Ethanol Enhances ADP-Ribosylation of Protein in Rat Hepatocytes B. EMMANUEL AKINSHOLA, SAVITRI SHARMA, JAMES J. POTTER AND ESTEBAN MEZEY Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Decreases in hepatocyte NAD' produced by ethanol not unexpected that the well-documented increases in are only partially explained by the increased con- free NADH during ethanol metabolism (3) are not version of NAD' to NADH and NADP+.The purpose of reflected in higher levels of total NADH. Also, the this study was to determine whether a mechanism for decrease in total NAD after exposure of hepatocytes to the ethanol-induced decrease in NAD+ is its increased ethanol can only be partially explained by the increase in use in ADP-ribosylation. Exposure of hepatocytes in NADP', which is of much smaller magnitude. The culture for 2 hr to 100 mmol/L ethanol increased the incorporation of 14C-ribosefrom prelabeled NAD into increase in NADP after 5 min exposure of hepatocytes 14C-ribosylated proteins. Poly (ADP-ribose) poly- to ethanol accounted for only 15% of the decrease in merase activity was increased by exposure of isolated NAD during the same time period (1).A major pathway hepatocytes to 100 mmol/L ethanol for 10 min. In of NAD' degradation is its use in ADP-ribosylation of hepatocyte culture, increases in poly (ADP-ribose) proteins (4).The purpose of this study was to determine polymerase were not detected after exposure to 100 whether a mechanism in the ethanol-induced decrease mmol/L ethanol for 10 min or 2 hr but rather occurred in NAD' is increased use in ADP-ribosylation of at 24 hr. Ethanol exposure of hepatocytes in culture for hepatocyte proteins. 2 hr, however, decreased the K, for NAD' of poly (ADP-ribose) polymerase. Both nicotinamide and MATERIALS AND METHODS 5-aminobenzamide,which are inhibitors of poly (ADPAnimals and Materials. Male Sprague-Dawley rats were ribose) polymerase, prevented the decrease in NAD obtained from the Charles River Breeding Laboratories (Wilproduced by 2-hr exposure of hepatocytes in culture to mington, MA). Collegenase (type I), EGTA, trypan blue, 100 mmol/L ethanol. The effectof ethanol in decreasing DNA synthesis on days 3 and 4 of culture was not $-methylpyrazole, nicotinamide, 3-aminobenzamide, hyreversed by the inhibitors of poly (ADP-ribose) poly- drazine, glutaric anhydride, m-aminophenylboronic acid and merase. These results indicate that increased ADP- snake venom phosphodiesterase were purchased from the ribosylation of hepatocyte proteins is a mechanism for Sigma Chemical Co. (St. Louis, MO). NAD' (grade I) was the effect of ethanol in decreasingNAD (HEPATOLOGYobtained from Boehringer Mannheim Diagnostics (Indianapolis, IN). Bio-Gel P-300 was purchased from Bio-Rad Labora1992;15471-476.) tories (Richmond, CA). Insulin and penicillin were purchased from E.R. Squibb & Sons, Inc. (Princeton, NJ). Streptomycin Acute exposure of hepatocytes to ethanol decreases was obtained from Eli Lilly (Indianapolis, IN). Dexamethasone total NAD' . This is associated with an increase in was purchased from Lypho Med, Inc. (Melrose Park, IL). NADP', a slight decrease in NADH but no change in Dulbecco's MEM was obtained from Flow Laboratories, Inc. NADPH (1).Intracellular NAD' and NADP' exist (McLean, VA). Supplementary growth factor (SGF-7) was principally in the free forms, whereas reduced NADH purchased from B & B Research Laboratory (Fiskeville, RI). and NADPH are mostly bound, This is reflected in Vitrogen 100 purified bovine dermal collagen (type I) was from Collagen Corporation (Palo Alto, CA). 3H-NAD measured cytosolic NAD +/NADH ratios of 2.4and 250 obtained (4.8 Ci/mmol) and 14C-D-ribose(51 mCi/mmol) were obtained for the total and free forms, respectively (2). Hence it is from DuPont New England Nuclear Research Products (Wilmington, DE). [MethyLSH]thymidine (87 Ciimmol) was purchased from Amersham Corp. (Arlington Heights, IL). The m-aminophenolboronic acid glutaryl hydrazine polyacrylReceived April 8,1991; accepted October 15, 1991. amide resin, which selectively binds proteins that contain This study was supported by Grant AA 00626 from the United States Public Health Service. covalently bound adenosine phosphoribose, was synthesized Dr. B. Emmanuel Akinshola was a postdoctoral fellow on training grant T 32 starting with the polyacrylamide resin Bio-Gel P-300 and AA07467 from the National Institute on Alcohol Abuse and Alcoholism. sequentially coupling it with hydrazine, glutaric anhydride and This study was presented in part as a poster at the Annual Meeting of the m-aminophenylboronic acid as described by Rosmaschin et al. American Association for the Study of Liver Diseases in Chicago, Illinois, (5). November 1990. Hepatocytezsolation and Cell Culture. Rats weighing 200 to Address reprint requests to: Esteban Mezey, M.D., 416 Hunterian, The Johns 250 gm were anesthetized with ether. The livers were perfused Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD in situ through the portal vein with HBSS containing 0.5 21205. mmol/L EGTA and 2.5 mmol/L tricine, followed by the 31/1/34662 +

