Stimulation of hepatic and renal diamine oxidase activity after acute ethanol administration

Biochimica et BiophysicaActa, 801 (1984) 285-289

285

Elsevier

BBA 21822

S T I M U L A T I O N OF HEPATIC A N D RENAL D I A M I N E O X I D A S E ACTIVITY AFTER ACUTE E T H A N O L A D M I N I S T R A T I O N ANGELA SESSA a, M. ALFONSINA DESIDERIO b and A N T O N I O PERIN b

a CNR Center for Research of Cell Pathology, and b Institute of General Pathology, University of Milan, Via Mangiagalli 31, 20133 Milan (Italy) (Received May l l t h , 1984)

Key words: Diamine oxidase; Ethanol," (Liver, Kidney)

The effect of a single administration of ethanol (2 g / k g body weight) on hepatic and renal diamine oxidase activity was studied in fasted rats. Diamine oxidase activity significantly increased in liver and kidney 6 h after ethanol intubation. Pyrazole (an inhibitor of alcohol dehydrogenase), cycloheximide or actinomycin D (inhibitors of macromolecular syntheses), as well as prior adrenalectomy, prevented the ethanol-induced stimulation of diamine oxidase in the liver, but not in the kidney. The results demonstrated that the enhancement of diamine oxidase activity in the liver was due to an enzyme induction mediated by alcohol metabolism as well as by adrenals. In contrast, the stimulation of diamine oxidase activity in the kidney did not depend on synthesis of new enzyme molecules and was not mediated by ethanol metabolism or adrenal hormones.

Introduction Diamine oxidase (EC 1.4.3.6), which catalyzes the oxidative deamination of histamine and other aliphatic diamines with 3 - 6 carbon atoms [1,2], is one of the enzymes that has focused the attention of many authors in the last years. In fact, it is rate-limiting in putrescine degradation in vivo [3,4] and may regulate in normal and growing tissues the levels of this diamine [5,6], which is considered a critical molecule for cellular growth [7]. Our studies demonstrated a de novo synthesis of diamine oxidase in some growing tissues correlated with an increase in putrescine formation [6,8-11]. It is noteworthy that partial hepatectomy, which is known to produce an enhancement in putrescine level owing to an increase in ornithine decarboxylase (EC 4.1.1.17) [12,131 and spermidine N lacetyltransferase [14,15] activities, causes an induction of diamine oxidase [9], which may contribute to regulate the intracellular pool of putrescine. 0304-4165/84/$03.00 © 1984 Elsevier Science Publishers B.V.

To our knowledge, the effects on hepatic diarnine oxidase activity of liver-damaging stimuli such as carbon tetrachloride, thioacetamide and ethanol, which increase putrescine biosynthesis and content in the liver [14,16-18], have not been studied. To gain more information in this regard, we report an investigation in which an acute dose of ethanol given to rats caused an induction of diamine oxidase in the liver, the principal organ for alcohol metabolism [19], that appeared to be mediated by ethanol oxidation as well as adrenal hormones. Diamine oxidase activity was also studied in kidney, an organ characterized by a very low rate of alcohol metabolism [20], and the results are discussed in comparison with those obtained in the liver. A preliminary report of part of this work has appeared in abstract form [21]. Materials Chemicals. [1,4-14C]Putrescine dihydrochloride (spec. act. 122 C i / m o l ) was obtained from the

