratase expression decreases in ratlivers injured by chronic thioacetamide ingestion

Inmaculada Lopez-Flores,1 Juan B. Barroso,1 Raquel Valderrama,1

Francisco J. Esteban,2 Esther Martınez-Lara,1 Francisco Luque,3

M. Angeles Peinado,2 Hirofumi Ogawa,4 Jose A. Lupianez5

and Juan Peragon1

1Biochemistry and Molecular Biology Section, Department of Experimental Biology, University of Jaen, Campus LasLagunillas, Jaen, Spain; 2Cell Biology Section, Department of Experimental Biology, University of Jaen, Jaen, Spain; 3GeneticSection, Department of Experimental Biology, University of Jaen, Jaen, Spain; 4Department of Biochemistry, Toyama Medicaland Pharmaceutical University Faculty of Medicine, Toyama, Japan; 5Department of Biochemistry and Molecular Biology,Centre for Biological Sciences, University of Granada, Granada, Spain

Received 30 March 2004; accepted 24 June 2004

Abstract

Serine dehydratase (SerDH) is a gluconeogenic enzyme involved in the catabolism of serine, which is regulated by the com-position of their diet and their hormonal status in rats. This study examines how chronic injury caused to the liver of rats bythe ingestion of thioacetamide (TAA) affects SerDH protein, mRNA levels, enzyme kinetics and its tissue location. After 97days’ oral intake of TAA, the activity of SerDH at all substrate concentrations assayed was about 60% lower than in controls.No significant differences in Km values were found between the treated group and controls. Immunoblotting and immunohis-tochemistry revealed a significant reduction in the level of SerDH protein in the livers of the treated rats. SerDH was detectedspecifically in the periportal zone of the hepatic acinus and this location did not change in response to TAA treatment. The levelof SerDH mRNA, quantified by reverse transcription and polymerase chain reaction, was significantly lower in treated rats thanin the controls. The present findings suggest that the SerDH expression is rendered to be down regulatory during chronic liverinjury induced by TAA. These results enhance our understanding about the biochemical mechanisms implied in the control andintegration of serine catabolism during liver injury in rat. (Mol Cell Biochem 268: 33–43, 2005)

Key words: serine dehydratase, thioacetamide, cirrhosis, RT-PCR, serine catabolism

Abbreviation: SerDH, serine dehydratase; TAA, thioacetamide; Km; Michaelis constant; cAMP, 3′,5′-cyclic adenosinemonophosphate; PLP, pyridoxal 5′-phosphate; ASAT, aspartate aminotransferase; ALAT, alanine aminotransferase; AP, alkalinephosphatase; γ -GT, gamma-glutamyl transferase; rGST-7-7, glutathione transferase isoenzyme 7-7; Vmax, maximum velocity;SDS-PAGE, polyacrylamide-gel electrophoresis with sodium dodecyl sulphate; TBS, Tris buffer solution; PBS, phosphatebuffer solution; RT-PCR, reverse transcription and polymerase chain reaction; dNTP, deoxynucleotide triphosphate; AMV-RT,avian-myeloblastosis virus reverse transcriptase; cDNA, complementary DNA; Tm, melting temperature; SDH3, SerDH cDNAprobe; S.E.M., standard error of the mean; bp, base pair(s)

Address for offprints: J. Peragon, Area de Bioquımica y Biologıa Molecular, Departamento de Biologıa Experimental, Universidad de Jaen, CampusLas Lagunillas, 23071 Jaen, Spain (E-mail: jperagon@ujaen.es)

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Introduction

Serine dehydratase (SerDH, EC 4.2.1.13) is the enzyme thatcatalyzes the PLP-dependent deamination of serine and thre-onine to produce pyruvate and α-ketobutyrate, respectively.It is a cytosolic enzyme [1], which is located primarily in theperiportal area of the rat liver [2]. Two indistinguishable im-munochemically dimeric isoforms composed of 34–35 kDamonomers have been identified in adult rat liver [3]. The en-zyme is involved in the regulation of liver gluconeogenesisfrom serine in different dietary and hormonal states [4]. Itsactivity tends to increase in gluconeogenic situations such asthose caused by starvation, high-protein diet uptake and dia-betes mellitus [5, 6], and is depressed when the high-proteindiet is supplemented with glucose [7] or on the administrationof insulin in the case of diabetes mellitus [6]. The increasein SerDH activity brought about under the conditions men-tioned above seems to be triggered by a stimulation of thetranscription of the SerDH gene mediated by glucagon [8],resulting in a rise in SerDH mRNA levels [9]. This stimula-tion is mediated by cAMP [8] and is maximal in the presenceof glucocorticoids [10]. Recently it has been demonstratedthat both quantity and quality of dietary protein also controlthe expression level of SerDH [11, 12].

