Cardnogenesis vol.14 no.5 pp.941-946, 1993

Microsomal sphingomyelin accumulation in thioacetamide-injuredregenerating rat liver: involvement of sphingomyelin synthaseactivity

Marfa-Jesiis MinS-Obradors, Jesus Osada1,Hortensia Aylagas, Inmaculada Sdnchez-Vegazo2 andEvangelina Palacios-Alaiz3

Instituto de Bioqulmica (Centro mixto UCM-CSIQ, Facultad de Farmacia,Universidad Complutense, Ciudad Universharia, E-28040, Madrid,'Departamento de Bioqulmica y Biologla Molecular y Celular, Facultad deVeterinaria, Miguel Servet, 177, E-50013 Zaragoza and 2Departamento deAnatonna Patologica, Hospital Puerta de Hierro, San Martin de Porres, 4,E-28035 Madrid, Spain

^ o whom correspondence should be addressed

The purpose of this work was to determine whetheralterations in the lipid composition of rat liver microsomalmembranes existed during thioacetamide-induced injury priorto the development of hepatic cancer and biochemicalmechanisms involved. Rats were injected intraperitoneallywith (50 mg/kg body wt per day) thioacetamide or diluentfor 8 days. Liver homogenates and microsomal membranesfrom liver homogenates were obtained. Incorporation of[^PJorthophosphate into whole liver lipids and hepaticmicrosomal lipids was evaluated 75 min after isotopeadministration. These determinations were made after twoseparate periods of treatment (3 and 8 days). Activity ofsphingomyelin synthase was assayed in rat liver homogenatesas well as in the purified microsomal fractions. Resultsdemonstrated a maintenance of liver and hepatic microsomalcontents of phosphatidylcholine during thioacetamide-inducedinjury even when the biosynthesis of this glycerophospholipidin both liver and their microsomal fractions appeareddecreased. Also observed was a considerable increase ofmkrosomal sphingomyelin, as well as an increased hepaticbiosynthesis of sphingomyelin caused by thioacetamide treat-ment. The microsomal sphingomyelin/phosphatidylcholineradioactivity ratio significantly increased. Sphingomyelinsynthase activity in liver homogenate appeared stimulated.In conclusion, our data are consistent with a thioacetamide-induced increase in microsomal sphingomyelin by a stimula-tion of sphingomyelin synthase. Based on this and previousstudies, accumulation of sphingomyelin in the microsomalpurified fraction is associated with the number of thioaceta-mide doses and is an early event clearly detected prior totumoral characteristics of hepatocytes.

IntroductionThioacetamide (TAA*) is a toxic agent that undergoes metabolicactivation within the liver to become a more active metabolite(1). Using TAA as a model of experimental hepatotoxicity, itis possible to demonstrate that necrosis, regenerating liver andcirrhosis are preneoplastic stages of cholangiocarcinoma orcarcinoma (2—5). This weak carcinogen reproduces the abovepatterns of hepatic injury depending on dosage and period oftreatment (2-5) .

•Abbreviations: TAA, thioacetamide; ER, endoplasmic reticulum; SPM,sphingomyelin; PC, phosphatidylcholine.

Considerable attention has been focused on the membrane lipidcomposition of microsomes in liver injury and its regeneration,in several hepatomas and in 3,4-benzopyrene-induced hepatoma(6—13). Special emphasis has been put on the fatty acidcomposition of phospholipids in tumors (14). Changes have beenreported prior to the development of colonic cancer in the lipidcomposition of brush border membranes of animals treated with1,2-dimethylhydrazine (15,16). We decided to study microsomalmembranes because many of the enzymes involved inphospholipid metabolism are located in the endoplasmic reticulum(ER) from which microsomes are essentially derived, and manyxenobiotics are further metabolized in this compartment (17).These theoretical considerations led us to suppose that microsomalmembranes could be a target in thioacetamide intoxication andthat some of the changes observed with omer carcinogens (12,18-20) could also be present in the regenerating liver followingTAA-induced injury. This early stage, prior to the developmentof hepatoma (5, 21,22), was induced following 8 days of TAAtreatment and was characterized by an absence of inflammatorycells. A study of microsomal lipid composition was made. Sinceresults from these experiments demonstrated an accumulation ofsphingomyelin (SPM) in the microsomal fraction, we measuredthe specific radioactivity of phosphatidylcholine (PC) and SPMfrom liver and its microsomal fractions after 75 min pulse of[•^PJorthophosphate, as well as sphingomyelin synthase activityin liver homogenates and in microsomal fractions from liverhomogenates.

