Wy-14,643 stimulates hepatic protein kinase C activity

Toxicology Letters, 62 (1992) 3 17-322 0

317

1992 Elsevier Science Publishers B.V. All rights reserved 0378-4274/92/$05.00

TOXLET 02784

Wy- 14,643 stimulates hepatic protein kinase C activity

Heidi K. Bojesa,b, Barbara J. Keller” and Ronald G. Thurmana “Laboratory of Hepatobiology and Toxicology, Department of Pharmacology and bCurriculum in Toxicology, The University of North Carolina at Chapel Hill, Chapel Hill. NC (USA)

(Received 15 April 1992) (Accepted 12 June 1992) Key words: Protein kinase C; Hepatic tumor; Wy-14,643; Rat; Peroxisome proliferators

SUMMARY The mechanism by which hypolipidemic drugs and industrial plasticizers cause hepatic tumors in rodents remains unknown. Protein kinase C is elevated during hepatic cell turnover, and sustained cellular replication has been shown to correlate with an increase in hepatic tumors. Therefore, the effect of [4-chloro-6(2,3-xylidino)-2-pyrimidinylthiolacetic acid (WY-14,643) on protein kinase C activity was examined. Female Sprague-Dawley rats were given 100 mg/kg Wy-14,643 in olive oil (ig.), while control rats received equal volumes of oil vehicle. After 24 h, the activity of protein kinase C was estimated in isolated hepatic fractions by measuring the binding of ‘H-phorbal-12,13-dibutyrate, a specific ligand for protein kinase C. Administration of Wy-14,643 significantly increased protein kinase C activity nearly 2-fold in microsomal fractions. Thus, it is possible that Wy-14,643 increases cell proliferation and causes tumors by mechanisms involving protein kinase C.

INTRODUCTION

Concern about the long-term exposure of man to peroxisome proliferators and their consequent effect on human health has heightened throughout the years. This concern is based on studies with rodents which show that compounds which induce hepatic peroxisomes also cause hepatocellular carcinoma [l]; however, the exact mechanism by which these agents cause tumors remains unknown. A prevalent theory states that peroxisome proliferators induce H202 production via

Correspondence to: Dr. Ronald G. Thurman, Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, CB No. 7365, Faculty Laboratory Office Building, The University of North Carolina at Chapel Hill, Chapel Hill, NC 2759997365, USA.

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P-oxidation [I]. Excess H,02 is then hypothesized to leak out of the peroxisome and generate reactive oxygen species which modify macromolecules such as DNA and thereby cause neoplasia. In support of this theory, lipofuscin accumulated after chronic exposure to phthalates [2] and 8-hydroxyguanosine was detected after treatment with ciprofibrate [3]. However, basal values were high in the latter study and other investigators failed to demonstrate an increase in Ghydroxyguanosine in DNA from isolated nuclei after treatment with nafenopin [4]. Taken together, these observations indicate that the mechanisms of action of peroxisome proliferators may be more complex than simple oxidative stress. In addition to inducing peroxisomes, this class of chemicals also uncouples oxidative phosphorylation in mitochondria in both parenchymal and Kupffer cells. Keller et al. demonstrated that several peroxisome proliferators including Wy-14,643 uncoupled oxidative phosphorylation in mitochondria roughly in proportion to their potency as hepatocarcinogens [5]. An outcome of uncoupling is a decrease in ATP which may lead to an increase in intracellular free Ca2’. In fact, the addition of p-trifluoromethoxyphenylhydrazone (FCCP), a classical uncoupler, increased Ca*+ in cultured parenchymal cells [6]. Similarly, the addition of Wy-14,643 increased Ca*+ in cultured Kupffer cells [7]. Sustained cellular replication is thought to be an important component necessary for the development of tumors [8]. Chronic increases in cell proliferation decrease the time available for DNA repair prior to replication and, therefore, can enhance the rate at which DNA lesions are converted into mutations as well as elevate the likelihood of clonal expansion of initiated cells [9]. For example, Ellwein and Cohen showed that saccharin-induced cell replication correlated directly with an increased incidence of bladder tumors in rats exposed to saccharin [lo]. Also, Marsman et al. demonstrated that Wy-14,643 stimulated hepatocyte replication in rats [l I]. Eacho et al. confirmed this finding by showing that rats treated with Wy-14,643 sustained high levels of hepatocellular DNA synthesis for 30 days [ 121. Protein kinase C is elevated during enhanced cellular turnover [8]. Therefore, in an attempt to understand the mechanism of tumor formation associated with peroxisome proliferators, the present work was designed to determine whether Wy14,643, a potent peroxisome proliferator which causes liver tumors, influences hepatic protein kinase C activity. MATERIALS

Animals

AND METHODS

and treatment

Female Sprague-Dawley rats (Zivic-Miller, Raleigh, NC) weighing 150-300 g were maintained on lab chow and tap water ad libitum and housed with a 12-h nightlday cycle. Treated rats were given 100 mgikg Wy-14,643 in olive oil (i.g.), while control rats received equal volumes of the vehicle alone.