Moreover, in rat, the SerDH has an important functionin maintaining appropriate levels of L-serine, an amino acidconsidered essential for cell proliferation and a source of onecarbon groups for nucleotide biosynthesis [13]. Serine is alsoneeded for the development and specific function of the cen-tral nervous system [13]. SerDH is considered a marker ofliver-maturation and is involved in the developmental regula-tion of different tissues. In this sense, it has been demonstratedthat SerDH is expressed in mature hepatocytes in which itsactivity and mRNA could be induced by glucagon and dex-amethasone [14]. A major increase liver-SerDH activity hasbeen detected in Ehrlich ascites tumor-bearing mice [15], theresearchers relating this increase to greater liver gluconeoge-nesis in the tumor-bearing host.

Chronic liver disease is associated with alterations in themetabolism of glucose, lipids and proteins, which can leadto a clinical state of caloric malnutrition [16]. Hepatic glu-cose metabolism may also be altered in patients with chronicliver injury, although the specific alterations caused remainunclear. Both normal and decreased production of glucose inthe liver has been reported under such conditions [17, 18].Moreover, the behaviour and contribution of the key enzymesthat regulate gluconeogenesis from amino acids in general,and from serine in particular, to the overall production ofglucose under these conditions is still poorly understood. Inthe light of the apparent importance of serine as a gluco-neogenic amino acid involved in developmental processesand of SerDH as a regulatory enzyme of this pathway [19],we have determined the regulation of rat-liver SerDH in re-

sponse to chronic liver injury. To induce liver injury, we usedTAA, a hepatotoxic compound that causes liver damage todifferent degrees according to dosage and length of admin-istration: an acute dosage causes centrilobular necrosis [20]while chronic administration results in cirrhosis and nodulartumors [21].

We found that in rats with liver injury induced by thechronic administration of TAA, levels of enzyme activity,protein and mRNA of SerDH were significantly lower thanin controls, clearly indicating the existence of the down-regulation of SerDH gene expression during chronic liverdisease.

Materials and methods

Chemicals

Substrates, coenzymes and other chemical compounds usedfor making buffers and assay media came from SigmaChemical Co. (St. Louis, USA) and Fluka Chemie GmbH(Buchs, Switzerland). The reagents used for immunohisto-chemistry and electrophoresis of proteins and nucleic acidswere from Bio-Rad Laboratories (Hercules, California, USA)and Roche Diagnostics Corporation (Indianapolis, USA).The β-actin primers were synthesized by Promega Corpo-ration (Madison, USA). Oligonucleotide primers were syn-thesized by Roche Diagnostics Corporation (Indianapolis,USA). Other specific reagents and kits were of research grade.

Animals, experimental design and liver-injury markers

Male Wistar rats were used in all experiments and receivedhumane care in compliance with national and internationalguidelines [22–24]. The rats were adapted to laboratory con-ditions for two weeks at a constant temperature of 22 ◦C ±2 ◦C and artificial light from 08.00 h to 20.00 h. They werefed ad libitum on a standard diet (Panlab -Barcelona, Spain-A04, D.G.P.A. 16867-CAT, 54.5% carbohydrate, 16.2% fishand meat protein, 2.8% fat, 3 kcal/g) and had free access towater. They were then separated into two groups of 12 speci-mens, each group being subdivided into three cages of 4 ratsper cage. For 97 days one group was given a solution of 300mg TAA/litre (TAA group) and the other tap water (controlgroup), both administered via water bottles. During the ex-perimental period all the rats were allowed ad libitum accessto the same pre-experimental diet and either to the water-plus-TAA treatment (TAA group) or water alone (controlgroup). For the determination of the degree of liver damagecaused by the TAA treatment the levels of activity of aspartateaminotransferase (ASAT), alanine aminotransferase (ALAT),alkaline phosphatase (AP) and gamma-glutamyl transferase

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(γ -GT) were measured in liver samples using Sigma diag-nostic kits. The level of hepatic glutathione transferase isoen-zyme 7 (rGST-7-7) was also determined to the same end. Thislatter enzyme is not present under control conditions but isinduced during liver neoplasia and cirrhosis [25]. The level ofrGST-7-7 was determined by Western blotting, as indicatedbelow for SerDH, using a specific rabbit antibody againstrGST-7-7 (Biotrin International, Dublin, Ireland).

Microscopy

Liver blocks were fixed in 2.5% glutaraldehyde in 0.1 Mcacodylate buffer at pH 7.4 for 4 h at 4 ◦C. Samples werewashed 3 times during 20 min and postfixed with 2% osmiumtetroxide from 2 h. After samples were washed and dehydra-tred with an alcohol series prior to being embedded in resin(Epon). Thin (1 µm) and ultrathin (70 nm) sections weremade with an ultramicrotome. Thin sections were stainedwith toluidine blue and examined under optical microscopy.Ultrathin sections were stained with uranyl acetate and leadcitrate and examined by transmission electronic microscopy.

