Effect of chlorophenoxyacetic acid herbicides on glycine conjugation of benzoic acid.

1. 2,4-Dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) (0.1-0.5 mmol/kg i.p.) delayed the disappearance of injected benzoate from blood and diminished the urinary excretion of the formed benzoylglycine, but elevated the blood levels of benzoylglycine in rat, suggesting that these herbicides interfere with both the formation and the renal transport of benzoylglycine. 2. Inhibition of the renal excretion of benzoylglycine by 2,4-D or 2,4,5-T (0.5 mmol/kg i.p.) was directly demonstrated in rat injected with benzoylglycine. 3. Inhibition of benzoylglycine formation from benzoic acid by 2,4-D or 2,4,5-T (0.5 mmol/kg i.p.) was directly demonstrated in renal pedicles-ligated rats injected with benzoate. 4. Neither 2,4-D nor 2,4,5-T influenced the hepatic concentrations of ATP, coenzyme A (CoA) or glycine; therefore, it is unlikely that they inhibit glycine conjugation of benzoic acid by diminishing the availability of co-substrates. 5. Although the chlorophenoxyacetic acids did not appear to be a substrate for the mitochondrial acyl-CoA synthetases, both 2,4-D and 2,4,5-T diminished the activity of benzoyl-CoA synthetase (but not that of benzoyl-CoA:glycine N-acyltransferase) in solubilized hepatic mitochondria. These findings suggest that 2,4-D and 2,4,5-T impair benzoylglycine formation in rat by inhibiting benzoyl-CoA synthetase.

Effects of fibrates on the glycine conjugation of benzoic acid in rats.

In the course of glycine conjugation, benzoic acid is successively converted into benzoyl-CoA and benzoylglycine by mitochondrial enzymes (i.e. benzoyl-CoA synthetase and benzoyl-CoA/glycine N-acyltransferase, respectively), utilizing ATP, CoA, and glycine. Large doses of benzoate deplete CoA from the liver, suggesting that the supply of CoA may limit the capacity for glycine conjugation. Because fibrates are known to increase hepatic CoA synthesis, we examined whether treatment with fenofibrate or bezafibrate enhanced the capacity of rats to conjugate benzoic acid with glycine. Dietary administration of fenofibrate or bezafibrate (2.5 mmol/kg of feed, for 10 days) increased hepatic CoA levels 8-10-fold, while not affecting hepatic ATP levels; only fenofibrate elevated, albeit moderately, the concentration of glycine in liver. Hepatic mitochondria isolated from fibrate-fed rats, compared with those from controls, exhibited unchanged benzoyl-CoA synthetase activity but higher benzoyl-CoA hydrolase and lower benzoyl-CoA/glycine N-acyltransferase activities. Feeding with either fibrate increased liver mass by 50-60%. Control and fibrate-fed rats were administered benzoate at different doses, one to produce a large demand for CoA (i.e. 2 mmol/kg, iv) and two others to produce smaller demands for CoA (i.e. 1 mmol/kg or 2 mmol/kg plus glycine, iv). Fenofibrate-fed rats, and to a lesser extent bezafibrate-fed animals, exhibited increased glycine conjugation capacity, as indicated by faster disappearance of benzoate from the blood and appearance of benzoylglycine in the blood and urine, compared with controls; however, fibrates were not more effective in rats receiving the benzoate dose that produced the greatest demand for CoA. In contrast, benzoylglycine formation from benzoate (0.1-1 mM) was not enhanced in liver slices from fibrate-fed rats; moreover, it was lower than control levels in slices from bezafibrate-fed animals. Bezafibrate, but not fenofibrate, given to rats in a single dose (0.5 mmol/kg, ip) decreased the elimination and glycine conjugation of benzoate, indicating that bezafibrate is a direct inhibitor of glycine conjugation. In summary, fibrates influence glycine conjugation in a complex manner. Some fibrate-induced alterations (i.e. increased benzoyl-CoA hydrolase and decreased glycine transferase activities and direct inhibition by bezafibrate) can potentially hinder conjugation of benzoate with glycine, thus precluding conclusions regarding whether increased CoA availability enhances glycine conjugation. Fibrate-induced hepatomegaly appears to significantly contribute to the increased glycine conjugation capacity of rats treated with fenofibrate or bezafibrate.

Does hepatic ATP depletion impair glycine conjugation in vivo?