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Isolation ofmicrosomes

and cytosol

Rats were anesthetized with sodium pentobarbital(75 mg/kg i.p.). Approximately 3 g of the left lateral lobe of the liver was removed and placed in ice-cold saline. The liver was then transferred to 3 volumes of ice-cold sucrose buffer containing the following: 0.25 M sucrose, 0.025 M Tris-HCl (pH 7.4), 2.5 mM EGTA, 2 mM dithiothreitol (DTT), 2.5 M MgCl, and 0.14 mgiml phenylmethanesulphonyl huoride in a final volume of 20 ml. After homogenization, the microsomal and cytosolic liver fractions were isolated by standard procedures of differential centrifugation [ 131and stored at -80°C in 1 ml microcentrifuge tubes. Protein kinase C activity

The activity of protein kinase C was determined by measuring the binding of 3Hphorbal-12,13-dibutyrate (E3H]PBDu) to the active form of the protein kinase C molecule. The reaction mixture contained 46 mM Tris-HCl (pH 7.4), 1 mM CaCl,, 0.1 mg/ml bovine serum albumin, 0.02 M MgCl,, 2 mM DTT, 0.04 mgiml phosphatidyl serine and [3H]PBDu (15.6 mCiinmo1). Sample protein (50 ~1) was incubated with an equal volume of reaction mixture for 30 min at room temperature. Non-specific binding was determined by displacement of label by incubation with excess phorbal myristate acetate (PMA; 1 PM). The binding reaction was terminated by the addition of 1 ml ice-cold 10 mM Tris-HCl (pH 7.4) to each tube. Samples were then passed through 1.2 pm Millipore filters and rinsed twice with ice-cold Tris-HCl. Dried filters were placed in scintillation vials containing 5 ml liquid scintillation cocktail (Ecolume, INC Biomedical, Inc., Irvine, CA), and radioactivity was measured. Each sample was assayed in duplicate, and values from reaction mixtures containing PMA were subtracted from those without PMA to determine specific binding. Protein concentration for each sample was determined by the method of Lowry et al. using bovine serum albumin as the standard [141.Total protein kinase C activity was calculated by dividing total counts by the appropriate protein concentration. Materials

[4-Chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (WY-14,643) was obtained from Chemsyn Science Laboratories, Lenexa, KA. 3H-Phorbol-12,13-dibutyrate (24 Ci/mmol) was purchased from Amersham, Arlington Heights, IL. All other chemicals and reagents were of highest available purity from standard commercial sources. Statistics

Data represent means 2 SE. Results were evaluated by Student’s t-test [15]. RESULTS AND DISCUSSION

Wy-24,643 increases mi&rosomai protein kinase C activity

As shown in Figure 1, background levels of hepatic protein kinase C activity were

320

0 .s

2.5

w

control

m

Wy-14,643

** 1

cytosol

microsomal

Fig. 1. Effects of in vivo administration of Wy-14,643 on protein kinase C activity in hepatic microsomal and cytosolic fractions. Female Sprague-Dawley rats were given 100 mg/kg Wy-14,643 in olive oil (i.g.), while control rats received equal volumes of the oil vehicle. After 24 h, hepatic microsomal and cytosolic liver fractions were isolated by standard techniques of differential centrifugation [13]. Protein kinase C activity was measured and calculated as described in Materials and Methods. Data represent mean f SE (n=7). ‘P
higher in microsomes than in the cytosol. In vivo administration of Wy-14,643 (100 mg/kg) tended to decrease enzyme activity in cytosolic fractions but increased activity significantly by approx. 2-fold in microsomal fractions. As activation of protein kinase C is known to stimulate cell turnover, it follows that increasing the rate of cell replication could augment the likelihood of conversion of an initiated cell into a transformed cell [9]. For example, Reuber observed that several chlorinated hydrocarbons which induce liver tumors in mice were also potent stimulators of protein kinase C activity [ 161.Furthermore, several dissimilar tumor promoters in skin, colon and liver have been shown to stimulate protein kinase C activity directly or indirectly P71. Recently, several investigators have shown a correlation between enhanced cell turnover and increased incidence of hepatocellular carcinoma [18]. Marsman et al. demonstrated that Wy-14,643, a potent nongenotoxic carcinogen, but not di(ethylhexyl)phthalate (DEHP), a weaker counterpart, caused significant elevation of hepatocyte DNA replication in rats [l 11.Wy-14,643 also caused many more tumors than DEHP [ll]. Eacho et al. confirmed the findings on cell replication by demonstrating that higher rates of cell turnover by Wy-14,643 could be sustained for 30 days [ 121.The observations from previous studies integrated with the findings of this report suggest a central role for protein kinase C in the mechanism of peroxisome proliferator-induced hepatocarcinoma. Peroxisome proliferators are highly lipophilic and may accumulate in mitochondrial membranes of either Kupffer or parenchymal cells. Uncoupling of oxidative phosphorylation diminishes the ATP supply for ion pumps which could increase intracellular levels of Ca*‘. Indeed, Hijioka et al. demonstrated recently that Wy-14,643