Preparation of liver homogenates

Rats were consistently killed at 10.00 h by cervical disloca-tion. Their livers were removed immediately and placed inice-cold saline solution (0.9% NaCl) for the preparation oftwo types of homogenates. The first (1/3 w:v) was made with5 g of liver and medium containing 50 mM Hepes, 100 mMKCl, 10 mM MgCl2, 10 mM NaH2PO4, 0.30 mg/ml of typeIII trypsin inhibitor at pH 7.4 to 15 ml of final volume. Thehomogenate was centrifuged at 105,000 × g for 1 h. The cy-tosolic supernatant was used for SerDH activity assays andWestern-blot analyses. The second homogenate was madewith 100 mg of liver and 2.0 ml of RNAzol B (Cinna BiotecxLaboratories, Inc., Houston, USA) and was used to isolatetotal RNA.

SerDH-activity assay

SerDH was assayed at 25 ◦C by spectrophotometry, as de-scribed in Sandoval and Sols [26], with some modifications.SerDH activity was determined at pH 7.4 in a medium con-taining 27.5 mM Hepes, 55.0 mM KCl, 5.50 mM MgCl2,NaH2PO4, 1 mM dithioerythritol, 0.1 mM PLP, 0.2 mMNADH, 0.01 mg lactate dehydrogenase, 2 mg of cytosolicprotein and L-serine at different concentrations (0.05, 0.075,0.100, 0.250, 0.375 and 0.5 M) to a total volume of 1 ml.The change in absorbance at 340 nm was recorded and af-ter confirmation of no exogenous activity the reaction was

started by the addition of substrate. One mU is defined asthe amount of enzyme needed to reduce 1 nmol of serineper min at 25 ◦C. The kinetic constants (Km and Vmax) andthe kinetic behaviour of SerDH were determined using twonon-linear regression-analysis programs: Enzfitter (ElsevierBiosoft) and Graphit (Erithacus Software Ltd., MicrosoftCorp). The protein concentration of the cytosolic extractswas determined using the Bradford method [27].

Determination of SerDH protein level by Western-blotanalysis

Polyacrylamide-gel electrophoresis with sodium dodecyl sul-phate (SDS-PAGE) and immunoblotting were performed asindicated by Barroso et al. [28]. Samples from high-speedsupernatant fractions were heated to 95 ◦C for 3 min in 62mM Tris-HCl buffer, pH 6.8, containing 2% (w:v) SDS,10% (v:v) glycerol, 2.5% 2-mercaptoethanol, 0.045 mMbromphenol blue and 10 mM 1,4-dithiothreitol. Polypep-tides were separated by 10% SDS-PAGE using a Bio-RadMini-Protean II apparatus and electroblotted onto 0.45 µmpolyvinylidene difluoride membrane (Immobilon-P, Milli-pore Corporation, Bedford, USA) using a semi-dry trans-fer apparatus with a 1.5 mA/cm2 membrane for 150 min in10 mM 3(cyclohexylamino)-1-propano-sulphonic acid and10% methanol (v/v), pH 11.0. The membranes were blockedwith 25 mM Tris-HCl, 100 mM NaCl, 2.5 mM KCl buffer(TBS) at pH 7.6, containing 5% non-fat dried milk and 0.05%Tween 20. The blots were then incubated overnight at 4 ◦Cwith rabbit anti-rat-liver-SerDH antiserum (diluted 1:25000in blocking solution). The anti-SerDH antiserum was de-veloped by Dr. H. Ogawa and previously characterized andused in analogous determinations [29–31]. The blots werewashed with TBS buffer containing 0.1% Tween 20. Im-munodetection was performed using an enhanced chemilu-miniscence kit (ECL-Plus, Amersham Pharmacia Biotech,Buckinghamshire, England). The blots were scanned with anAGFA Horizon ultra scanner, photographed and analysed byvideodensitometry using Bio-1D 97 computer software fromBio-Profil.

Immunohistochemistry

Immunohistochemical studies were made as described else-where [28]. The rats were anaesthetized by an intraperitonealinjection of equitensin (0.36 mg/kg body weight) and the liverwas perfused through the portal vein with 50 ml of 10 mMcarbogenated phosphate buffer solution (PBS) followed by4% paraformaldehyde in PBS. Fixed livers were removed,cut into 8–10 mm3 cubes and post-fixed for 3 h at roomtemperature with the same fixative. Liver blocks were later

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immersed in 30% sucrose-0.1 M PB (4 ◦C). Serial sectionsof 30 µm were obtained using a cryostat (2800 Frigocut E,Reicher-Jung, Vienna, Austria). Endogenous peroxidase wasinhibited on free-floating sections with 0.03% H2O2 in PBSfor 30 min, and after several washes they were then incu-bated overnight at 4 ◦C with rabbit anti-rat SerDH serumdiluted 1:5000 in PBS containing 0.2% Triton X-100. Thesections were subsequently washed in PBS and incubatedwith biotinylated goat anti-rabbit IgG (Vector Laboratories,Burlingame, USA), diluted 1:100 for 1 h, washed and laterincubated with peroxidase-linked avidin-biotin complex atroom temperature for 90 min. Peroxidase activity was de-tected by the nickel-enhanced diaminobenzidine procedure[32] and the sections were then mounted on slides usingDPX mountant for histology. Control procedures were car-ried out with the primary antibody either being omitted orreplaced with an equivalent concentration of pre-immuneserum.