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AKINSHOLA ET AL.

TABLE 1. Effects of ethanol on the activities of NAD' glycohydrolase and poly (ADP-ribose)polymerase in isolated hepatocytes Poly (ADP-ribose) polymerase) NAD ' glycohydrolase pmolimg nmolimg proteinimin proteinimin

Treatment ~

Control Ethanol (8 mmol/L) Ethanol (100 mmoUL)

~~

26.2 t 1.2 23.7 i- 1.5 33.0 t 1.1"

81.7 76.7 128.9

* 2.6 ?

2

2.4 10.1"

Hepatocytes were incubated in the presence and absence of ethanol for 10 min. The activities of NAD ' glycohydrolase and poly (ADPribose) polymerase were determined in hepatocyte homogenates and in isolated nuclei respectively as described in "Materials and Methods." All values are expressed as means 2 S.E. of four to eight determinations. "Significant.lydifferent from control at p < 0.01.

TABLE 2. Effects of ethanol and acetaldehyde on the activity of poly (ADP-ribose)polymerase in isolated hepatocytes

Treatment

Poly (ADP-ribose) polymerase) pmolimg proteinimin

~~

Control Ethanol (100 mmol/L) Ethanol (100 mmol/L) + 4 MP (4mmoVL) Acetaldehyde (10 FrnolL) Acetaldehyde (100 pmol/L) Acetaldehyde (1,000 pmol/L)

*

84.0 13.6 152.1 t 15.6" 153.8 t 21.2" 123.2 * 15.7 109.0 t 20.4 131.8 i 18.5

Hepatocytes were incubated in the presence or absence of ethanol or acetaldehyde for 10 min; 4-methylpyrazole (MP) was added 5 rnin before the addition of ethanol. All values are expressed as means k S.E.M. of six determinations. "Significantly different from control a t p < 0.05.