Materials and methodsInbred male Wistar rats weighing 200-250 g, aged 2 months, were injected i.p.with a daily dose of 0.15 M NaCl solution or TAA at a dose of 50 mg/kg bodywt for 8 days. Animals had free access to water and food. Control and TAA-treated animals were killed after fasting for 18 h. For each experiment, six animalswere used. European Community procedures for care and use of laboratory animalsin research were followed. Livers were perfused with cold 0.15 M NaCl solu-tion through porta venae. Subcellular fractions were obtained as previouslypublished (23,24), and the method described by Coleman a al. (25) was appliedto obtain the plasma membrane fractions from the 750 g pellet.

A known aliquot was removed for determination of proteins, and assessmentwas made of the purity of microsomal fractions. The remainder was used forlipid composition determinations. Protein was measured by the method of Lowryet aL (26) using bovine serum albumine as standard. The purity of microsomalpreparations was assessed by the marker enzymes: NADPH: cytochrome P450reductase and succinic dehydrogenase which were measured by the Parkes andThompson method (27); acid phosphatase as described by Fishman and Lemer(28); 5'-nucleotidase as described by Cammer a aL (29). Alkaline phosphataseand pbosphodiesterase as tnHK-gt<vl by American Association for Clinical Chemistry(30) and by Razzell (31) respectively. Ghicagon srimnlatnd adenylate cyclase wasassayed according to Schultz and Jakobs (32). Electron microscopy was doneaccording to Tjkmg and Debuch (33).

Lipid composition studiesLipids were extracted from the microsomal membranes by the method of Folcha aL (34). Phospholipids were separated by two-dimensional thin layerchromatography on silica gel G-60 plates by the modified Rouser method (35).Lipid spots were visualised with iodine vapours and scraped into tubes forphosphorus analysis following the method of Rouser a aL (36). Total cholesterolwas measured by the method of Huang et al. (37).

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Histological studies

Hepatic fragments from rats of each of the two experimental groups wereimmediately fixed in 10% formaldehyde. Fixed specimens were then embeddedin paraffin and stained with hematoxylin and eosin (H & E) for light microscopicexamination.

Radioactive studies

In order to evaluate die incorporation of ^ P into hepatic and microsomalphospholipids, each rat received an i.p. injection of 7.4 or 15 MBq of[^Plorthophosphate 75 min before killing (38). Livers were removed andimmediately processed. Microsomal membranes and their lipids were obtainedas described above. Hepatic lipids were extracted with chlorofonn-metnanol-water(1:2:0.4, v/v) (39). Lipid extracts were purified by chromatography on sephadexLH-20 (40). Separation of phospholipids was performed as described above. Thegel from the thin layer chromatography plates at the location of die lipid spotswas scraped off into scintillation vials containing 10 ml of 5% naphthalene and0.4% P.P.O. in toluene solution. Radioactivity was measured, using a Packard,Tri-Carb 2425 liquid scintillation spectrometer.

Sphingomyelin synthase activity

The phosphatidylcholine ceramide cholinephosphotransferase (sphingomyelinsynthase) was determined by toeasuring the quantity of [KC]-sphingomyelinproduced from dipalmitoyl L-a-phosphatidyl [methyl-14C] cboline substrate asdescribed by Margraff and Kanfer (41,42). Briefly, incubation mixtures contained7.2 nmol prtosphatidyl[14C]choline (0.20 /tCi), 1.2 mg defatted serum albumin,0.2 mmol/1 ceramide as a mixture of brain bovine ceramide type IV andphosphatidylethanolamine (1:1.5 w/w), 180 nmol MnCl2, 600 nmol imidazolebuffer and the enzyme source in a total volume of 60 /d. Incubation was carriedout for 3 h at 38°C.