286

Radiochemical Centre, Amersham, U.K.; acetaldehyde from Fluka A G Chemische Fabrik, Buchs, Switzerland; cycloheximide and actinomycin D from Aldrich Chemical Co., Milwaukee, WI, U.S.A.; pyrazole from Schuchardt, Munich, F.R.G. Animals and treatments. Male Wistar rats (250-300 g body weight) were maintained in temperature-controlled rooms on a 12 h dark/light illumination cycle and were starved overnight before the treatments. A first group of rats was tube fed under light ether anesthesia with saline or ethanol (2 g / k g body weight) as a 20% (w/v) solution in saline between 9 and 10 a.m. Some animals were given intraperitoneally pyrazole (250 m g / k g body weight) 1 h before ethanol treatment, or cycloheximide (2 m g / k g body weight) or actinomycin D (2.5 m g / k g body weight) at the time of ethanol intubation. Pyrazole and cycloheximide were dissolved in saline, and actinomycin D in propylene glycol in saline. The animals were killed 1 or 6 h after ethanol treatment, and livers and kidneys were used for diamine oxidase determination. A second group of rats was bilaterally adrenalectomized or sham-operated through dorsal incisions under light ether anesthesia and was given saline as the only drinking fluid. After 48 h, animals were intubated with saline or ethanol (2 g / k g body weight) as a 20% (w/v) solution in saline between 9 and 10 a.m., killed 6 h later, and livers and kidneys were used for diamine oxidase activity determination. Diamine oxidase activity determination. Diamine oxidase activity was determined in liver and kidney homogenates as reported in our previous papers [9,10] with the method of Okuyama and Kobayashi [22], modified by Kusche et al. [23], which measures the Al[14C]pyrroline formation from [14C]putrescine used as substrate. The incubation mixture contained, as previously described [9,10], 10 mM acetaldehyde to exclude the interference of tissue aldehyde-metabolizing enzymes in the yield of N-pyrroline [8,10,11,24]. Diamine oxidase activity was expressed as nmol or pmol of Al-pyrroline formed per h per gram of fresh tissue or mg of tissue protein. The amount of proteins was determined by the biuret method with bovine serum albumin as standard [25]. Determination of ethanol. At 1 and 6 h after

ethanol intubation, tail blood samples were placed immediately in an ice-cold solution of 0.6 M perchloric acid containing 25 mM thiourea, and livers, frozen with solid CO z and weighed, were homogenized (20% w / v ) in the same solution. All these samples were centrifuged at 4000 × g for 15 rain, and ethanol was assayed in supernatants by gas chromatography using the head-space technique [26]. Statistical analysis. The significance of the differences between the means was evaluated by Dunnett's test [27]. The results were considered significant when P < 0.05. Results

Effect of ethanol on hepatic and renal diamine oxidase activity of rat Hepatic diamine oxidase activity and ethanol levels in blood and liver are reported in Table I. Ethanol did not modify diamine oxidase activity 1 h after intubation, whereas at 6 h it caused a significant, approx. 3-fold increase in this enzyme activity, as compared to that of the saline group. Ethanol levels in blood and liver were higher at 1 h than at 6 h. An increase in diamine oxidase activity, about 2-fold that of the saline group, was observed also in the kidney 6 h after ethanol administration (Table II). For an evaluation of the mechanism(s) responsible for diamine oxidase stimulation in both studied tissues, we determined whether ethanol itself or its oxidation as well as de novo synthesis of enzyme were implicated in enzyme increase. As shown in Table II, pyrazole, and inhibitor of alcohol dehydrogenase [28], did not influence hepatic or renal diamine oxidase activity of rats treated with saline. When given 1 h before ethanol, it completely abolished in the liver, but not in the kidney, the increase in diamine oxidase activity observed 6 h after ethanol administration. Cycloheximide (which inhibits the translational phase of protein synthesis) [29] or actinomycin D (an inhibitor of m R N A synthesis) [30], given to rats at the same time as ethanol, completely prevented the ethanol-induced stimulation of diamine oxidase activity in the liver. In contrast, the increase in renal enzyme activity was insensitive to cyclo-

287 TABLE I EFFECT OF ETHANOL A D M I N I S T R A T I O N ON RAT LIVER D I A M I N E OXIDASE ACTIVITY Saline or ethanol (2 g / k g body weight), as a 20% ( w / v ) solution in saline, was given by gastric intubation to fasted rats and the animals were killed 1 or 6 h later. Diamine oxidase activity was measured in homogenates and expressed as Al[14C]pyrroline formation from [14C]putrescine. The values are the means _+S.E. of five animals for each group. Treatment

Diamine oxidase activity

Saline Ethanol 1 h before death Ethanol 6 h before death

Ethanol concentration

( n m o l / h per g wet wt.)