complete culture medium containing 0.03% collagenase. The livers were removed, and the hepatocytes were separated from nonparenchymal cells by centrifugation at 60 g for 2 min, a process that was repeated three times, each time after washing and resuspending the cells in cool collagenase-free and serum-free medium. The medium was Dulbecco's MEM with dexamethasone (0.1 pmol/L), and insulin (250 IUIL), and the following supplemental growth factors (SG-7): transferrin, selenium, epidermal growth factor, fetuin and BSA-oleic acid and aluminum-linoleic acid complexes. It also contained penicillin (1 x 10" IUIL) and streptomycin (0.13 mmolIL). The cell suspensions were saturated with 95% 0, and 5% CO,. Only hepatocyte suspensions with a viability greater than 95% determined by trypan blue exclusion were used for experiments with isolated hepatocytes or for cell culture. The procedure of hepatocyte isolation was approved by the Animal Care and Use Committee of the Johns Hopkins University. The hepatocytes were cultured in petri culture dishes previously coated with collagen with the Dulbecco's MEM containing dexamethasone (0.1 Fmol/L) and SGF-7 as described above. The cultures were incubated at 37" C in a humidified atmosphere of 95% 0, and 5% CO,. Poly (ADP-ribose) Polymerase. This enzyme activity was determined in hepatocyte nuclei. For isolation of nuclei, freshly isolated hepatocytes or hepatocytes in culture were washed twice with PBS, placed in 2.0 mol/L sucrose, 10% glycerol, 0.025 mol/L KCl, 0.005 m o l b MgCl, and 0.05

HEPATOLOGY

Tris-HC1 (pH 7.5) and homogenized with two passes in a Dounce homogenizer (Wheaton Scientific, Millville, NJ) followed by five passes in a French press. The homogenate was layered over an equal volume of homogenizing buffer and centrifuged at 25,000 rpm in a Beckman SW-40 rotor (Beckman Instruments Corp., Palo Alto, CA) for 30 min. The nuclear pellet was then reconstituted in 0.1 mol/L Tris-HC1 buffer containing 10 mmol/L MgC1, and 1 mmol/L dithiothreitol. The enzyme activity was determined as described by Alvarez-Gonzalez (6). The reaction mixture was 0.5 ml and consisted of 0.1 moVL Tris-HC1, pH 8.0, containing 10 mmol/L MgC1, and 1 mmol/L dithiothreitol, 0.5 mmol/L "H-NAD+ (4 pCi/pmole) and 0.1 ml of the nuclear pellet corresponding to approximately 2 million cells. The reaction was incubated with agitation of 37" C and terminated at 10 min by the sequential addition of 500 pg albumin and 20% trichloroacetic acid (wtlvol). The precipitates were collected on GF/C filter papers (Whatman International, Ltd., Maidstone, UK) and then washed four times with trichloroacetic acid and twice with diethyl ether, dried and counted for radioactivity. NAD Glycohydrolase. Hepatocytes were incubated in media with and without ethanol (8and 100 mmol/L) for 10 min at 37" C. The cells were then sonicated and the activity of NAD glycohydrolase determined by measurement of nicotinamide released from NAD+ (7). The reaction mixture consisted of 0.1 molIL potassium phosphate buffer (pH 7.5), 0.5 mmol/L NAD' and 0.5 ml of the hepatocyte suspension (8 million cells/ml) in a total volume of 5.0 ml at 37" C. A blank reaction contained no cell suspension. Aliquots of the reaction mixture removed at zero time and at 5-min intervals for 20 min were treated with 5% perchloric acid. The aliquots were then neutralized with 2 N KOH containing 0.3 N MOPS, centrifuged, and the supernatant was stored at -70" c. The nicotinamide released was assayed by isocratic reverse-phase HPLC using the same Excellopack column used for determination of the pyridine dinucleotides (see below). The rate of formation of nicotinamide was determined by linear regression analysis using the method of least squares. NAD'. Hepatocytes in culture (2.5 million cells) were washed with and suspended in HBSS followed by freezing in liquid nitrogen. The frozen cell suspensions were extracted with 3 moliL perchloric acid, sonicated, neutralized with KOH containing 0.3 N MOPS and centrifuged to remove insoluble material. NAD was determined by isocratic reverse-phase HPLC using an Excellopack ODS C,, (4.6 x 150 mm) column (R.E. Gourley Co., Laurel, MD) as described previously (8).In some experiments when NAD was labeled, the resolved peak was collected with a fraction collector and counted (1). ADP-ribosylation of Hepatoeyte Proteins. NAD - in hepatocyte culture was prelabeled by the addition to the culture media of 10 pCi 14C-Dribose/culture plate (2.5 million cells) for 24 hr between days 2 and 3 of culture, after which the cells were exposed to ethanol (8 mmol/L or 100 mmol/L) or no ethanol for 2 hr before harvesting. Twenty culture plates were pooled into four sets for each condition for determinations of 14C-ribosylated proteins performed as described by Hussain, Ghani and Hunt (9). The media were removed, and the cells were rinsed with PBS and homogenized in 50 mmol/L sodium acetate (pH 2.5) containing 1mmoliL dithiothreitol and 0.05% Triton X-100. The homogenate was dialyzed to remove unbound radioactivity and precipitated with 10% perchloric acid. The precipitate was washed five times with 5% perchloric acid and dissolved in 6 mol/L guanidine HC1 in 200 mmol/L morpholine buffer (pH 8.2). The ADP-14C-ribosylatedproteins were then placed on an m-aminophenylboronic acid glutaryi hydrazine polyacrylamide column, washed with the same buffer, eluted with 6 mol/L guanidine HC1 in 200 mmol/L +