Statistical analysis

The Shapiro—Wilk test was applied to establish the behaviour of distributions.Whenever the Shapiro-Wilk test rejected the hypodiesis of normal distribution,or when the Bartlett test for homogeneity of variances was significantly different,die overall significance of differences was calculated with the Kruskal- Wallis(one way analysis of variance) test If the differences were significant (P < 0.05),we tested the differences between the groups pair-wise using the Mann-WhitneyC/-tesL Association between parameters was studied using the Pearson correlationcoefficient and regression analysis (43).

Materials

[32P]Orthophosphate free carrier and dipalmitoyl phosphatidyl(14C]choljne werepurchased from The Radiochemical Center, Amersham. Thioacetamide, silkagelG and organic solvents were obtained from Merck (Damstadt). Cytochrome cwas supplied by Boehringer Manheim. All reagents for enzymatic analysis andphospholipid standards were supplied by Sigma Chemical Co. (St Louis, MO).

ResultsBody weightIncrease in body weight, after 8 days, did not show significantdifferences between control and TAA-treated rats.Histological analysisFigure 1 shows the liver pattern after eight thioacetamide doses.The hepatic structure was not different from that observed incontrol animals. At this time period, an absence of leukocyteswas observed. This observation confirms that inflammation wasnot responsible for the biochemical alterations noted in themembranes from TAA-treated rats, and hepatocyte regenerationwith resistance against the necrotic effect of the xenobiotic wasdeveloped.

Purity of microsomal preparationsEnrichment of this fraction was about 5-fold according to thespecific activities of NADPH cytochrome P450 reductase inhepatic microsomes and homogenates from control and TAA-treated rats (Tables I and II). Recovery of total activity of thisenzyme was similar for control and TAA-treated animals, (70and 62% as mean values respectively) (Tables I and II).Microsomal protein recovery was the same for the two groups(13%). Contamination of microsomal fractions by mitochondriawas about 2.5% for control and TAA-treated animals, based onsuccinate dehydrogenase activity (Tables I and H). The recovery

Fig. 1. liver section of a rat treated with thioacetamide for 8 days. Itsappearance resembles diat of normal liver. Animals received a dailyinjection of 50 mg/kg body wt/day of TAA intraperitoneally. (H & ElOOx).

of this mitochondrial marker enzyme in microsomal fractionsranged between 1.5% for control liver and 2.2% for the liversof TAA-treated animals with respect to the total homogenateactivity. Contamination of microsomal fractions by plasmamembrane was 1 % (control) and 2.2% (TAA), based on activityof the canalicular marker enzyme 5'-nucleotidase; 0.7% (control)and 1.2% (TAA) based on alkaline phosphatase activity; 1.1%(control) and 1.6% (TAA), based on the sinusoidal markerenzyme alkaline phosphodiesterase; 0.9% (control) and 0.2%(TAA), based on the basolateral plasma membrane markerglucagon stimulated adenylate cyclase activity (Tables I and IT).Recovery of these plasma membrane markers in microsomes withrespect to total homogenate activities (Table I) was always similarin control and TAA-treated animals: 7% 5'-nucleotidase; 12%alkaline phosphatase; 13% (control) and 11% (TAA) alkalinephosphodiesterase and 10% (control) and 12% (TAA) glucagonstimulated adenylate cyclase. Contamination of microsomes bylysosomes was estimated to be 13 ± 3% for control and treatedanimals, based on acid phosphatase acivities (Table IT). Recoveryof this lysosomal marker enzyme ranged between 9—10% withrespect to the total homogenate activity. Electron microscopyconfirmed these results except for lysosomal contamination whichwas 2 ± 0.5%.Lipid composition analysis