( p m o l / h per mg protein)

1.52 + 0.09 1.61 + 0.13 4.34+0.41 a

5.87 + 0.22 6.55 + 0.53 16.38_+1.70 a

Blood (t~ m o l / m l )

Liver ( # m o l / g wet wt.)

23.0 ± 3.0 6.05:0.8

30.0 + 3,5 8.0_+0.9

P < 0.01 (Dunnett's test).

TABLE II EFFECT OF PYRAZOLE, CYCLOHEXIMIDE, AND A C T I N O M Y C I N D ON HEPATIC A N D RENAL D I A M I N E OXIDASE ACTIVITY AFTER ETHANOL A D M I N I S T R A T I O N TO RATS Saline or ethanol (2 g / k g body weight), as a 20% ( w / v ) solution in saline, was given by gastric intubation to fasted rats 6 h before killing. Pyrazole (250 m g / k g body weight) was given intraperitoneally 1 h prior to ethanol administration. Cycloheximide (2 m g / k g body weight) or actinomycin D (2.5 m g / k g body weight) was given intraperitoneally at the time of ethanol administration. The values are the means 5: S.E., with number of cases in parentheses. Diamine oxidase activity ( n m o l / h per g wet wt.)

Treatment

Liver Saline Ethanol Pyrazole + saline Pyrazole + ethanol Ethanol + cycloheximide Ethanol + actinomycin D

1,40 + 0.08 3,745:0.34 1.39 + 0.12 1.33 -i-0.13 1,05 + 0.12 1.56 + 0.19

Kidney (8) (8) a (8) (8) (8) (8)

3.54 + 0.19 7.71 -t-0.61 4.08 + 0.38 8.03 + 0.88 6.62 + 0.74 7.58 + 0.77

(6) (6) ~ (6) (6) a (6) a (6) a

a p < 0.01 (Dunnett's test).

TABLE III EFFECT OF ETHANOL ON HEPATIC A N D RENAL D I A M I N E OXIDASE ACTIVITY IN A D R E N A L E C T O M I Z E D RATS Intact or 48-h adrenalectomized rats were intubated with saline or ethanol (2 g / k g body weight), as a 20% ( w / v ) solution in saline, 6 h before killing. The values are the means_+ S.E. of five animals for each group. Treatment

Diamine oxidase activity ( n m o l / h per g wet wt.)

Saline Ethanol Adrenalectomy + saline Adrenalectomy + ethanol a

p < 0.01 ( D u n n e t t ' s

test).

Liver

Kidney

1.32 5:0.12 4.25 + 0.46 a 1.34 5:0.23 1.84 5:0.31

3.50 + 0.21 7.60 5:0.80 a 4.28 + 0.62 7.01 + 0.77 a

288 heximide and to actinomycin D treatments (Table II).

Effect of ethanol on hepatic and renal diamine oxidase activity in adrenalectomized rats In view of the role of ethanol in activating the pituitary-adrenal axis [31,32], the effects of this drug on hepatic and renal diamine oxidase activities in adrenalectomized rats were examined. The results reported in Table III indicate that adrenalectomy did not modify the enzyme activities of tested tissues compared to those of intact animals. Interestingly, the ethanol-induced increase in diamine oxidase activity was prevented by prior adrenalectomy in liver, but not in kidney. Discussion

The present study was undertaken to investigate the effect that acute ethanol treatment exerts on rat hepatic and renal diamine oxidase activity. It is well known that this enzyme in rodents is present in a wide variety of tissues with different degrees of activity. In the rat, diamine oxidase activity is particularly high in gastroduodenal tract, thymus and placenta, whereas in kidney, liver and heart this enzyme activity is low [33]. We here provide evidence that hepatic and renal diamine oxidase activities are stimulated by acute ethanol exposure. It is of interest to understand the mechanism(s) responsible for this enzymatic increase, and it is opportune to examine first those implicated in the elevation of diamine oxidase activity in the liver. Since in this organ the highest values of diamine oxidase activity (6 h after intubation) did not coincide with the maximal levels of ethanol (1 h after intubation), the stimulation of diamine oxidase activity probably is not due to ethanol per se. In addition, pretreatment with pyrazole, an inhibitor of alcohol dehydrogenase [28], prevented the ethanol-induced increase in diamine oxidase activity and indicated that the metabolism of ethanol or products of its oxidation are involved in the stimulation of hepatic diamine oxidase activity. It is noteworthy that ethanol, acetaldehyde or acetate does not have a direct activator effect on the enzyme, since the activity of a commercial preparation of hog kidney diamine oxidase was unmodified by these subs-