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ETHANOL AND ADP-RIBOSYLATION

sodium acetate (pH 4.0) and counted. The specific activity of the radiolabeled NAD was determined in additional culture plates as described above. Chain lengths of the ADP-ribose polymers formed were determined by releasing the poly (ADP-ribose) moieties from protein with 1.0 mol/L KOH, followed by digestion with snake venom phosphodiesterase, which releases 5‘ AMP from the distal terminus and phosphoribosyl-AMP (PR-AMP) from the interior polymer chain (10). Both products were quantitated by anion exchange chromatography on a Partisil 10 SAX column (Alltech Associates Inc, Deerfield, IL) (lo), with a linear gradient starting with 7 mmol/L potassium phosphate (pH 5.8) and eluting with 150 mmoliL potassium phosphate, 0.25 mol/L potassium chloride (pH 6.5). The resolved peaks of radioactive AMP and PR-AMP were collected with a fraction collector and counted. AMP and ADP were used as standards. The average polymer chain length was calculated from the formula (AMP + PR-AMP)/(AMP) (11). DNA Synthesis. DNA synthesis was determined from the incorporation into hepatocyte DNA of 5.0 pCi of 3H-thymidine added to the culture media 24 h r before harvesting the cells (2.5 million/plate). At the time of harvesting, the cells were washed twice with 10 mmol/L Tris-HC1 plus 1mmol/L EDTA, incubated for 1 hr at 37” C in 0.1 mmoVL Tris-HC1 (pH 8.0) containing 0.1 mol/L EDTA, 20 pglml RNase and 0.5% SDS and then incubated for 3 hr at 50” C after the addition of proteinase K at a final concentration of 100 pg/ml (12). The solution was extracted with phenol and DNA in the aqueous phase precipitated with 0.1 vol 3 mol/L sodium acetate and 2 vol ethanol. The DNA was then dissolved in 0.1 mmol/L Tris-HC1 buffer (pH 8.0) and quantitated from its absorption at 260 nm and counted. The number of hepatocytes in culture was calculated from the activity of lactate dehydrogenase in the hepatocytes as described by Jauregui, Hayner and Driscoll (131, using a standard plot of enzyme activity vs. cell counts obtained in the fresh hepatocyte preparation before plating. Lactate dehydrogenase activity was determined by the method of Plagemann, Gregory and Wrobleski (14). DNA strand breaks in isolated hepatocyte nuclei were determined by the fluorometric alkaline DNA strand unwinding assay of Birnboim and Jevcak (15). Protein determination was determined by the method of Lowry et al. (16) with BSA used as a standard. Statistical Analysis. The data were analyzed by ANOVA. The Duncan new multiple-range test was used to estimate differences between the means. The Dunnett procedure was used to compare multiple treatment means with control (17). +

*

T

*

150r

1

3

2

Days of Culture FIG. 1. Effect of ethanol on poly (ADP-ribose) polymerase in hepatocyte culture. Hepatocytes were cultured in the absence (dark bars) or presence fstrzped bars) of 100 mmoliL ethanol. Poly (ADPribose) polymerase activity was determined in the isolated nuclei after 1to 3 days in culture as described in “Materials and Methods.” Values are expressed as mean S.E.M. of eight determinations. *p < 0.05 as compared with corresponding value on the same day in the absence of ethanol.