Lipid composition of microsomal membranes is shown in TableHI. The molar cholesterol/total phospholipid ratio did not changesignificantly after treatment with TAA. The total phospholipidcontent of microsomes from TAA-treated rats was within therange found in microsomes from control animals. The resultsin each phospholipid class are expressed as a molar percentageof the total phospholipids. The only phospholipid to show asignificant variation was SPM, the mean value of which in hepaticmicrosomes from TAA-treated rats was 52% higher than incontrols. Other phospholipids, such as PC, phosphatidyl-ethanolamine, phosphatidylserine + phosphatidylinositol andlysolecithin, did not show appreciable changes. As a result ofthe increase in the SPM molar percentage and the unchangedlevels of the major phospholipid, PC, the SPM/PC ratio waselevated.

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Table I

Marker

. Enzyme

enzyme

activities in homogenate and

HomogenateSpecific Total

subcellular fractions

MitochondriaSpecific Total

MkrosomesSpecific Total

Plasma membraneSpecific Total

NADPH cytochrome P450 reductase*-0

Control 13 ± 2 1691 ± 200TAA 13 ± 2 1491 ± 160

5 ± 1 (0.4) 77 ± 816 ± 2* (1.2) 200 ± 25*

S'-nucleotidase*-0

Control 17TAA* 54

± 2± 6

Alkaline phosphodiesterase*'c

Control 29 ± 1 0TAA* 9 ± 2

2210 ± 986022 ± 678

3757 ± 1000999 ± 90

15 ± 4 (0.9)78 ± 6 (1.4)

245 ± 29990 ± 71

Glucagon-stimulated adenylate cyclasebid

Control 1.4 ± 0.6 182 ± 18TAA* 5.5 ± 0.8 607 ± 62

Alkaline phosphatase*10

Control 3TAA* 8

Succinate dehydrogenase*10

Control 71TAA 36

± 0.5 292 ± 30± 0.5 885 ± 90

9 ± 4 (0.3) 139 ± 50ad ad

1 ± 0.1 (0.7) 16 ± 220 ± 1 1 (3.6) 138 ± 15

2.6 ± 0.3 (0.9) 41 ± 76.6 ± 0.2 (0.8) 84 ± 9

70 ± 10 (5)66 ± 10 (5)

9 ± 2 (0.5)30 ± 3 (0.5)

30 ± 6 (1)8 ± 1 (0.9)

1 rb 0.1 (0.7)5 ± 0.4 (0.9)

1191 ± 120924 ± 100

16 ± 1 (0.1)23 ± 2* (1.8)

0.2 ± 0.10.4 ± 0.1

145 ± 2 1 864 ± 143 (51) 11 ± 2420 ± 40 1320 ±323 (24) 22 ± 6

510 ± 32 2668 ± 900 (92) 32 ± 9109 ± 12 485 ± 62 (54) 8 ± 2

18 ± 2 118 ± 11 (84) 1.4 ± 0.275 ± 6 2922 ± 537 (531) 50 ± 6

2 ± 0.3 (0.7) 35 ± 4 301 ± 31 (100) 3.6 ± 0.67 ± 1 (0.8) 98 ± 10 565 ± 21 (71) 9.6 ± 1

± 12 9301 ± 9 5 0 336 ± 40 (5) 5389 ± 600± 6* 3960 ± 450* 291 ± 30 (8) 3703 ± 405*

8 ± 2 (0.1)6 ± 2 (0.1)

143 ± 15 nd90 ± 10* nd

ndnd

Specific activity is expressed as *nmol/min '/mg ' protein, bpmol/min Vmg ' protein.Total activity is expressed as cnmol/min~1/g~1 wet tissue, dpmol/min~'/g~1 wet tissue. Relative enrichment is shown in brackets and is the relation ofspecific activities between the specific fraction and homogenate.Not detected, nd.Data are means ± standard deviation of three independent experiments assayed in duplicate (six rats each preparation). TAA animals received an i.p. injectionof 50 mg/kg body wt/day of TAA for 8 days.Statistical analysis was performed using the Mann-Whitney U-test (P > 0.05, not significant; *P < 0.01 versus control).

