Effects of pyrazole, 4-bromopyrazole and 4-methylpyrazole on mitochondrial function

Pyrazole, an inhibitor of alcohol dehydrogenase, has been widely used in studies of ethanol metabolism. Since its specificity has recently been questioned, we studied the effects of pyrazole, methylpyrazole and bromopyrazole on mitochondrial function. These compounds inhibited oxidative phosphorylation, the ATP^?32P exchange reaction, and energy dependent and independent calcium uptake. With α-ketoglutarate as substrate, state 3 (coupled) respiration was inhibited, whereas state 4 (resting) respiration was not affected. By contrast, state 4 respiration was stimulated when succinate or ascorbate served as the substrate, while state 3 respiration was slightly inhibited. Regardless of the substrate, the respiratory control ratio was depressed. The activities of succinic dehydrogenase and cytochrome oxidase were stimulated by pyrazole and its derivatives, which may explain the stimulation of succinate and ascorbate oxidation. The inhibitory effects of these compounds were reversed by washing the mitochondria, indicating that no permanent damage to mitochondria had occurred. This is supported by the lack of stimulation of latent ATPase activity and the unchanged barrier to the penetration of NADH. Pyrazole and its derivatives decreased the uptake of citrate and glutamate, but stimulated that of phosphate and malate. Methylpyrazole and bromopyrazole inhibited the transport of reducing equivalents into the mitochondria, as catalyzed by the malate-aspartate, fatty acid and α-glycerophosphate shuttles. The data mandate caution in advocating the therapeutic use of pyrazole or its derivatives in man, and suggest that the use of pyrazole to assess ethanol metabolism and its sequelae in vivo may have limitations.     

Activating and anesthetic effects of general depressants

The long-sleep (LS) and short-sleep (SS) lines of mice were derived by selective breeding with respect to ethanol sleep time. We found that in current generations LS mice also have longer sleep times than SS mice to trichloroethanol and paraldehyde. Two subsequent experiments tested our hypothesis that mice that are relatively insensitive to the hypnotic effects of depressant drugs might be relatively activated by low doses of these drugs. Both experiments failed to support the hypothesis. First, although SS mice were more activated than LS mice by subhypnotic doses of paraldehyde, the lines did not differ in the degree of activation produced by low doses of trichloroethanol. Second, among mice from a genetically heterogeneous population (HS), there was no relation between the degree of activation induced by a low dose of ethanol and sensitivity to the hypnotic effects of a higher dose.     

Human liver alcohol dehydrogenase--inhibition of methanol activity by pyrazole, 4-methylpyrazole, 4-hydroxymethylpyrazole and 4-carboxypyrazole.

With human liver alcohol dehydrogenase of high purity at pH 7.0 and 500 μM NAD the K_m for methanol is 7.0 mM (ten times greater than the K_m for ethanol) and the turnover number 1.4/active site/min (about one-tenth of the turnover with ethanol in the same conditions). From secondary kinetic plots it can be calculated that at saturating concentrations of both substrates, namely methanol and NAD, these constants do not change appreciably: the K_m for methanol is somewhat lower (5.2 mM) and the turnover number slightly higher (1.7/active site/min). The difference in turnover numbers with methanol and ethanol as substrates suggests that the kinetic mechanism for methanol is different from that for ethanol dehydrogenation. The dissociation constant between human alcohol dehydrogenase and NAD, determined kinetically with methanol as substrate, is 127 μM. The K_i values for pyrazole, 4-methylpyrazole and 4-hydroxymethylpyrazole are 0.54, 0.09 and 6.6 μM respectively; 4-carboxypyrazole (100 μM) at 3mM methanol does not inhibit human ADH. The inhibitory effect of 4-methylpyrazole is therefore not likely to be enhanced by a possible metabolic conversion to 4-hydroxymethylpyrazole and 4-carboxypyrazole.     

Effect of 4-methylpyrazole and pyrazole on the induction of fatty liver by a single dose of ethanol

Pyrazole (272 mg/kg), 4-methylpyrazole (4-MP; 200 mg/kg) or saline was injected intraperitoneally into fasted male and female rats. Ten min later, ethanol (4 or 6 g/kg) or an equicaloric dose of sucrose was given by stomach tube. Hepatic triglyceride (TG) levels were measured at 6, 12 or 16 hr after the gavage. With a 4 g/kg dose of ethanol, pyrazole reduced the accumulation of TG at 6 hr in females, but not at 12 and 16 hr. In males, ethanol gave relatively little TG accumulation at 6 hr and pyrazole did not affect this, but at 16 hr the TG levels in the ethanol-pyrazole group had not risen as much as in the ethanol-saline group. In contrast to pyrazole, 4-MP by itself increased liver TG content, and significantly increased the TG accumulation caused by a 4 g/kg dose of ethanol in both males and females at 16 hr. However, 4-MP caused a significantly smaller TG accumulation in females at 6 hr after the ethanol, but not in males. With a larger dose of ethanol (6 g/kg), both pyrazole and 4-MP decreased the accumulation of TG at 16 hr in males. It is concluded that ethanol per se, ethanol as a metabolic substrate, and pyrazoles as pharmacological agents with complex actions may all contribute to the development of acute fatty liver. Therefore, pyrazole and 4-MP do not appear to be suitable tools for resolving the controversy about the mechanism of production of alcoholic fatty liver.     

