Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by 99.243.105.24 on 01/09/15 For personal use only.
Effect of Pyrazole on Ethanol Metabolism in Ethanol-TolerantRats Depar-tment of Pllarmucology, LTniversityof Toronto, anti Addiction Research Foundation yfOntario, Toronto, Onturio M5S PA8 Received October 30. 1974
KALANT,H., K ~ P A N N.A I.,M., and ENDRENYI, 1,. 1975. Effect of pyrazole on ethanol ~netabolismin ethanol-tolerant rats. Can. 9. Physiol. Pharmaco). 53.416422. Adult male rats were pair-fed liquid diets, providing 37% of calories as ethanol or sucrose, for 1 month. Alcohol dehydrogenase (ADH) activity in the cytosol fractions of liver homogenates from the two groups did not differ with respect to total activity per 100 g body weight, Kin for ethanol, or Ki for pyrazole. Other rats, fed in the same way, were fasted for 18-24 h, then given an intraperitoneal injection sf pyrazole followed I later by an injection of ethanol, 3 g/kg. Blood alcohol curves showed an unexplained slower rise to maximum Bevel in the chronic alcohol group. Both groups showed a period of several hours in which the blood alcohol stayed at the respective maximum concentrations, which were higher in the control group. After 7-8 h the alcohol concentration began to fall in both groups, significantly more rapidly in the chronic alcohol-fed animals. A kinetic ainalysis shows that the results are adequately explained by the known effects of pyrazole on the AI9H-mitochondria! system. The results are interpreted as evidence against the function of any microsoma1 ethanol oxidizing system in vivo. KALANT. M.,K H A N N A J . ,M. et ENDMENYI, k. 1975. Effect of pyrazole o n ethanol metabolism in ethanol-tolerant rats. Can. J . Physiol. Pharmacol. 53,416422. Des rats males adultes ont requ, pendant un mois, des dietes liquides dont 34% des calories provenaient soit d'ethanol, soit de sucre. L'activite de B'alcool deshydrogenase (ADH) a ete mesuree dans la fraction surnageante 21 185 008 x g obtenue B partir des homogenats de foie. 11 y a eu Ba mkme activite totale de I'ADH par rapport au poids corporel, la mGme valeur de K , , pour l'ithanol et le mZme K ; pour pyrazole, chez les rits traites qaae les contr6les. D'autres rats, alimentes de paire avec Bes m2mes dietes, apres I8 B 24 h h jeun ont requ une injection intraperitoniale cBe pyrazsle, stmivie I h plus tard d'uame injection d16thano1(3g/kg). I,es courbes d'alcoo%emieont rnontre une ascension plus lente chez les animaux alcooliques que les contsoBes, ce qui reste encore sanns explication. Chez les deux groupes, Bes courbes sont restees pendant quelqames heur-es a leurs niveaux nnaximum respectifs, le maximaam etannt plus eleves chez les contrciles. Apres 7-8 h. il y avait une chute de l'alcoolemie chez les deux grmpes, mais la pente etait significativement plus rapide chez le group aicoolique. Selon B'analyse cinetique, les resultats sont susceptibles d'une explication ridequate B base des actions bien connues d u pyrazole sur le systeme ADH-mitochondries, et ne soutiennent pas le concept d9unr61e biologique dai phknonnene d'oxydatirsn de l'ethanol par les microsomes hepatiques.
Hla$rsduction Since the description of a hepatic microsoma1 ethanol-oxidizing system ( bfEOS) by Lieber and DeCarli (19781, there has been a continuing debate in the literature as to whether or not this system is an in viao artifact, and what, if any, role it plays in ethanol metabolism in vhvo (for references see Hawkins and Kalant 1972). It has been found repeatedly that chronic feeding of ethanol-containing liquid diets to rats results in significant increases in MEOS activity (Lieber and DeCarli 11970, Tobon and Mezey 1971, Khanna e l al. 1972). However, this change does not always occur in parallel with changes in the rate of cthanol metabolism in viva (Mezey 1971, Khanna et al 1972).