Table n . Biochemical characteristics of microsomal fractions

Control TAA

NADPH cytochrome P450 reductasesp. act.Microsomal/homogenate relative enrichmentRecovery of total activityMitochondrial contamination(succinate dehydrogenase)Canalicular plasma membrane contamination(alkaline phosphatase)Canalicular plasma membrane contamination(5'-nucleotidase)Basolateral plasma membrane contamination(glucagon stimulated adenylate cyclase)Sinusoidal plasma membrane contamination(alkaline phosphodiesterase)Lysosomal contamination(acid phosphatase)

70 ± 105.3 ± 0.4

70 ± 6%

2.5 ± 0.3%

0.66 ± 0.2%

1 ± 0.2%

0.9 ± 0.1%

1.1 ± 0.02%

13 ± 3%

66 ± 1 05 ± 0.3

62 ± 6%

2.2 ± 0.2%

1.2 ± 0.2%

2.2 ± 0.3%

0.18 ± 0.02%

1.6 ± 0.02%

12 ± 3%

Specific activity of NADPH cytochrome P45O reductase is expressed as nmol/min '/mg ' protein.Data are means ± standard deviation of three independent experiments assayed in duplicate (six rats each preparation). TAA animals received an i.p. injectionof 50 mg/kg body wt/day of TAA for 8 days.Degree of contamination has been calculated as:

microsomal sp. act, of marker enzyme x 100sp. act. of organelle the most enriched in this marker

Radioactive studiesIncorporation of [^PJorthophosphate in whole liver PC (Figure2A) and SPM (Figure 2B) showed a decrease in the former and

an increase in the sphingohpid synthesis after treatment of animalswith TAA. Changes in isotopic dilution can be discarded becauseof lack of variations in hepatic PC and SPM contents. Hepatic

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Table HI. Lipid composition

Cholesterol/phospholipklsPhospholipids/proteinsIndividual pbospbolipids:PhosphatidylcholinePhosphatidylethanolaminePhosphatidylinositol +phosphatidylserineSphingomyelinLysophosphatidylcholine

of microsomal membranes

Control

0.31352

5923.3

941.4

(10)

± 0.06± 50

± 1 '± 1.3

± 1.3± 0.9± 0.6

TAA

0.33320

5823

96.11

± 0.03± 40

=fc 1± 0.2

± 0.9± 0.9*± 0.2

Data are means and standard deviation of three preparations assayed induplicate (six rats each preparation) unless another number is specified inbrackets. Rats were treated with TAA for 8 days.Cholesterol/phospholipids are expressed as mol/mol, phospholipids/proteinsas nmol/protein rag and individual phospholipids are expressed as molarpercentage of total phospholipids.Statistical analysis was performed using the Mann-Whitney U-test.(P > 0.05, not significant; *P < 0.01 TAA versus control).

o

g 100-

o

Per

cen

A Control• TAA

I A

t 1I3 8

:on

tro

l 1

O

^ too-coa

t • "

3 8

Days of treatment

Fig. 2. Radioactive incorporation of [32P]orthophosphate in liverphosphatidylcholine (A) and sphingomyelin (B) during TAA treatment.Results (d.p.m./jig of inorganic phosphorus) are expressed as percentage ofcontrol values and represent means ± standard deviation obtained with sixrats after 75 min administration of 7.4 MBq of [32P]orrhophosphate.Statistical analysis was performed using the Marm-Whitney U-tcsx(P > 0.05, not significant; ***P > 0.01).

PC content was 450 ± 35 /tg/g for control and 410 ± 40 forTAA- treated rats. Hepatic SPM content was 65 ± 5 /tg/g forcontrols and 62 ± 5 for TAA-treated rats. Incorporation of[•^PJorthophosphate in microsomal PC also showed a significantdecrease by the effect of TAA (Figure 3A). Incorporation of thisradiolabelled compound in microsomal SPM was decreased(Figure 3B) when expressed as specific radioactivity. When ratioof specific radioactivities of both phospholipids was analyzed,a significant increase in SPM/PC was evident by the TAAtreatment (Figure 3Q.