*

0

8 Ethanol (mM)

100

FIG.2. Effect of ethanol on ADP-ribosylation of hepatocyte proteins. Hepatocytes in culture were incubated with 10 pCi of 14C-D-ribosefor 24 hr between days 2 and 3 of culture to label NAD . The hepatocytes were then exposed to ethanol or no ethanol for 2 hr before harvesting. The ‘*C-ribosylatedproteins were isolated and counted as described in “Materials and Methods.” Values are expressed as mean t S.E.M. of four determinations. *p < 0.05 vs. values without ethanol. +

RESULTS Exposure of freshly isolated hepatocytes to 100 mmol/L ethanol for 10 min resulted in increases in cytosolic NAD glycohydrolase and in nuclear poly (ADP-ribose) polymerase activities (Table 1).Ethanol (8 mmol/L) did not alter either enzyme activity. The effect of 100 mmol/L ethanol in enhancing poly (ADP-ribose) polymerase was not blocked by 4 mmol/L 4-methylpyrazole and could not be reproduced by acetaldehyde in concentrations ranging from 10 kmol/L to 1 mmol/L (Table 2). The exposure of hepatocytes to 100 mmol/L ethanol for 10 min did not cause formation of DNA strand breaks in isolated nuclei. In contrast to the findings in isolated hepatocytes, short-term exposure of hepatocytes in culture to 100 mmoVL ethanol for 10 min or 2 h r had no effect on the activity of poly (ADP-ribose) polymerase (data not +

shown). However, continued long-term exposure of the hepatocytes in culture to 100 mmol/L ethanol increased poly (ADP-ribose) polymerase activity (Fig. 1). The enzyme activity decreased between days 1and 3 in the control hepatocyte culture, whereas 100 mmol/L ethanol increased the enzyme activity on days 1, 2 and 3 of culture. The K, of NAD’ for poly (ADP-ribose) polymerase was decreased after a 2-hr exposure of the hepatocytes to 100 mmol/L ethanol from 311 pmol/L in the control to 127 pmol/L in the ethanol-treated cultures. Nicotinamide (5 mmol/L) and 3-aminobenzamide (5 mmol/L), which are inhibitors of poly (ADP-ribose) polymerase activity, prevented decreases in NAD induced by a 2-hr exposure of hepatocytes in culture to 100 mmol/L ethanol (Table 3). +

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TABLE 3. Effect of inhibitors of poly (ADP-ribose) polymerase on ethanol-induced decreases in NAD '

