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.
318
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.
319
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
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.
318
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.
319
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