Lieber and DeCarli (1972) have more recently presented additional evidence in support of their contention that MEOS plays a significant role in vivo, particularly in subjects treated chronically with ethanol. Rats which had received an ethanol-containing liquid diet for 24 days showed the usual increase in rate of alcohol disappearance, when compared with pair-fed, sucrose-treated controls. When both groups were pretreated with pyrazole, the chronic alcohol group showed a stceper slope of blood alcohol disappearance, and it was argued that this indicated a significant role of a pyrazoIe-insensitive pathway, i.e., one other than alcohol dehydrogenase (ADH) . Lieber and DcCarli further claimed that the apparent K,,, for alcohol disappearance in vivo following
417
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by 99.243.105.24 on 01/09/15 For personal use only.
KALANT ET AP..: PYKAZOLE EFFECT ON ETHANOL ME'TABOLISM
the pyrazole treatment had a value of approximately 8.5 mM, almost identical with the K,,, for MEQS activity in vitro. On this basis, they suggested that the additional alcohol metabolism in the chronically treated animals was almost entirely explainable on the basis of MEOS activity. Two objections to their interpretation are based on their choice of results for these ealculations. The first is that they ignored the blood alcohol levels during the first 12 h after ethanol administration (i.e., the first 17 h after pyrazole administration). Pyrazole has a halflife of 14 h in the rat, increasing to about 21 h in the presence of ethanol (Rydberg et nl. 1972). Therefore, the inhibition by pyrazole would be far from complete during their period of observation, and the difference in slopes of blood ethanol disappearance in the alcohol and sucrose treated groups could well reflect differences in the levels of ADH activity. The second objection is that K,,, values for the in vivo disappearance of ethanol were calculated from points which included a number on the linear portion of the descending blood-alcohol curve. Since, by definition, K,,, can be calcuIated only during periods of first-order metabolism, the apparent resemblance between the Kt,, values for alcohol disappearance in vivo and MEOS activity in vitro might well be
erroneous. The following studies were, therefore, conducted with attention to these details. &laterialsand Methods Male Wiqtar strain rats of about 145 g initial body weight were pair-matched on the basis of weight, so that pair-mates did not differ by more than 3 g. They were kept in individual cages and fed liquid diets based on those described by Lieber et al. (19631, slighlty modified as described elsewhere (Khanna et a / . 1967). The ethanol diet provided 35% of the total calories as ethanol, 41% as fat, 19% as protein hydrolysate, 5% as sucrose, and was nutritionally adequate in all other respects. The voluntary intake of this diet was suficient to maintain the daily dosage of alcohol at 10-12 g/kg. Pair-fed controls received equal volumes of diet in which the ethanol was replaced by an equicaloric concentration of sucrose. The diets were replaced by tap water for both groups 18-24 h before tests of alcohol metabolism. The liquid diet treatment lasted 28 days. Two separate irz vivo studies were carried out. In the first, each animal received a single dose of pyrazole, 4 mmol/kg intraperitoneally (i.p.1, followed 1 h later by a 6 5 mmol/kg dose of ethanol ( 3 g/kg i.p.,
as a 20% v / v solution in isotonic saline). Samples of capillary blood from the tip of the tail were taken at 3, 4, 5, and 6 h after the ethanol injection, and every 3 h thereafter until 24 h post-injection. The second experiment was essentially the same as the first, except that the dose of pyrazole was 4.4 mmol/kg and the blood samples were taken at hourly intervals for the first 8 h, every 2 h thereafter until the 28th h, and a final sample at 51 h post-ethanol. The blood samples were deproteinized and analyzed for their ethanol content by gas-liquid chromatography, with n-butanol as an internal standard (LeBlanc 1968 ) . For in vifro studies, the animals were sacrificed by decapitation, the liver was removed as rapidly as possible, rinsed free of blood and weighed. Weighed portions of each liver were used for preparation of the 105 000 X g supernatant fraction of homogenates in distilled water. for measurement of liver ABH activity as described previously (Hawkins et al. 1966). For each preparation, activity was measured with initial ethanol concentrations of 0.5, 1.8, 2.0, and 4.0 mM, each at pyrazole concentrations of 0, 10-5 and 10" mM.