Increased sensitivity of the microsomal oxidation of ethanol to inhibition by pyrazole and 4-methylpyrazole after chronic ethanol treatment

Pyrazole and 4-methylpyrazole, inhibitors of the oxidation of ethanol by alcohol dehydrogenase, also inhibit microsomal metabolism of ethanol. The inhibitory effectiveness of these agents was increased in microsomes isolated from rats treated chronically with ethanol as compared to microsomes from pair-fed controls or from rats treated with other cytochrome P-450 inducers such as phenobarbital or 3-methylcholanthrene. Pyrazole and 4-methylpyrazole produced type II binding spectra with all the microsomal preparations. However, there was an increased affinity (lower Ks value) for these agents by the microsomes from the ethanol-fed rats. A correlation between Ks values and inhibitory effectiveness against ethanol oxidation by the various microsomal preparations could be observed. This suggests that an increase in affinity, which may reflect the induction of an alcohol-preferring isozyme of cytochrome P-450, is responsible for the increased inhibitory effectiveness of pyrazole and 4-methylpyrazole towards ethanol oxidation by microsomes after chronic ethanol treatment. One difference between pyrazole and 4-methylpyrazole was the increased affinity and inhibitory effectiveness of the latter but not the former with microsomes from rats treated with 3-methylcholanthrene. This could be due to the ability of 4-methylpyrazole, compared to pyrazole, to interact with and induce several isozymes of cytochrome P-450. Pyrazole and 4-methylpyrazole are often utilized to evaluate ethanol metabolism by alcohol-dehydrogenase-dependent and -independent pathways. However, the sensitivity of microsomal ethanol oxidation to inhibition by these agents, especially after chronic ethanol treatment, would suggest that their use in this regard is complex and could tend to underestimate the contribution of the microsomal pathway towards the metabolic tolerance found after ethanol treatment.     

Suppression of acetaldehyde accumulation by 4-methylpyrazole in alcohol-hypersensitive Japanese

Alcohol-sensitive Japanese subjects with facial flushing and an increase in heart rate during ethanol intoxication exhibited marked individual variation in accumulation of acetaldehyde. This variation correlated well with the intensity of the above mentioned physiological responses. Oral pretreatment with 10 mg/kg 4-methylpyrazole, which inhibited the ethanol elimination rate by 15-25%, strongly suppressed both acetaldehyde accumulation and the associated responses. Under this condition, the sensitivity to acetaldehyde appeared to be reduced, and the correlation between the acetaldehyde level and the physiological responses disappeared. The effectiveness of even a low dose of 4-methylpyrazole suggests its clinical usefulness for alleviation of acute acetaldehyde toxicity in alcohol-hypersensitive Japanese individuals as well as in disulfiram-treated alcoholics.     

Comparison of the effects of ethanol and 4-methylpyrazole on the pharmacokinetics and toxicity of ethylene glycol in the dog

The purpose of this investigation was to compare the effects of ethanol and 4-methylpyrazole (4MP) on the toxicity and pharmacokinetics of ethylene glycol (EG) in the dog. All dogs received 173 mmol/kg EG, p.o. Dogs were randomly assigned to 3 groups: EG-treated only, EG + ethanol (19.3 mmol/kg, i.v. 3, 7, 14 and 24 h after EG) and EG + 4MP (0.24 mmol/kg, i.v. 3 h after EG, 0.18 mmol/kg at 24 h and 0.06 mmol/kg at 36 h). EG produced a rapid onset of metabolic acidosis (within 3 h) and acute oliguric renal failure (after 48 h), whereas administration of ethanol or 4MP greatly attenuated acidosis and prevented renal toxicity. The administration of ethanol, however, severely increased the central nervous system (CNS) depression that existed after ingestion of EG. The half-life of FG in serum was 10.8 +/- 0.7 h in the EG-only treatment group, 6.8 +/- 0.7 (P less than 0.05) in the EG + ethanol group and 9.8 +/- 0.9 h in the EG + 4MP group. Approx. 10% and 48% of the dose of EG was excreted unchanged in the urine at the 0-3 and 3-72 h periods, respectively. Treatment with 4MP increased the amount of EG excreted in the urine (71% from 3-72 h), whereas ethanol did not (51%). However, both ethanol and 4MP increased the rate constant of EG excretion into urine approx. 70%. These data demonstrate the utility of 4MP over ethanol for the treatment of EG-induced toxicity in dogs and indicate that ethanol and 4MP cause an increase in the rate constant of EG excretion in the urine and not a prolongation in EG half-life.     