tances (Ref. 8; and Perin, A., Sessa, A. and Desiderio, M.A.~ unpublished data). An important aspect of our results is that the enhancement in the activity of hepatic diamine oxidase, although very rapid, reflects an enzyme induction with new synthesis of m R N A s that code for diamine oxidase. In fact, cycloheximide or actinomycin D, specific inhibitors of protein or m R N A synthesis, respectively [29,30], completely abolished such an enhancement. In line with the importance of ethanol as a stressor that activates the hypothalamo-pituitaryadrenal axis [34], the induction in hepatic diamine oxidase after ethanol was almost completely prevented by prior bilateral adrenalectomy. Since adrenalectomy does not seem to influence liver alcohol dehydrogenase activity [35], our results suggest that adrenal hormones are implicated in the new synthesis of diamine oxidase. In this regard, acetaldehyde, which is derived from ethanol oxidation, independently of ACTH, is capable of stimulating the adrenal gland, probably by an enhancement in cAMP production [36]. The renal increase in diamine oxidase activity, in constrast with liver findings, was not the result of an enzyme induction, nor was it mediated by alcohol metabolism or adrenal hormones. In fact, cycloheximide, actinomycin D, pyrazole or prior adrenalectomy did not prevent the ethanol-induced enhancement in diamine oxidase activity. In these experiments, the doses of cycloheximide and actinomycin D used were known to give a similar pattern of inhibition in liver and kidney [9,10,37], and pyrazole was certainly sufficient to saturate alcohol dehydrogenase, whose activity is very low in rat kidney [19]. One of the factors that may contribute to the elevation of renal diamine oxidase activity may be an increased supply of enzyme from the gastroduodenal tract, where ethanol has been found to cause a marked diminution in this enzyme activity [38]. Moreover, taking into account the fact that the molecular weight of diamine oxidase is about 180000, the enzyme may accumulate in kidney because it is neither filtered nor excreted. Although these observations show the complexity of the mechanisms implicated in the changes in tissue diamine oxidase activity, they confirm that this enzyme is inducible under circumstances in

289 which a s t i m u l a t i o n of o r n i t h i n e decarboxylase activity also occurs, as in the case of the liver of ethanol-treated rats [18]. Thus, as a consequence of its de n o v o synthesis, d i a m i n e oxidase activity can assume, is a n organ with low enzyme activity such as rat liver, a new biological significance as a regulatory m e c h a n i s m to reduce the c o n t e n t of putrescine formed in excess. I n s u p p o r t of this hypothesis, when a n i n d u c t i o n of d i a m i n e oxidase similar to that f o u n d after ethanol occurs, as it the case in growing tissues, a m i n o g u a n i d i n e administ r a t i o n inhibits enzyme activity a n d causes a prop o r t i o n a l e n h a n c e m e n t in tissue putrescine levels [6,39]. F u r t h e r experiments are necessary to establish the relative c o n t r i b u t i o n of each metabolic p a t h w a y of putrescine in the regulation of the hepatic levels of this d i a m i n e after ethanol exposure. More i n f o r m a t i o n is also required to decide whether the described s t i m u l a t i o n of d i a m i n e oxidase activity in the liver reflects only a mechan i s m to m a i n t a i n cellular homeostasis or whether it is implicated, b y degradative products of polyamines, in the pathogenesis of some hepato-renal illnesses due to alcohol, i n spite in the difficulty in correlating the experimental models to clinical states.

Acknowledgements This study was supported i n part b y grant Nos. 1201 R a n d 1202.123 C from the Ministero della P u b b l i c a Istruzione, Rome. T h e authors t h a n k Dr. A d a Gasparoli, Stazione Sperimentale Olii e Grassi, Milan, for the gas-chromatographic ethanol determination.

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