Results Eflect of Pyrccaole on in Vitrs Ethanol Oxidation Individual nonlinear least-squares calculations (Cleland 1967, Endrenyi and Kwong 1972) for each animal, bascd on a hyperbolic relation between substrate concentration and velocity, were made separately at each pyrazole concentration. The results indicated that gyrazole caused competitive inhibition of NADdependent ADH activity. In both the controls and the chronic ethanol group, the values of C',,, were not affected by pyrazole (F = 1.59, d.f.= 2 and 36, p > 0.05 in an analysis of variance involviiag the logarithm of the estimated parameters), while the apparent R,, values changed very substantially ( F 51.8, $.I. = 2 and 36. p < 0.001). Individual values of R ,,,, V ,,,, and Riwere estimated also on pooled data at all pyrazole concentrations for a given animal, by nonlinear regression based on the model for competitive inhibition (Cleland 1967) . The mean values for K,, and K i were not significantly different in the two treatment groups (Table 1 ). As in previous experiments (Khanna et al. 1972), the liver weight was increased significantly in the chronic ethanol group. For equal volumes of supernatant, representing equal weights of liver, as used in thc standard in vitro reaction mixture, the value for V"',,,,was significantly greater in the sucrose controls
-
418
CAN. J . PHYSIOL. PHARMACOL. VOL.
53. 1975
TABLE 1. In uitro enzyme kinetic parameters with and without pyrazole in ethanol treated rats and pair-fed controls Ethanol-treated (n = 7)
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by 99.243.105.24 on 01/09/15 For personal use only.
Parameter
Controls (n = 7)
+ 0-04 + 0.67
0.78 i 0.05 4.50 0.32 0.186 k 0.010 4.66 Q .08
K", (mM) K j for pyraaole ( p M )
0.77 5.16 0.262 _+ 3.42 k
+ +
%ax*
Liver weight g per 100 g body weight V,,,,* x liver weight per I00 g body weight
P
21.67 k 1 . 0 6
N.S. N.S.
Effect of Pyrazole on Ethanol Metabolism in Ethanol-TolerantRats Depar-tment of Pllarmucology, LTniversityof Toronto, anti Addiction Research Foundation yfOntario, Toronto, Onturio M5S PA8 Received October 30. 1974
KALANT,H., K ~ P A N N.A I.,M., and ENDRENYI, 1,. 1975. Effect of pyrazole on ethanol ~netabolismin ethanol-tolerant rats. Can. 9. Physiol. Pharmaco). 53.416422. Adult male rats were pair-fed liquid diets, providing 37% of calories as ethanol or sucrose, for 1 month. Alcohol dehydrogenase (ADH) activity in the cytosol fractions of liver homogenates from the two groups did not differ with respect to total activity per 100 g body weight, Kin for ethanol, or Ki for pyrazole. Other rats, fed in the same way, were fasted for 18-24 h, then given an intraperitoneal injection sf pyrazole followed I later by an injection of ethanol, 3 g/kg. Blood alcohol curves showed an unexplained slower rise to maximum Bevel in the chronic alcohol group. Both groups showed a period of several hours in which the blood alcohol stayed at the respective maximum concentrations, which were higher in the control group. After 7-8 h the alcohol concentration began to fall in both groups, significantly more rapidly in the chronic alcohol-fed animals. A kinetic ainalysis shows that the results are adequately explained by the known effects of pyrazole on the AI9H-mitochondria! system. The results are interpreted as evidence against the function of any microsoma1 ethanol oxidizing system in vivo. KALANT. M.,K H A N N A J . ,M. et ENDMENYI, k. 1975. Effect of pyrazole o n ethanol metabolism in ethanol-tolerant rats. Can. J . Physiol. Pharmacol. 