nmol/million hepatocytes in 2 hr. The chain length of protein-bound poly (ADP-ribose) formed increased from 1.56 -I 0.13 in the absence of ethanol to 2.24 +- 0.21 in NAD' nmol/million the presence of 100 mmol/L ethanol. This 44% increase Treatment cells in poly (ADP-ribose)chain length approximates the 52% increase in ADP-ribosylation of proteins observed with Experiment 1 Control 2.50 -t 0.16 100 mmol/L ethanol in the same experiments. 1.54 2 0.31" Ethanol (100 mmol/L) Exposure of hepatocytes on day 2 of culture to 100 2.51 ? 0.30 Nicotinamide (5 mmoVL) mmol/L ethanol for 2 h r had no effect on DNA synthesis 3.38 f 0.61 Ethanol (100 mmol/L) + nicotinamide (data not shown). By contrast, exposure of the hepato(5 mmol/L) cytes for 24 h r between day 2 and 3 of culture to 100 Experiment 2 mmol/L ethanol decreased DNA synthesis (Table 4). Control 3.05 t 0.34 DNA synthesis increased between days 1 and 4 of 1.73 k 0.15' Ethanol (100 mmoVL) hepatocyte culture (Table 5). Continuous exposure of 3.66 t 0.15 3-Aminobenzamide (5 mmoliL) the hepatocytes in culture to 100 mmol/L ethanol Ethanol (100 mmol/L) + 3-aminobenzamide 3.63 2 0.16 depressed DNA synthesis only on days 3 and 4 of culture. (5 mmoliL) The continuous presence of 5 mmol/L nicotinamide or of Hepatocytes in the third day of culture were preincubated for 10 0.1 mmol/L aminobenzamide in the media did not affect min with either nicotinamide or 3-aminobenzamide and then exposed to ethanol for 2 hr before harvesting. NAD' was determined as DNA synthesis on days 1 to 4 of culture and did not reverse the inhibitory effect of ethanol on DNA syndescribed in "Materials and Methods." All values are expressed mean ? S.E.M. of eight determinations. thesis on days 3 and 4 of culture (Table 6). Statistical significant difference from respective controls. "p i0.05. F'p < 0.01.

DISCUSSION

ADP-ribosylation is a posttranslational modification of proteins that consists of covalent attachment of ADP-ribose moieties originating from NAD to proteins (18). Either mono or poly ADP-ribosylation can occur with the formation of chain lengths of various sizes. Poly (ADP-ribose) polymerase, an enzyme located in the nucleus, catalyzes the synthesis of protein-bound homopolymers of ADP-ribose (19). Various proteins such as histones, nuclear enzymes and even poly (ADPribose) polymerase are known to undergo ADPribosylation (20). This study shows that ethanol enhances the transfer of ADP-ribose moieties from NAD' to ribosylated hepatic proteins. The preventive effects of nicotinamide and 3-aminobenzamide, which are inhibitors of poly (ADP-ribose) polymerase activity, on the ethanolinduced decrease in NAD' indicate that the transfer of ADP-ribose moieties from NAD to protein is a principal mechanism for the depressant effect of ethanol on NAD . Nicotinamide is a very effective inhibitor of poly (ADP-ribose) polymerase and a weaker inhibitor of NAD glycohydrolase (211, whereas 3-aminobenzamide is a more potent inhibitor of poly (ADP-ribose) polymerase with no effect of NAD glycohydrolase (22).The observed ethanol-induced enhancement of the incorporation of ADP-ribose moieties into hepatocyte protein of 2.6 nmol/million cells in 2 h r exceeds the fall in NAD' of 0.96 to 1.32 nmol/million cells during the same period. Continued synthesis of NAD', which has a rapid turnover with a half-life of 1hr determined in a human cell line derived from HeLa cells (23), most likely explains the lesser decrease in NAD as compared with the use of NAD' in the ribosylation of proteins. The mechanism for the ethanol-induced increase in the use of NAD in ADP-ribosylation of proteins may be caused by a decreased K , of poly (ADP-ribose) polymerase for NAD' demonstrated 2 hr after exposure of hepatocytes in culture to ethanol. The enzyme activity appears to be a rate-limiting factor for protein ribosy+

TABLE4. Effect of ethanol on DNA synthesis in hepatocyte culture DNA synthesis dpmimillion cellsi24 hr Treatment

Control Ethanol (100 mmol/L)

( X

98.4 64.6

lo-=) & 3-

4.9 8.3"

dpmlwz DNAl24 hr ( X 10-8)

17.5 ? 1.7 9.6 2 1.3"

Hepatocytes were exposed to 100 mmol/L ethanol and 5 pCi "-thymidine for 24 hr between 48 and 72 hr of culture before harvesting. All values are expressed as mean t S.E.M. of eight determinations. "Significantly different from control at p < 0.01.