Determination of SerDH-mRNA levels by reversetranscription followed by polymerase chain reaction(RT-PCR)

Extraction and isolation of RNATotal RNA was isolated from rat liver by the modified acidguanidium-phenol-chloroform extraction method [33] us-ing RNAzol B (Cinna Biotecx Laboratories, Inc. Houston,USA).

Reverse transciption (RT)Samples of 2 µg of total RNA were subject to reverse tran-scription with oligo(dT) as a primer in a medium containing10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 1mM of each dNTP, 1.6 µg of oligo(dT)15, 50 units of ribonu-clease inhibitor and 20 units of avian myeloblastosis virusreverse transcriptase (AMV-RT) in a final volume of 20 µl.The reaction mixture was incubated for 1 h at 42 ◦C to syn-thesize corresponding cDNAs and then heated to 99 ◦C for5 min.

Polymerase chain reaction (PCR)The reverse-transcribed mixture was amplified using spe-cific primers for SerDH and β-actin DNA sequences. Theoligonucleotide primers were designed from the SDH3 cDNAsequence [34] using the program Oligo 4.1 (National Bio-sciences Inc., Cascade, USA). The sense and antisense se-quences were: 5′-GCTGTAAACATTTCGTCTGC-3′ and 5′-CAGCATCTCTCCCACCACTT-3′. These sequences annealat 55.5 ◦C and are the complements of two segments of therat-liver SDH3 probe between the 279–298 and 432–451 po-sitions, respectively. The amplification product was 173 bpin size. Primers for β-actin were used as a positive control for

PCR and were designed against a consensus β-actin sequenceto amplify a product of 296 bp.

The primers were: actin sense: 5′-TCATGAAGTGTGACGTTGCATCCGT-3′, and actin antisense 5′-CCTAGAAGCATTTGCGGTGCCGATG-3′. These sequences have a Tm of59.3 ◦C. Sets of primers were chosen with similar annealingtemperatures to give clean results when co-amplification wasperformed.

The amplification medium contained 10 mM Tris-HCl (pH8.3), 50 mM KCl, 1.5 mM MgCl2, 2.5 units of Taq poly-merase, 1 pmol of each SerDH cDNA primer and 20 µl ofRT mixture to a final volume of 100 µl. The reaction mixturewas first denatured for 5 min at 95 ◦C and then subjected to30 cycles of amplification in a DNA thermal cycler. Each cy-cle consisted of a heat-denaturing step at 95 ◦C for 1 min, anannealing step at 60 ◦C for 1 min, and a polymerisation stepat 72 ◦C for 1.5 min, except for the final polymerisation stepof the thirtieth cycle, which was maintained for 5 min. Afterthe first 10 cycles, 1 pmol of β-actin cDNA primer was addedto the reaction mixture and 20 additional amplification cycleswere run. To avoid masking the amplification product of theSerDH mRNA sequence when the SerDH and β-actin cDNAprimers were added simultaneously, the β-actin primers wereadded 10 cycles after the SerDH cDNA primers, thus achiev-ing the optimum amplification of both sequences (Fig. 1).

Two control reactions were set up for each experimentalsituation: one tube without AMV-RT to check for the presenceof any contaminating cDNA template, and another withouttotal RNA to detect any contamination in the reaction mix-tures. Both controls were consistently negative.

The PCR products were separated by electrophoresis on4% agarose. DNA was visualised by ethidium bromide usingan ultraviolet transilluminator and then photographed. Bandintensity was quantified by densitometry assisted by the Bio-1D 97 program.

To demonstrate the identity of RT-PCR products we madeSouthern-blot analyses using a specific SerDH cDNA probe(SDH3) [34] labelled by random priming with digoxigenin.The DNA fragments from 4% agarose electrophoresis wereblotted onto a nylon membrane (Hybond-N+, AmershamPharmacia Biotech, Buckinghamshire, England) by salinecapillary transfer and detected using a DIG nucleic acid detec-tion kit (Roche Diagnostics Corporation, Indianapolis, USA).

Statistics

The results are expressed as the means ± S.E.M. The dif-ferences between means were analysed using an unpairedStudent’s t-test. Any possibility of cage effect was tested foreach parameter, also using the unpaired Student’s t-test. Nodifferences were found between cages of the same experi-mental group (data not shown).