321

elevated intracellular Ca*’ in Kupffer cells [7]. Kupffer cells release mitogenic stimuli which in turn may be responsible for the increased protein kinase C activity observed in this study [19]. Phthalates and lipid-lowering drugs could also influence parenchymal cell protein kinase C activity by affecting lipid metabolism. Peroxisome proliferators could aggregate in the mitochondrial membranes of parenchymal cells, uncouple oxidative phosphorylation and consequently diminish cellular energetics [20]. A decreased supply of ATP could lead to diminished rates of synthesis of acyl CoA compounds and thereby raise the level of intracellular free fatty acids. In fact, in the perfused rat liver, ethylhexanol inhibited ketone body production by over 60% [21]. As unsaturated free fatty acids are known activators of protein kinase C activity in vitro [22], it follows that they could increase protein kinase C activity in vivo. Recently, Cannon and Eacho demonstrated that various peroxisomal proliferators, including Wy-14,643, associate with fatty acid binding protein (FABP) and displace an endogenous ligand from its binding site [23]. Although the significance of displacement of fatty acids from FABP by peroxisome proliferators is not clear at this time, it is possible that compounds displace fatty acids from FABP rendering them free to stimulate protein kinase C. Despite the well-established hepatocarcinogenicity of Wy-14,643 [24], its mechanism remains to be elucidated. This report establishes clearly that Wy-14,643 can stimulate protein kinase C activity. Taken together with results from previous studies, this observation suggests that activation of protein kinase C by members of this class of chemical agents may be involved in the mechanism by which peroxisome proliferators cause tumors. ACKNOWLEDGEMENTS

This study was supported, in part, by a grant from NIEHS (ES-04325). HKB was supported by a predoctoral traineeship (National Research Service Award 5 T32 ES-07 1260) from NIEHS. REFERENCES 1 Reddy, J.K. and Lalwani, N.D. (1983) Carcinogenesis by hepatic peroxisome proliferators: Evaluation of the risk of hypolipidemic drugs and industrial plasticizers to humans. Crit. Rev. Toxicol. 12, l-58. 2 Conway, J.G., Tomaszewski, K.E., Olson, M.J., Cattley, R.C., Marsman, D.S. and Popp, J.A. (1989) Relationship of oxidative damage to the hepatocarcinogenicity of the peroxisome proliferators di(2ethylhexyl)phthalate and Wy-14,643. Carcinogenesis 10, 513-519. 3 Kasai, H., Okada, Y., Nishimura, S., Rao, M.S. and Reddy, J.K. (1989) Formation of S-hydroxydeoxyguanosine in liver DNA of rats following long-term exposure to a peroxisome proliferator. Cancer Res. 49,2603-2605. 4 Hegi, M.E., Ulrich, D., Sagelsdorff, P., Richter, C. and Lutz, W.K. (1990) No measurable increase in thymidine glycol or 8-hydroxydeoxyguanosine in liver DNA of rats treated with nafenopin or cholinedevoid low-methionine diet. Mutat. Res. 238, 3255329. 5 Keller, B.J. and Thurman, R.G. (1990) Non-genotoxic carcinogens are uncouplers of mitochondrial

322

oxidative

phosphorylation.

6 Jewell, S.A., Bellomo, cytes during

The Pharmacologist

G., Hjordis,

drug metabolism

32, 333.

T., Orrenius,

S. and Smith, M.T. (1982) Bleb formation

caused by disturbance

in thiol and calcium

in hepato-

ion homeostasis.

Science 217,

1257-1259. 7 Hijioka,

T., Keller, B.J. and Thurman,

lular free calcium 8 Cohen,

in cultured

R.G. (1992) Wy-14,643

Kupffer

cells. Toxicol.