Sphingomyelin synthase activityThioacetamide induced an increase in homogenate activity whileno change was observed in microsomal fraction (Table IV). The

a.

fin,

io

o

PC R

AT

a.

3000-

2000-

1000-

1200-

800'

« » •

0 '

o •

TAA-3

» » *

flTAA-3

• i•TAA-3

.ATAA-8

•TAA-8

•TAA-8

T••CONTROL

t •••LJHt

CONTROL

1LLACONTROL

A

B

C

Fig. 3. Radioactive incorporation of [32P]orthophosphate in hepaticmicrosomal phosphatidylcholine (A), sphingomyelin (B) and specificradioactivity ratio (C) during TAA treatment. Results are expressed asd.p.m./jig of inorganic phosphorus and represent means ± standarddeviation obtained from six rats after 75 min administration of 15 MBq of[•^Plorthophosphate. Statistical analysis was performed using theMann-Whitney U-tesl. (**P < 0.02, ***P < 0.01).

Table IV. Specific activities of sphingomyelin synthase

Days Experimentalcondition

Homogenate Microsomes

3

8

ControlTAAControlTAA

277 ± 30625 ± 62*308 ± 30783 ± 80*

11 ± 413 ± 312 ± 314 ± 4

Data are expressed as pmol/h x mg protein and are means ± standarddeviation of three independent preparations assayed in triplicate. *P < 0.01TAA versus control using Mann-Whitney i/-test.

low activity in microsomes is consistent with a residual activityas has been reported (44,45).

DiscussionChemical hepatocarcinogenesis appears as a multistage process(5). Praet and Roels reported that administration of7 mg/day X rat of TAA for 4 months induced a hepatic cirrhosis.After 15 months, all TAA-exposed animals developed tumors,some with pulmonary metastases of a ductular carcinoma (2).We used a 50 mg/kg/day dose (10 mg TAA/day x rat) whichproduced a maximum level of necrosis after 3 days, as previouslydemonstrated according to histological and biochemical data (21).After 8 days, levels of aminotransferases were in the range ofcontrol animals (21,22) and histological analysis showed that thehepatic structure did not differ from controls (Figure 1). Absenceof inflammatory cells justified our choice of this stage to studymembrane changes during chemical carcinogenesis.

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Microsomal sphingomyelln accumulation in rat liver

Characteristics of the microsomal fraction shown in Tables Iand II support that membranes from the control and treated groupswere of similar purity and lipid changes cannot be attributed toartifacts in preparation. These fractions were enriched in ER andpractically free of Golgi and plasma membrane according to theassayed marker enzymes NADPH cytochrome P450 reductasefor the former (27,46) and 5'-nucleotidase for the two lattermembranes (29, 47). The molar percentage of SPM in thepurified microsomal fractions increased during TAA treatment(Table HI). Similar behaviour has been reported in fetal liverand in several hepatoma cell lines (7—13). Likewise, inductionof colonic neoplasia by dimethylhydrazine resulted in an increasein SPM content of membranes (15). The SPM/PC ratio foundin the present study for TAA animals was 0.1, a value that iswithin the range observed in different hepatomas (7-13). Altera-tion of this value cannot be due to the stage of regenerating liverbecause of the constancy of lipid composition of membranesreported by several authors for regenerating liver after surgicalhepatectomy (9, 48). In addition we found a more strikingalteration in SPM/PC ratio than in TAA regenerating liver aftera long term treatment with TAA, when pathological features werecompatible with liver cirrhosis (49). Based on lipid compositionstudies, Bergelson et al. (11) and Upreti et al. (10) proposed thatthe subcellular fractions from normal cells retain specificity inthe lipid composition and that differentiation accompanies aloss in specificity. It has been suggested that changes in lipidmetabolism may be among the primary events in thetransformation of normal to neoplastic cells (50). In our investiga-tion, most cells resembled normal hepatocytes; however, theirmicrosomal membranes presented characteristics of hepatomamicrosomal membranes, particularly the increased SPM/PC ratio.The exact role of these membrane lipid alterations in the malig-nant transformation process in liver remains unclear.