Kinetic disposition of ethanol in the neonatal piglet and hemodynamic effects in the presence and absence of 4-methylpyrazole

The hemodynamic effects of acute ethanol intoxication and the kinetic disposition of ethanol are reported for the first time in neonatal piglets under nitrous oxide anesthesia. Two hours after a single dose of ethanol (1.4 g/kg), blood pressure decreased from 76 +/- 4 to 71 +/- 4 mm Hg (p less than 0.05) and heart rate increased from 194 +/- 10 to 227 +/- 8 beats/min (p less than 0.05; means +/- SE). By 5 hr, blood pressure dropped to 67.5 +/- 4 mm Hg and heart rate increased to 239 +/- 8 beats/min. In piglets pretreated with 4-methylpyrazole, an alcohol dehydrogenase inhibitor, there was a transient increase in blood pressure (p less than 0.05) and a decrease in heart rate (p less than 0.05) immediately after the end of the ethanol infusion. However, the hemodynamic alterations observed 2 hr after ethanol treatment alone were prevented with 4-methylpyrazole. These findings indicate that ethanol metabolites play a significant role in hemodynamic alterations observed after acute ethanol intoxication. The mean ethanol metabolic rate derived from plasma data was 94 +/- 9 mg/liter/hr. This corresponded to an apparent Km of 68 +/- 3 mg/liter and a Vm of 123 +/- 11 mg/liter/hr. The Vd was 0.966 +/- 0.031 liter/kg. The metabolic rate for ethanol, derived from plasma data, correlated with in vitro alcohol dehydrogenase activity at pH 7.4 and 25 and 37 degrees C. The optimum pH for hepatic alcohol dehydrogenase activity was 9.9.     

Distribution of oral 4-methylpyrazole in the rat: inhibition of elimination by ethanol

4-Methylpyrazole (4-MP), a potent competitive inhibitor of alcohol dehydrogenase activity, is being studied as a therapeutic agent for methanol and ethylene glycol poisoning. In order to evaluate the distribution of 4-MP using doses in the potentially therapeutic range, male Sprague-Dawley rats were administered 4-MP orally at zero time in doses of 5, 10, or 20 mg/kg. Half of the rats were also treated orally at 0, 1, 2, and 3 h with ethanol (1 g/kg each h) and half with glucose in isocaloric amounts. At doses of 10 and 20 mg/kg, 4-MP elimination appeared to be saturated, with an elimination rate of 10 mumol/L/h. Elimination at 5 mg/kg was non-conclusive as to the order. The rate of 4-MP elimination was decreased about 50% by concomitant administration of ethanol. Urinary excretion of unchanged 4-MP accounted for only about 1% of the dose; the amount excreted unchanged was significantly increased by ethanol administration. The results demonstrate the mutual inhibition of metabolism by ethanol and 4-methylpyrazole, which may explain why the inhibition of ADH by 4-MP can be longer than that predicted by the elimination rate of 4-MP alone.     

Effect of ethanol and 4-methylpyrazole on the immunotoxicity of ethylene glycol

The results of experiments on Wistar rats showed that acute poisoning with 1.0 LD50 of ethylene glycol decreased the nonspecific resistance of the organism (manifested by increased lethality caused by experimental infection, decreased average lethal dose of E. coli, and decreased mean effective lifetime of experimental animals) and reduced the antibody formation to predominantly a thymus-dependent antigen, natural and antibody-dependent cell cytotoxicity, and delayed type hypersensitivity reaction. 4-Methylpyrazole and (to a greater extent) ethanol potentiated the immunotoxic action of ethylene glycol.     

Analysis of 4-methylpyrazole in plasma and urine by gas chromatography with nitrogen-selective detection

A simple, sensitive, and specific gas chromatographic method for the quantitation of 4-methylpyrazole in plasma and urine is described. Samples containing 4-methylpyrazole, with 3-methylpyrazole as the internal standard, are extracted into ether and the concentrated ethereal extracts are chromatographed on a Carbowax 20M column using nitrogen-selective detection. Standard curves are linear and reproducible over the range of 25-1000 ng/mL for plasma and 0.5-5 micrograms/mL for urine. Recovery of 4-methylpyrazole is complete from plasma and urine, and the overall between-day coefficient of variation is within 6.0%. No interference is observed from the extractive constituents of plasma and urine. The assay method is suitable for an examination of 4-methylpyrazole disposition in animals and humans.     