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Fig. 1. Co-amplification of SerDH and β-actin sequences with the simultaneous use of primers for both sequences. Panel (A). Suppression of SerDH mRNAamplification. Reverse transcription was made from 1 µg of total RNA from the livers of rats starved for 6 days using oligo (dT) primers; cDNA was thenamplified using specific primers: SDH sense 5′-GCTGTAAACATTTCGTCTGC-3′ and SDH antisense 5′-CAGCATCTCTCCCACCACTT-3′, for SerDH, andβ-actin sense 5′-TCATGAAGTGTGACGTTGCATCCGT-3 and β-actin antisense 5′-CCTAGAAGCATTTGCGGTGCCGATG-3′, for β-actin. Symbols + and– over the gel lanes indicate the results of RT-PCR with or without the primers. The arrows point to bands of RT-PCR products for SerDH (173 bp) and β-actin(296 bp). Panel (B). Decrease in the quantity of product by delaying the addition of β-actin primers. Reverse transcription was made from 1 µg of the total RNAfrom the livers of rats starved for 6 days using oligo(dT) primers; cDNA was then amplified using specific primers for both sequences. The numbers over thegel lanes refer to the cycle in which β-actin primers were added. Lane 0 represents the sample in which the primers for β-actin and SerDH were simultaneouslyadded before amplification started. In all lanes 20 µl of reverse transcription mixture was added.

Results

Induction of chronic liver injury in rats via the oraladministration of thioacetamide

The chronic administration of TAA resulted in a significantdecrease in the growth of the rats. The daily whole-bodyand liver-weight gains in the TAA-group were 71% and 42%lower than those of the control group, respectively. After 97days the body weight of the TAA group was significantlylower (about 20%) than that of the control group (Table 1),although the difference between the two groups in final ab-solute liver weight was not significant. These facts explainthe high value of the liver somatic index in the TAA-group(Table 1). Throughout the 97 days of the experiment the av-erage intake of diet and water in the TAA group was signifi-cantly lower than in the control group (Table 1). The averageTAA consumption was 15.32 ± 0.72 mg per day per rat.

The livers of the rats treated with TAA were yellowish andcontained some relatively large, hard nodules. Histologicalmicrographs of liver structure in TAA treated rats are shownin Fig. 2, in which the formation of fibrotic septa (Fig. 2A)and the aspect of a hepatocyte (Fig. 2B) are evident. An in-filtration of neutrophils (Fig. 2C) and mastocytes, which isnormal in liver cirrhosis, is also visible. In the TAA-group,

all the enzyme activities used to test liver damage (ASAT,ALAT, AP, γ -GT) were significantly higher than in the con-trol group (Table 2). The greatest differences were detectedin AP and γ -GT activity: in the TAA-group AP activity was241% higher and γ -GT activity was 118% higher. The rGST-7-7 levels were determined by SDS-PAGE and immunoblotanalysis (Fig. 3). One immunoreactive 23 kDa polypeptide,coinciding with the monomeric form of the protein, was de-tected. Samples from TAA-treated rats showed a significantrise in the level of this polypeptide (Fig. 3). All these datademonstrate that under our experimental conditions the oralintake of TAA for 97 days induced chronic liver injury.

Kinetic behaviour of liver SerDH

The kinetic behaviour of rat-liver serine dehydratase was ex-amined in cytosolic supernatants by determining its activityat different serine concentrations. A typical hyperbolic curveappeared both in control and in TAA-group samples. In bothcases the values of the Hill coefficient showed no evidenceof sigmoidicity (Table 3). Vmax and specific activity at satu-rated substrate concentration was about 60% lower in treatedrats than in the controls (Table 3). Similarly, at all substrateconcentrations assayed the specific activity of SerDH in the

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Table 1. Weight, growth and intake values for rats exposed to chronictreatment with TAA

Control TAA

Body weights (g)Initial 372.00 ± 4.30 375.00 ± 5.40

Final 521.30 ± 16.70 419.00 ± 13.8∗

Liver weights (g)Initial 14.00 ± 0.25 14.10 ± 0.30

Final 15.58 ± 0.78 15.07 ± 0.61

Daily weight gain (percentage per day)a

Whole body 0.41 ± 0.02 0.12 ± 0.01∗

Liver 0.12 ± 0.01 0.07 ± 0.01∗

Liver somatic index (%)b 2.99 ± 0.15 3.60 ± 0.16∗

Intakec (per day per rat)Diet (g) 28.61 ± 0.76 22.52 ± 0.94∗

Water (ml) 45.86 ± 1.52 21.41 ± 1.00∗

TAA (mg) 0 6.42 ± 0.30∗

TAA (mg per day 0 15.32 ± 0.72∗per kg body weight)

Significant differences between control and TAA-treated rats are shownas ∗ p < 0.05.aThe daily weight gain of whole body and liver was calculated as [(finalweight – initial weight)/initial weight] × (100/97) and is expressed aspercentage per day.bLiver somatic index was calculated as 100 × liver weight/body weight).cDiet and water intake are expressed as g of diet or ml of water ingestedper rat per day throughout the experimental period. Data are means ±S.E.M. of at least seven values.

Table 2. Activity level of liver-injury marker enzymes in rats exposed tochronic treatment with TAA

Control TAA

ASAT 3.42 ± 0.1 5.04 ± 0.31∗

ALAT 4.01 ± 0.03 5.23 ± 0.31∗

AP 0.97 ± 0.05 3.31 ± 0.13∗

γ -GT 0.11 ± 0.18 0.24 ± 0.18∗

Results are expressed as milliunits per milligram of protein. Data are means± S.E.M. of seven values. Significant differences between controls and theTAA-treated rats are shown as ∗p < 0.05.