53,416422. Des rats males adultes ont requ, pendant un mois, des dietes liquides dont 34% des calories provenaient soit d'ethanol, soit de sucre. L'activite de B'alcool deshydrogenase (ADH) a ete mesuree dans la fraction surnageante 21 185 008 x g obtenue B partir des homogenats de foie. 11 y a eu Ba mkme activite totale de I'ADH par rapport au poids corporel, la mGme valeur de K , , pour l'ithanol et le mZme K ; pour pyrazole, chez les rits traites qaae les contr6les. D'autres rats, alimentes de paire avec Bes m2mes dietes, apres I8 B 24 h h jeun ont requ une injection intraperitoniale cBe pyrazsle, stmivie I h plus tard d'uame injection d16thano1(3g/kg). I,es courbes d'alcoo%emieont rnontre une ascension plus lente chez les animaux alcooliques que les contsoBes, ce qui reste encore sanns explication. Chez les deux groupes, Bes courbes sont restees pendant quelqames heur-es a leurs niveaux nnaximum respectifs, le maximaam etannt plus eleves chez les contrciles. Apres 7-8 h. il y avait une chute de l'alcoolemie chez les deux grmpes, mais la pente etait significativement plus rapide chez le group aicoolique. Selon B'analyse cinetique, les resultats sont susceptibles d'une explication ridequate B base des actions bien connues d u pyrazole sur le systeme ADH-mitochondries, et ne soutiennent pas le concept d9unr61e biologique dai phknonnene d'oxydatirsn de l'ethanol par les microsomes hepatiques.
Hla$rsduction Since the description of a hepatic microsoma1 ethanol-oxidizing system ( bfEOS) by Lieber and DeCarli (19781, there has been a continuing debate in the literature as to whether or not this system is an in viao artifact, and what, if any, role it plays in ethanol metabolism in vhvo (for references see Hawkins and Kalant 1972). It has been found repeatedly that chronic feeding of ethanol-containing liquid diets to rats results in significant increases in MEOS activity (Lieber and DeCarli 11970, Tobon and Mezey 1971, Khanna e l al. 1972). However, this change does not always occur in parallel with changes in the rate of cthanol metabolism in viva (Mezey 1971, Khanna et al 1972).
Lieber and DeCarli (1972) have more recently presented additional evidence in support of their contention that MEOS plays a significant role in vivo, particularly in subjects treated chronically with ethanol. Rats which had received an ethanol-containing liquid diet for 24 days showed the usual increase in rate of alcohol disappearance, when compared with pair-fed, sucrose-treated controls. When both groups were pretreated with pyrazole, the chronic alcohol group showed a stceper slope of blood alcohol disappearance, and it was argued that this indicated a significant role of a pyrazoIe-insensitive pathway, i.e., one other than alcohol dehydrogenase (ADH) . Lieber and DcCarli further claimed that the apparent K,,, for alcohol disappearance in vivo following
417
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by 99.243.105.24 on 01/09/15 For personal use only.
KALANT ET AP..: PYKAZOLE EFFECT ON ETHANOL ME'TABOLISM
the pyrazole treatment had a value of approximately 8.5 mM, almost identical with the K,,, for MEQS activity in vitro. On this basis, they suggested that the additional alcohol metabolism in the chronically treated animals was almost entirely explainable on the basis of MEOS activity. Two objections to their interpretation are based on their choice of results for these ealculations. The first is that they ignored the blood alcohol levels during the first 12 h after ethanol administration (i.e., the first 17 h after pyrazole administration). Pyrazole has a halflife of 14 h in the rat, increasing to about 21 h in the presence of ethanol (Rydberg et nl. 1972). Therefore, the inhibition by pyrazole would be far from complete during their period of observation, and the difference in slopes of blood ethanol disappearance in the alcohol and sucrose treated groups could well reflect differences in the levels of ADH activity. The second objection is that K,,, values for the in vivo disappearance of ethanol were calculated from points which included a number on the linear portion of the descending blood-alcohol curve. Since, by definition, K,,, can be calcuIated only during periods of first-order metabolism, the apparent resemblance between the Kt,, values for alcohol disappearance in vivo and MEOS activity in vitro might well be