Conjugation with glycine, a reaction important in the elimination of carboxylic acids (e.g. benzoic and salicylic acids), takes place in hepatic mitochondria and uses ATP, coenzyme A, and glycine. Although normal ATP supply does not limit glycine conjugation in vivo (Gregus, Z., et al., Drug Metab. Dispos. 20, 234, 1992), ATP deficiency may impair it. This hypothesis was tested by examining the effect of ATP depletors (oligomycin, fructose, and ethionine) on glycine conjugation and elimination of benzoic acid in rats. Pretreatment with the mitochondrial ATP synthesis inhibitor oligomycin (0.5-2 mg/kg, intraportally) decreased glycine conjugation of benzoic acid markedly and in a dose-dependent manner, as indicated by the delayed elimination of benzoate and delayed appearance of benzoylglycine in blood. Oligomycin also dramatically diminished urinary excretion of benzoylglycine, because it inhibited not only formation of benzoylglycine from benzoate, but also the renal transport of benzoylglycine. Treatment with fructose (a consumer of both cytosolic and mitochondrial ATP) or ethionine (a consumer of cytosolic ATP) depleted hepatic ATP from approximately 2.5 micromol/g to levels comparable with those observed after administration of 1 mg/kg oligomycin (approximately 1.2 micromol/g). Despite this, elimination of benzoate and formation of benzoylglycine were decreased less by fructose than by oligomycin and only negligibly by ethionine. ATP depletors did not influence hepatic glycine levels, and only oligomycin lowered coenzyme A levels in liver. However, the oligomycin-induced decline of hepatic coenzyme A levels was delayed, contrary to impairment of glycine conjugation, which was almost immediate. In summary, impairment of benzoylglycine formation by ATP depletors apparently correlates with their capacity to diminish ATP levels in hepatic mitochondria (i.e. at the site of glycine conjugation). These observations suggest that limited availability of mitochondrial (but not cytosolic) ATP reduces glycine conjugation capacity. Therefore, mitochondrium toxic agents and pathological mitochondrial injuries acting in liver may compromise glycine conjugation by decreasing ATP supply.

Lipoic acid impairs glycine conjugation of benzoic acid and renal excretion of benzoylglycine.

Glycine conjugation of benzoic acid is catalyzed by the mitochondrial enzymes benzoyl-coenzyme A(CoA) synthetase and benzoyl-CoA: glycine N-acyltransferase and requires ATP, CoA, and glycine as cosubstrates. Lipoic acid (LA), an important endogenous and also therapeutic compound, depletes hepatic CoA; therefore, it may interfere with glycine conjugation. To test this hypothesis, LA (0.5-1.5 mmol/kg ip) was given to anesthetized, glycine-loaded rats 1 hr before administration of benzoic acid (1 mmol/kg iv). LA inhibited glycine conjugation of benzoic acid in a dose-dependent manner as indicated by: 1) reduced clearance of benzoic acid from blood; 2) delayed appearance of benzoylglycine in blood; and 3) decreased excretion of benzoylglycine in urine. LA also decreased urinary excretion of injected benzoylglycine, indicating that reduced excretion of this metabolite after benzoic acid injection is caused by diminished formation and impaired renal transport of benzoylglycine. Urine formation was decreased by LA in a dose-dependent fashion, and acute renal failure was evident in rats receiving the highest dose. LA depleted hepatic CoA, carnitine, and glutathione, but not ATP, whereas it increased the hepatic concentration of glycine. In isolated and solubilized rat liver mitochondria, LA inhibited both benzoyl-CoA synthetase (IC50 approximately 1.5 mM) and benzoyl-CoA:glycine N-acyltransferase (IC50 approximately 0.3 mM). Thus, depletion of CoA and inhibition of the pertinent enzymes seem responsible for impairment of glycine conjugation of benzoic acid by LA. LA may also impair renal tubular cell function, compromising the tubular secretion of benzoylglycine and causing acute renal failure.

ANP(1-28), BNP(1-32) and CNP(1-22) increase the severity of picrotoxin-kindled seizure syndrome in rats.

The administration of rANP(1-28) in doses of 1.0 and 2.0 nmol (but not 0.2 nmol) into the lateral cerebral ventricle (i.c.v) of rats preliminarily kindled with picrotoxin resulted in an increase of the severity of picrotoxin-kindled convulsions 24 hrs after injection of the peptide. I.c.v. injection of pBNP(1-32) also resulted in a proepileptic effect when it was applied in the same doses with a similar time course; the increased seizure severity was observed 48 hrs after injection of pBNP in a dose of 2 nmol. I.c.v. administration of CNP(1-22) in a dose of 2 nmol induced an increase in the severity of picrotoxin-kindled convulsions 24 and 48 hrs after application of the peptide. None of the peptides influenced the seizure syndrome immediately after the injections. It is presumed that the delayed proepileptic properties of the three natriuretic peptides could be caused by some of their stable fragments which accumulate during their metabolism.

Anticonvulsive effects of galanin administered into the central nervous system upon the picrotoxin-kindled seizure syndrome in rats.

Galanin (2000, 1000 ng) administered into the lateral brain ventricle decreased the severity of picrotoxin-kindled convulsions in rats. The bilateral injection (200, 100 and 50 ng) of galanin into the hippocampus also evoked an anticonvulsive effect. When administered into the caudate nuclei or substantia nigra reticulata, galanin exerted anticonvulsive action only in a high dose (200 ng), whereas in the nucleus accumbens it did so in a low dose (50 ng). When administered into the ventral tegmental area in a dose of 50, 100 or 200 ng galanin failed to reduce the manifestations of picrotoxin-kindled seizures.