TAA group was significantly lower than in controls. Thus,there was no significant difference in the Km values of eitherexperimental group (Table 3). Catalytic efficiency (Vmax/Km)and total activity in the livers of the TAA-group were 66.6%and 74% lower than in the controls (Table 3).

SerDH protein levels

The level of liver SerDH protein in both experimental groupswas determined by immunoblot assays using a rabbit anti-rat

Table 3. Kinetic behaviour of liver-serine dehydratase in rats exposed tochronic treatment with TAA

Control TAA

Specific activity (mU · mg protein−1)a 29.21 ± 1.17 9.98 ± 0.9∗

Km (mM)b 177.61 ± 6.65 182.00 ± 9.10

Vmax (nmol · min−1)c 63.36 ± 2.54 26.32 ± 2.37∗

Hill coefficient 1.12 ± 0.12 0.78 ± 0.15

Activity ratio (Vss/Vmax)d 0.32 ± 0.01 0.30 ± 0.02

Catalytic efficiency (Vmax/Km)e 164.46 ± 6.37 54.83 ± 3.29∗

Total activity (Units)f 46.53 ± 2.10 12.02 ± 0.66∗

Significant differences between controls and TAA-treated rats are shown as∗ p < 0.05.aSpecific activity was determined at 0.5 M of L-serine.bKm = Michaelis constant for L-serine.cVmax = maximum velocity.dThe activity ratio (Vss/Vmax) was determined as the ratio between the spe-cific activity obtained at 0.1 M of L-serine and Vmax.eCatalytic efficiency (Vmax/Km) is expressed as milliunits (mU) of enzymemg of protein · mM−1.fTotal activity is expressed as enzyme units (U) in the whole organ. Resultsare expressed as means ± S.E.M. of seven values.

SerDH antiserum ( Fig. 4). One immunoreactive polypeptideof 34.1 kDa was detected, corresponding to the monomericform of the enzyme. In TAA-treated rats the level of thisimmunoreactive polypeptide was 37.5% below the level ofthe control rats (Fig. 4).

Immunohistochemistry

The specific SerDH immunoreaction product was only de-tected in the hepatocytes located at the periportal level of thehepatic acinus (Fig. 5). Immunoreactivity was not detectedin perivenous areas or in the bile ducts. A similar distributionwas observed in TAA-treated rats, although these samplesshowed a lower amount of immunoreactiviy when comparedto control (Fig. 5). In each case, the amount of immunore-action product was measured by three independent qualifiedobservers examining the specimens in a blind assay, and af-terwards an image-analysis program was used to compare thetwo types of samples. The lower amount of immunoreactivityfound in TAA rat livers indicates a lower level of SerDH inthese samples.

Estimation of SerDH-mRNA level by RT-PCR

Figure 6 shows the results of RT-PCR of total liver RNA sam-ples for both control and TAA-treated rats, made using cDNAprimers for SerDH and β-actin. The amplification level of theSerDH sequence observed in TAA samples was significantly

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lower than in the controls. The intensity of the band obtainedin samples of TAA-treated rats was 75% lower than that of thecontrol rats (Fig. 6A, Table 4). Blot analysis of the PCR prod-ucts gave the similar results (Fig. 6B). These results indicatethat the level of SerDH mRNA in the livers of TAA-treatedrats was significantly lower than in untreated rats.

Discussion

The chronic administration of thioacetamide to rats has beenused as an experimental model to reproduce the stages of liverinjury leading to cirrhosis that are likely to be induced in hu-mans by the consumption of alcohol or other toxic agents[35, 36]. Zimmermann et al. [35] have demonstrated that theintake of 300 mg of TAA/l of water by rats for two months

Fig. 2. Photomicrographs of liver sections from rats receiving TAA (300 mg/l) from 97 days. Panel (A). The general aspect of liver under optical microscopy(Bar = 50 µm). Panel (B). The appearance under electronic microscopy of a hepatocyte of TAA-rat liver (Bar = 10 µm). Panel (C). The appearance underelectronic microscopy of a neutrophyl found in the liver of TAA rats (Bar = 10 µm).