S.M. and Ellwein, L.B. (1990) Cell proliferation

9 Butterworth, ly induced

B.E., Loury, D.J., Smith-Oliver, hyperplasia

in the carcinogenic

(ed.), Recent Advances

in Phthalate

in carcinogenesis.

T. and Cattley,

activity

but not 2-ethylhexanol

increases

intracel-

Lett. 59, 239-244.

of the hypolipidemic

Esters Research,

Science 249, 1007-101 I.

R.C. (1987) The potential carcinogens.

role of chemicalIn: B.F. Schneider

Princeton

Scientific

Publishing

Co., Inc., Prince-

risks of saccharin

revisited.

Rev. Toxicol.

ton, NJ, pp. 129-148. 10 Ellwein, L.B. and Cohen, 11 Marsman,

SM. (1990) Health

D.S., Cattley,

proliferation

R.C., Conway,

and replicative

J.G. and Popp, J.A. (1988) Relationship

DNA synthesis

tors di(2-ethylhexyl)phthalate

to the hepatocarcinogenicity

Carcinogenesis 13 Remmer,

O.H.,

Folin phenol 15 Steel, R.G.D. McGraw-Hill,

C.A. (1991) Hepatocellular DNA synthesis in rats given of Wy-14,643 to clofibric acid, nafenopin and LY171883.

J., Estabrook,

R.W.,

Sasame,

0. (1966) Drug interaction

Rosebrough, reagent.

N.J., Farr,

J. Biol. Chem.

H., Gillette,

with hepatic

A.L. and Randall,

W.M.,

and the National

McConnell,

and Procedures

Cancer

Institute.

Mol. Pharma-

measurement

in Statistics:

A Biometric

and other lesions of the liver in mice ingesting

E.E., Huff, J.E., Haseman,

testing of phthalate

S., Cooper,

cytochrome.

R.J. (1951) Protein

17 Roghani, M., Da Silva, C. and Castagna, M. (1987) Tumor promotor kinase C activator. Biochem. Biophys. Res. Commun. 142,738-744. Carcinogenicity

J., Narashimhulu,

microsomal

with the

193, 265-275.

and Torrie, J.H. (1980) Principles New York.

16 Reuber, M.D. (1978) Carcinomas cides. Clin. Toxicol. 13, 231-256.

18 Kluwe,

prolifera-

acid (WY-14,643)

12, 1557-1561.

H., Schenkman,

D.Y. and Rosenthal, col. 2, 187-190. 14 Lowry,

of the peroxisome

and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic

in rats. Cancer Res. 48,6739-6744. 12 Eacho, PI., Lanier, T.L. and Brodhecker, peroxisome proliferating agents: comparison

20, 31 l-326.

of hepatic peroxisome

J.K.,Douglas,

esters and related compounds Environ.

Health

Perspect.

chloroform

organochlorine is a potent

J.F. and Hartwell,

by the National

Approach. pestiprotein

W.V. (1982)

Toxicology

Program

45, 129-133.

19 Buckley, A.R., Crowe, P.D. and Russell, D.H. (1988) Rapid activation of protein kinase C in isolated rat liver nuclei by prolactin, a known hepatic mitogen. Proc. Natl. Acad. Sci. USA 85,8649-8653. 20 Keller, B.J., Liang, D. and Thurman, R.G. (1991) 2-Ethylhexanol uncouples oxidative phosphorylation in rat liver mitochondria. Toxicol. Lett. 57, 113-120. 21 Keller, B.J., Yamanaka, H., Liang, D., Kauffman, F.C. and Thurman, R.G. (1990) Oxygen-dependent hepatotoxicity due to ethylhexanol col. Exp. Ther. 252, 1355-l 360. 22 McPhail,

L.C., Clayton,

in the perfused

C.C. and Snyderman,

rat liver: Mitochondria

R. (1984) A potential

as a site of action. J. Pharma-

second messenger

role for unsatu-

rated fatty acids: activation of Ca2’-dependent protein kinase. Science 224, 6222624. 23 Cannon, J.R. and Eacho, P.I. (1991) Interaction of LY 171883 and other peroxisome proliferators with fatty-acid-binding protein isolated from rat liver. Biochem. J. 280, 387-391. 24 Reddy, J.K. and Qureshi, S.A. (1979) Tumorigenicity of the hypolipidemic peroxisome proliferator ethyl-a-p-chlorophenoxyisobutyrate

(clofibrate)

in rats. Br. J. Cancer

40,476482.