Among the several processes that can contribute to the observedincrease in microsomal content of SPM are an increased biosyn-thesis, an accelerated transfer between membranes or a blockeddegradation of the sphingolipid (51). To investigate the firstpossibility, we tested the incorporation of [^PJorthophosphatein PC and SPM from liver and hepatic microsomal fractions after75 min of isotope administration. Thioacetamide treatment causeda diminished incorporation of [32P]orthophosphate in whole liverPC. The incorporation of ^P into microsomal pool of PC wasalso strongly decreased by TAA. These results indicate that thebiosynthesis of PC is diminished after treatment with TAA whichis consistent with the inhibition of the two regulatory enzymesof PC biosynthesis (52), cytidylyltransferase and phospholipidmethyltransferase by the effect of TAA (24,53).

The increased ^P incorporation in liver SPM (Figure 2B)observed following TAA treatment suggests a stimulation of PC:ceramide cholinephosphotransferase activity. This mechanismcould partly account for the decreased P incorporation inhepatic PC after 8 days of TAA treatment considering that PCis the phosphocholine donor in SPM biosynthesis (54-56). Evenwhen a significant decrease in 32P incorporation in PC was notobserved afer 3 days, the mean decrease in hepatic PCincorporation (1.5 nmol ^P/min/g liver) may explain theobserved increase in radioactivity of SPM (0.5 nmol P/min/gliver) because of the turnover rate of PC which is higher thanthat of SPM. Three subcellular compartments are involved inbiosynthesis of PC and SPM. While PC is mainly synthesizedin ER (52), SPM formation is located in the cis Golgi (44, 45)and plasma membranes (57,58). The TAA increased values forthe microsomal SPM/PC ratio of specific radioactivities (Figure

3C) suggest: Firstly, a stimulation of SPM biosynthesis. Theincreased incorporation of ^P into hepatic SPM is accompaniedby an increase in the liver homogenates of SPM synthase activity(Table IV). Secondly, SPM synthase preferentially uses the newlysynthesized PC species as can be inferred from the decreased

P incorporation in microsomal SPM and the correlationbetween microsomal PC and SPM specific radioactivities(r = 0.93, P < 0.0001). These arguments indicate that theincrease in content of SPM in the microsomal membranes is partlycaused by a stimulation of hepatic SPM biosynthesis. SPMaccumulation can be rather dramatic considering that during SPMformation diacylglycerol is generated, a lipid involved in signaltransduction (59,60). SPM turnover occurs over a longer periodthan phosphatidylinositol and may be involved in longer termcell changes, such as has been observed in HL-60 celldifferentiation (61) and in response to tumour necrosis factor (62).During SPM catabolism, ceramide is generated. This compoundstimulates protein phosphatases (63). The accumulation of SPMalso causes changes in membrane biophysical properties (64, 65).The relevance of the SPM role and its special cellular distribu-tion during TAA treatment make this drug an exciting model inthe study of cellular control of phospholipid transport andmetabolism in hepatic carcinogenesis.

AcknowledgementsThe authors thank Dr Millan for her advice and statistical evaluation of data andDrs Ordovas and Cebrian for their critical reading and suggestions. Gratitudeis also expressed to Aurora Osada for her help in drawing graphics and to SandraKennelly and Erik Lundin for their help in preparing the manuscript. Thisinvestigation was supported by grant SM90-0002 awarded by the DGICYT(M.E.C.), by grant 87/1336 awarded by FISS (INSALUD) and byEUROPHARMA.

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Received on October 12, 1992; revised on February 8, 1993; accepted on February11, 1993

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