The effect of cyanamide and 4-methylpyrazole on the ethanol-induced locomotor activity in mice

To assess the role of cyanamide and 4-methylpyrazole (4-MP) in mediating ethanol-induced locomotor activity in mice, they were pretreated with cyanamide (12.5, 25, or 50 g/kg) prior to one ethanol injection (2.4 g/kg) and showed significantly depressed locomotor activity compared with control groups. Cyanamide (25 mg/kg) also cancelled out the biphasic action of ethanol (0, 0.8, 1.6, 2.4, 3.2, or 4 g/kg) on locomotor activity. The action of cyanamide and 4-MP in combined administration was also tested. Our data show that pretreatment with 4-MP alone does not change the spontaneous or ethanol-induced locomotor activity. Conversely, when mice were pretreated with cyanamide and 4-MP, the depressive effect of cyanamide on the locomotor activity induced by ethanol disappeared, and the locomotor activity rose to levels similar to those of the control group, recovering the biphasic ethanol effect. These effects cannot be attributed to peripheral elevated blood acetaldehyde levels, as pretreatment with 4-MP prevents accumulation of acetaldehyde. These data might suggest some influence of brain catalase and aldehyde dehydrogenase (ALDH) on the effects of ethanol.     

Oxidation of pyrazole to 4-hydroxypyrazole by intact rat hepatocytes

4-Hydroxypyrazole has been identified as a major metabolite found in the urine of rats and mice after in vivo administration of pyrazole, a potent inhibitor of alcohol dehydrogenase and of ethanol metabolism. The locus and the enzyme systems responsible for the oxidation of pyrazole have not been identified. In the current report, isolated hepatocytes from fed rats were shown to oxidize pyrazole to 4-hydroxypyrazole. An HPLC procedure employing UV and electrochemical detection was utilized to separate and quantify the 4-hydroxypyrazole. The apparent Km for pyrazole by intact hepatocytes was about 2 mM, whereas the apparent Vmax was about 0.06 nmol 4-hydroxypyrazole per min per mg liver cell protein. The production of 4-hydroxypyrazole was inhibited by carbon monoxide and metyrapone, as well as by competitive drug substrates such as aniline or aminopyrine. These results implicate a role for cytochrome P-450 in the oxidation of pyrazole by the hepatocytes. Ethanol was an effective inhibitor of pyrazole oxidation. Hepatocytes were also isolated from rats treated with acetone and 4-methylpyrazole, to attempt to evaluate whether pyrazole oxidation is induced. The rate of 4-hydroxypyrazole production by hepatocytes after acetone and 4-methylpyrazole treatment was actually lower than that of controls. Kinetic assays suggested the presence of an endogenous inhibitor (perhaps the inducer itself) in the induced hepatocytes. In contrast, hepatocytes isolated from rats fasted for 48 hr showed a 2-fold increase in the oxidation of pyrazole to 4-hydroxypyrazole. The Km for pyrazole was the same in hepatocytes from fasted and fed rats, whereas Vmax was increased after fasting. The locus and enzyme system responsible for the oxidation of pyrazole to 4-hydroxypyrazole, and the site of sensitivity to ethanol, appears to be the cytochrome P-450 system of the hepatocyte.     

Interaction effects of ethanol and pyrazole in laboratory rodents

1. Interactions of pyrazole and ethanol were studied in three laboratory test procedures. They included sleeping time in mice, rotor rod balance in rats and lever pressing behaviour of rats.2. Equimolar concentrations of pyrazole and 3-methylpyrazole were compared for effects on enhancement of ethanol's activity on rotor rod holding time of rats.3. Minimally effective doses of pyrazole, the LADH inhibitor, and 3-methylpyrazole, a non-inhibitor of LADH, when administered before ethanol, resulted in an increased behavioural depression. These interaction effects are probably not caused by inhibition of LADH but rather by an increase in the direct depressant action of either one or both of the compounds.     

The effects of stimulants and depressants on cocaine self-administration behavior in the Rhesus monkey

The effects of acute intramuscular pretreatment with several dosages of a variety of centrally acting compounds on intravenous cocaine self-administration behavior were ascertained. Pretreatment with morphine and pentobarbital produced no change in this behavior until dosages (2.0 mg/kg and 15.0 mg/kg respectively) were administered which grossly depressed grooming, exploratory, and locomotor activity behaviors, d-amphetamine (0.5–4.0 mg/kg) and phenmetrazine (2.0–12.0 mg/kg) pretreatment produced a dose-related decrease in cocaine self-administration. Trifluoperazine in dosages of 0.01–0.1 mg/kg increased the frequency of this behavior; whereas, higher dosages (0.2, 0.4 mg/kg) grossly depressed behavior. Imipramine (10–50 mg/kg) produced a dose-related decrease in cocaine self-administration. Potential mechanisms of these drug—behavior and drug—drug interactions are discussed.This study was supported by NIMH Grant No. MH-12084 and by NIMH Grant No. MH-18245.