erroneous. The following studies were, therefore, conducted with attention to these details. &laterialsand Methods Male Wiqtar strain rats of about 145 g initial body weight were pair-matched on the basis of weight, so that pair-mates did not differ by more than 3 g. They were kept in individual cages and fed liquid diets based on those described by Lieber et al. (19631, slighlty modified as described elsewhere (Khanna et a / . 1967). The ethanol diet provided 35% of the total calories as ethanol, 41% as fat, 19% as protein hydrolysate, 5% as sucrose, and was nutritionally adequate in all other respects. The voluntary intake of this diet was suficient to maintain the daily dosage of alcohol at 10-12 g/kg. Pair-fed controls received equal volumes of diet in which the ethanol was replaced by an equicaloric concentration of sucrose. The diets were replaced by tap water for both groups 18-24 h before tests of alcohol metabolism. The liquid diet treatment lasted 28 days. Two separate irz vivo studies were carried out. In the first, each animal received a single dose of pyrazole, 4 mmol/kg intraperitoneally (i.p.1, followed 1 h later by a 6 5 mmol/kg dose of ethanol ( 3 g/kg i.p.,
as a 20% v / v solution in isotonic saline). Samples of capillary blood from the tip of the tail were taken at 3, 4, 5, and 6 h after the ethanol injection, and every 3 h thereafter until 24 h post-injection. The second experiment was essentially the same as the first, except that the dose of pyrazole was 4.4 mmol/kg and the blood samples were taken at hourly intervals for the first 8 h, every 2 h thereafter until the 28th h, and a final sample at 51 h post-ethanol. The blood samples were deproteinized and analyzed for their ethanol content by gas-liquid chromatography, with n-butanol as an internal standard (LeBlanc 1968 ) . For in vifro studies, the animals were sacrificed by decapitation, the liver was removed as rapidly as possible, rinsed free of blood and weighed. Weighed portions of each liver were used for preparation of the 105 000 X g supernatant fraction of homogenates in distilled water. for measurement of liver ABH activity as described previously (Hawkins et al. 1966). For each preparation, activity was measured with initial ethanol concentrations of 0.5, 1.8, 2.0, and 4.0 mM, each at pyrazole concentrations of 0, 10-5 and 10" mM.
Results Eflect of Pyrccaole on in Vitrs Ethanol Oxidation Individual nonlinear least-squares calculations (Cleland 1967, Endrenyi and Kwong 1972) for each animal, bascd on a hyperbolic relation between substrate concentration and velocity, were made separately at each pyrazole concentration. The results indicated that gyrazole caused competitive inhibition of NADdependent ADH activity. In both the controls and the chronic ethanol group, the values of C',,, were not affected by pyrazole (F = 1.59, d.f.= 2 and 36, p > 0.05 in an analysis of variance involviiag the logarithm of the estimated parameters), while the apparent R,, values changed very substantially ( F 51.8, $.I. = 2 and 36. p < 0.001). Individual values of R ,,,, V ,,,, and Riwere estimated also on pooled data at all pyrazole concentrations for a given animal, by nonlinear regression based on the model for competitive inhibition (Cleland 1967) . The mean values for K,, and K i were not significantly different in the two treatment groups (Table 1 ). As in previous experiments (Khanna et al. 1972), the liver weight was increased significantly in the chronic ethanol group. For equal volumes of supernatant, representing equal weights of liver, as used in thc standard in vitro reaction mixture, the value for V"',,,,was significantly greater in the sucrose controls
-
418
CAN. J . PHYSIOL. PHARMACOL. VOL.
53. 1975
TABLE 1. In uitro enzyme kinetic parameters with and without pyrazole in ethanol treated rats and pair-fed controls Ethanol-treated (n = 7)
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by 99.243.105.24 on 01/09/15 For personal use only.
Parameter
Controls (n = 7)
+ 0-04 + 0.67
0.78 i 0.05 4.50 0.32 0.186 k 0.010 4.66 Q .08
K", (mM) K j for pyraaole ( p M )
0.77 5.16 0.262 _+ 3.42 k
+ +
%ax*
Liver weight g per 100 g body weight V,,,,* x liver weight per I00 g body weight
P
21.67 k 1 . 0 6
N.S. N.S.