Table 4. Relative quantification of hepatic SerDH protein and mRNA levelsin control and TAA-treated rats by densitometry of Western-blotting andRT-PCR products

SerDH protein SerDH mRNA

Control TAA Control TAA

Arbitrary units 100.00 ± 0.00 37.53 ± 4.30* 100.00 ± 0.00 24.99 ± 1.25∗

In the quantification of RT-PCR products, the area of SerDH-RT-PCR productwas normalized with the area of β-actin. Results are expressed as means±S.E.M. of five values. Significant differences between controls and TAA-treated rats are shown as ∗ p < 0.05.

provokes liver cirrhosis but not death. The administration ofTAA for a period of four months causes micronodular cir-rhosis in which fibrotic septa and severe alterations of hep-atocytes [37]. We have reproduced these conditions and the

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Fig. 3. Hepatic levels of rGST-7-7 in rats exposed to chronic treatment withthioacetamide. Immunoblot of liver rGST-7-7 was made using a rabbit poly-clonal antiserum versus rat GST-7-7 (50 µg protein per lane), which revealedthe existence of one immunoreactive polypeptide of 23.0 kDa (Panel B),corresponding to the monomeric form of the protein. The quantification ofrGST-7-7 levels by densitometric analysis is shown in Panel A. The resultsare means ± S.E.M. of five independent experiments, expressed as arbi-trary units of integrated optic density compared to control rats. Significantdifferences between controls and TAA-treated rats are shown as ∗ p < 0.05.

results from liver-injury markers and other tests demonstratethat the treatment significantly damaged to the liver and ifcontinued, would have led to cirrhosis.

The kinetic behaviour of liver SerDH under our experimen-tal conditions agrees with those described by other authors[4, 26]. SerDH shows a hyperbolic kinetic with a high Km

value for L-serine [4]. The Km values found under our ex-perimental conditions are very similar to those described bySnell [4]. Among the regulatory enzymes involved in serinemetabolism in adult rat liver, SerDH shows the highest ac-tivity [4] and, although it has a high Km value for L-serine, ithas also been shown to be controlled at transcriptional levelunder several gluconeogenic conditions [9], indicating that itmight be important in the regulation of gluconeogenesis fromserine [4]. Recently, SerDH has been shown to be the mostactive of various enzymes involved in the serine catabolismin rats [19].

Here, we have shown by kinetic and immunological anal-yses that both SerDH protein levels and its activity decreasesignificantly in the livers of rats damaged by the oral intake ofTAA. These results lead us to conclude that the lower SerDH

Fig. 4. Immunoblot of rat-liver SerDH in rats chronically exposed to thioac-etamide. Proteins from liver cytosolic supernatants were electrophoresed (30µg per lane) and then blotted to polyvinylidene difluoride (PVDF). The 34.1-kDa polypeptide, corresponding to a monomeric form of the enzyme, wasdetected with specific polyclonal anti-SerDH rabbit serum (Panel A). Thequantification of SerDH levels by densitometric analysis is shown in Panel B.The results are means ± S.E.M. of five independent experiments, expressedas arbitrary units of integrated optic density compared to control rats. Sig-nificant differences between controls and TAA-treated rats are shown as∗ p < 0.05.

activity is due to the presence of smaller quantities of the en-zyme itself. This implies that when the liver undergoes injury,in this case induced by thioacetamide, the catabolism of serinevia SerDH decreases and probably reducing the use of ser-ine for gluconeogenesis. This situation might be explained interms of the pattern of carbohydrate metabolism found during

41

Fig. 5. Immunohistochemical analysis of liver SerDH in rats chronicallyexposed to thioacetamide. Light microphotographs of SerDH immunoreac-tivity in 30-µm-thick sections from (A) control and (B) cirrhotic rat liver.(CV = central vein; PT = portal triade; Bar = 170 µm). The arrow pointsto the location of the nickel deposits. The micrographs shown in this figureare representative of five independent determinations.

liver injury and cirrhosis, in which carbohydrate metabolismis generally altered and hyperglycemia, hyperinsulinemia andmuscle insulin resistance have all been reported [38]. Theliver acts as a buffer organ in carbohydrate metabolism, tend-ing to maintain blood-glucose levels stable under differentphysiological conditions; thus, when blood-glucose level ishigh, it is reasonable to assume that liver gluconeogenesis ingeneral, and especially via serine, will be reduced. The be-haviour we have observed in response to treatment with TAAis the same as that shown by this system in situations wheresufficient quantities of glucose are available for the rest ofthe body’s tissues and the liver is not required to synthesizefurther supplies from amino acids. Thus, the inhibition ofboth SerDH-protein levels and its activity in rats treated withTAA may be due to the single direct effect of high levels ofglucose and/or insulin caused by chronic liver disease [38].It is noteworthy that the administration of insulin to rats withexperimentally induced diabetes has partially reversed hep-atic SerDH levels [8]. Nevertheless, it is unclear whetherthe effect of insulin is due to the direct action of this hor-mone or to the indirect action of glucose, because evidencealso exists to show that glucose suppresses the induction ofenzymes produced by a high-amino-acid diet or glucagonintake [8].

Fig. 6. RT-PCR amplification of liver SerDH mRNA in rats chronicallyexposed to thioacetamide. Panel (A). Electrophoresis on 4% agarose of co-amplification of SerDH and β-actin mRNA. Reverse transcription was per-formed from 2 µg of the total RNA from rat-liver using oligo (dT) primers.The cDNAs were amplified by PCR with specific primers for SerDH and β-actin. β-actin primers were added after 10 amplification cycles. SerDH andβ-actin RT-PCR products are indicated by an arrow over the gel lane. In lane1, 4 µl of markers were added. This standard was composed of 15 DNA frag-ments of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,1400 and 1500 bp. (Panel B). Southern analysis of RT-PCR products. The re-sults of electrophoresis were blotted onto nylon membrane (Hybond-N∗) bysaline capillary blotting. The amplification product of SerDH was detectedby incubating the membrane with a specific cDNA probe anti-SerDH gene.