+

Exposure of the hepatocytes on day 3 of culture for 2 hr to 100 mmol/L ethanol increased the formation of ADP-ribosylated proteins determined from the incorporation of 14C-ribose from prelabeled NAD' into ADP'*C-ribosylated proteins (Fig. 2). Ethanol (8mmol/L) did not increase the formation of ADP-ribosylated proteins. The specific activity of the NAD' that had been prelabeled with I4C-ribose for 24 hr was not affected by subsequent exposure of the hepatocytes to ethanol for 2 hr. The specific activities were 2.51 +- 1.36, 3.13 k 0.89 and 2.62 +- 0.91 dpminmol NAD+ ( x 1 0 - 9 after exposure to 0, 8 mmol/L and 100 mmol/L ethanol, respectively. From the specific activities, it was determined that ADP-ribosylation of proteins was increased from 6.2 t 0.39 to 9.4 t 0.42 nmollmg of protein (p > 0.05) or by a mean of 3.2 nmollmg protein in the presence of 100 mmol/L ethanol for 2 hr. Similar changes were obtained when the data were expressed per microgram of DNA. The mean increase of 3.2 nmol in ADP-moieties per milligram of hepatic protein after ethanol exposure is equivalent to an increase of 2.58

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ETHANOL AND ADP-RIBOSYLATION

lation. The mean maximal activity in the control hepatocytes in culture of 65 pmol/mg protein/min or 49 pmol/million cells/min would allow for a maximal ribosylation of 5.9 nmol/million cells/2 hr. This value is in the same range as the mean increase in endogenous ribosylation of 2.6 nmol/million hepatocytes observed after exposure to ethanol for 2 hr. Furthermore, the K,,, of NAD.' for the enzyme of 311 pmol/L and 127 pmol/L in the control and ethanol-treated cultures are in the range of hepatic NAD' concentrations of 600 t o 800 pmol/L (24). In other studies, histones (25) or spermine (26)were shown to decrease the& for NAD of purified rat liver poly (ADP-ribose) polymerase, and this was associated with increased in uztro ADP-ribosylation. Although exposure of isolated hepatocytes for 10 min to ethanol increased poly (ADP-ribose) polymerase activity, the increase in ADP-ribosylation of proteins demonstrated after 2 h r exposure of hepatocytes in culture to ethanol was not preceded or accompanied by an increase in enzyme activity. Rather, in hepatocyte culture, the increase in poly (ADP-ribose) polymerase was only demonstrated after several hours of exposure to ethanol. Differences in the metabolic state of freshly isolated as compared with cultured hepatocytes may be a reason for the dissimilar responses of poly (ADPribose) polymerase activity to ethanol. Previously, a very high ethanol concentration of 7% was found to increase poly (ADP-ribose) polymerase during a 30-min incubation (27). However, lower concentrations of ethanol were not tested. More recently, acetaldehyde injected intraperitoneally into 6-wk-old to 8-wk-old mice in doses of 133 and 400 pglkg of body weight increased the activity of poly (ADP-ribose) polymerase in hepatocytes obtained 5 h r after the treatment (28). The rapid increase in poly (ADP-ribose) polymerase after exposure of isolated hepatocytes to ethanol is not unique because ethanol was also observed to result in a rapid increase in NAD' kinase activity. However, in the latter case, the effect of ethanol was blocked by 4-methylpyrazole and could be reproduced by acetaldehyde. In other studies, ethanol was found to result in rapid increases in other enzymes, such as glucose-6phosphatase (291, 5-aminolevulinate synthase (30) and pyruvate kinase activities (31).Poly (ADP-ribose) polymerase activity increases after exposure of cells to cytotoxic agents such as y-irradiation (32) or N-methylnitroso guanidine (33). A major role of ADP-ribosylation in these instances appears to be DNA excision repair because inhibitors of poly (ADP-ribose) polymerase prevent the rejoining of DNA strand breaks induced by the cytotoxic agents (33). The increase in hormonally stimulated DNA synthesis on days 3 and 4 in control-cultured hepatocytes and the effect of ethanol in attenuating this effect confirms prior observations by other investigators (34). ADPribosylation has been postulated to be involved in the initiation and termination of DNA synthesis (35). Increases in the activity of poly (ADP-ribose) polymerase precede increases in DNA synthesis in the cell cycle (36) and in regenerating liver (371, whereas the lowest activity of poly (ADP-ribose) polymerase occurs during maximal DNA synthesis in the cell cycle (36). In our +