Another consequence of lower liver SerDH in TAA-liverinjury should be that serum levels of serine rise, and in fact asignificant increase in blood-serine levels in TAA-treated ratshas been reported elsewhere [37]. In our study, the high levelof serine in blood serum could be attributed to the lower levelsof liver SerDH found, and thus the levels of this enzyme maywell control the levels of serine in the blood. This is reflectedin a similar way in the behaviour of tyrosine and tyrosineaminotransferase, another amino-acid catabolic regulatoryenzyme, in which the activity of tyrosine aminotransferasealso decreases in rats treated with TAA [39] concomitantlywith a significant increase in the serum levels of tyrosine [37].High levels of serine have been shown to have an importantrole in the development of nervous disorders and neurologi-cal abnormalities [40], and serine has an important functionin brain development and function [13]. The low levels ofSerDH found in our work in response to liver injury couldexplain the high level of serine previously reported [36] andcould be related to the nervous disorders that arise during thelast stages of cirrhosis.

42

Our immunohistochemical results show that SerDH is lo-cated mainly in the periportal area of the hepatic acinus (bothin control and TAA-treated rats). These results agree withthose published elsewhere by Ogawa and Kawamata [2],who first described the location of SerDH and its mRNAin the periportal zone of the hepatic acinus in normal ani-mals. It is important to bear in mind that this is the mor-phofunctional zone of the liver parenchyma, where the keyenzymes for gluconeogenesis and the catabolism of aminoacids, among others, are located [41]. This location is inaccordance with the gluconeogenic role of SerDH in ratliver.

Above, we have mentioned that TAA-induced liver injurydid not bring about any changes in the location of hepaticSerDH. Ogawa and Kawamata [2] made similar findingsconcerning the distribution of SerDH mRNA in the liversof rats which were suffering from streptozotocin-induceddiabetes mellitus, which had been fed glucagon and dex-amethasone, or which had undergone laparotomy, all ofwhich lead to a significant increase in SerDH mRNA levels.Our results indicate that the level of the immunoreactionproduct to SerDH decreases in the liver of TAA-treated ratscompared to control animals.

To determine the level of SerDH mRNA we have used RT-PCR and our results have indicated that SerDH mRNA levelscan be satisfactorily estimated by this technique. In the liversof TAA-treated rats we found a lower level of activity andspecific SerDH protein concomitant with a lower quantity ofspecific SerDH mRNA, which can be ascribed to a decreasein the expression of the SerDH gene in the liver due to in-jury induced by the chronic intake of TAA. This reflects thebehaviour observed in this system under other experimentalconditions in which SerDH protein levels and activity valueschange as a consequence of transcriptional regulation mech-anisms [9, 42]. Thus, according to Ogawa et al. [9], hepaticSerDH activity and its mRNA levels show a concomitantincrease with the protein content of the diet, and in a sim-ilar way these values decline in response to a protein-freediet. Kanamoto et al. [8] have shown that the simultaneousadministration of glucagon and dexamethasone in adrenalec-tomized rats prompts a parallel increase in the activity andrate of SerDH and mRNA synthesis in the liver. The sameauthors found in other experiments [42] that starvation anddiabetes induces hepatic SerDH activity associated with in-creased SerDH protein and mRNA quantities, and that whenglucose or insulin were administrated to starved or diabeticrats this tendency was reversed and there was a marked re-duction in SerDH mRNA. These authors concluded that theregulation of SerDH in rat liver in vivo is controlled predomi-nantly at the transcriptional level [42]. Our results agree withthis conclusion in that we found that the administration ofTAA caused parallel changes in SerDH activity and proteinand mRNA levels, thus offering further evidence to support

the hypothesis that the SerDH synthesis is regulated at thetranscriptional level.

It also bears noting, concerning our findings on chronicliver injury, that SerDH activity decreases or even disappearsin conjunction with several types of liver cancer, in particularwith transplantable Morris hepatomas, Novikoff hepatomaand with a primary hepatoma arising during azodye-inducedcarcinogenesis [4]. In all these cases SerDH is practicallyabsent from the tissue. Thus the decrease in SerDH geneexpression that we have observed could well be an early stagein this development, which if continued would result in evenfurther decline in SerDH levels.

Acknowledgements

This work was supported by a research grant (CVI-157) fromthe Plan Andaluz de Investigacion (PAI), Junta de Andalucıa,Spain. The authors extend their thanks to Dr. J. Trout for hisrevision and comments on the text. The authors also thankElectronic Microscopy Unit of the Scientific InstrumentationCentre of the University of Granada for their technical assis-tance in sample preparation for electron microscopy.

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