TABLE 5. Effect of continuous exposure of hepatocytes in culture to ethanol DNA synthesis dpm/*g DNAl24 hr

dpm/million cellsi24 hr ( X 10-3) Day

Control

1 2 3 4

1.9 f 0.3 9.6 f 0.2 34.4 t 1.3 121.1 2 10.8

(X

Ethanol

1.9 9.4 30.4 51.6

Control

t 0.5 t 1.0

0.6 t 0.1 2.0 f 0.2 7.8 0.5 25.6 f 4.8

*

t 0.8" t 3.96

10-9

Ethanol 0.5 t 1.7 t 5.4 k 11.4 ?

0.06 0.2 0.7" 4.8"

Hepatocytes in culture were exposed to 100 mmol/L ethanol continuously with changes in the culture media every 24 hr; 5 pCi of 3H-thymidine was added 24 h r before harvesting on the different days. All values are expressed as means t S.E.M. of six determinations. "Significant differences from control p < 0.05. bSignificant differences from control p < 0.0 1.

TABLE6. Effect of inhibitors of poly (ADP-ribose) polymerase on DNA synthesis Treatment ~~~

~

DNA synthesis dpm/pg DNAl24 hr ( X

lo-')

~

Experiment 1 Control Ethanol (100 mmoVL) Nicotinamide (5 mmoVL) Ethanol (100 mmol/L) + nicotinamide (5 mmol/L) Experiment 2 Control Ethanol (100 mmoUL) 3-Aminobenzamide (0.1 mmol/L) Ethanol (100 mmoliL) + 3-aminobenzamide (0.1 mmoliL)

Day 3

Day 4

7.8 ? 0.5 5.4 k 0.7" 8.8 t 2.8 5.5 t 0.36

25.6 f 4.8 11.4 f 4.5" 18.4 2 3.3 10.4 t 6.6"

19.1 t 1.4 8.7 t 0.56 16.8 k 1.4

48.6 2 7.6 31.0 ? 4.1" 36.8 2 8.1

5.9

2

1.26

21.3 f 3A6

Hepatocytes in culture were exposed continuously to ethanol 100 mmol/L and either nicotinamide or 3-aminobenzamide; 5 pCi of 3H-thymidine was added for 24 hr between 48 and 72 hr (day 3) and 72 and 96 hr (day 4) before harvesting. All values are expressed as mean of four to six determinations. "Statistically significant differences from control: p < 0.05. bStatistically significant differences from control: p < 0.01.

study, the increase in DNA synthesis in control hepatocyte cultures was associated with a steady fall in poly (ADP-ribose) polymerase activity, whereas the inhibition by ethanol of DNA synthesis was associated with an increase in the enzyme activity. Despite these associations, our study shows that poly (ADP-ribose) polymerase was not involved in the regulation of DNA synthesis because inhibitors of the enzyme did not affect either the increase in DNA synthesis in control cultures or its attenuation by ethanol. Another recent study also shows that the inhibition of poly (ADP-ribose) polymerase by 3-aminobenzamide did not affect hormonally stimulated initiation of DNA synthesis in cultured hepatocytes (38). By contrast, unscheduled DNA synthesis produced by DNA-damaging agents such as W radiation or N-methyl-N'-nitroso-N-nitrosoguanidine is further enhanced by inhibitors of poly (ADP-ribose)

AKINSHOLA ET AL.

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polymerase (39, 40). The apparent mechanism for this effect is the preservation of NAD’ pools and the continued generation of ATP required for protein and DNA synthesis (39).

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