Time course and role of extracellular Ca2+ in veratridine-induced glutamate release.

The veratridine-induced release of glutamate in the striatum of anaesthetized rats was studied using regional brain microdialysis to elucidate the role of extracellular Ca2+ and to investigate the exact time course of the glutamate response. In a first series of experiments with Ca(2+)-depletion starting simultaneously with the veratridine pulse no significant differences were found under Ca(2+)-replete and Ca(2+)-free conditions. Strikingly, under both conditions glutamate efflux followed a biphasic time course. When Ca2+ depletion preceded veratridine stimulation a significant inhibition of the initial glutamate release was found, indicating that the first glutamate portion originates from Ca(2+)-dependent vesicular exocytosis. The nature of the second and longer lasting phase of glutamate efflux is not clear, but it may be mediated by reversal of the glutamate uptake system of the plasma membrane located both on neurones and on astrocytes. This points to a possibly important contribution of glial cells to glutamate overflow under pathological conditions and should be subject to further investigations.

Effects of exogenous factors on the cerebral glutathione in rodents.

Since glutathione is thought to be involved in cerebral functions, changes in the glutathione level imply modulations of the neurotransmission in addition to all the known effects of GSH. It was investigated whether alterations of the cerebral glutathione can be induced by consumption of GSH, by inhibition or stimulation of the synthesis of GSH, or by an inhibition of the re-reduction of the oxidized glutathione. Aminophenazone, propyphenazone, acetaminophen, phenytoin, morphine and nitrofurantoin, known to deplete hepatic GSH, had no effects on cerebral GSH. Diethyl maleate (0.6 ml/kg) decreased the cerebral content of GSH and GSSG in adult rats as well as in fetuses. The depletion of the cerebral GSH caused by diethyl maleate treatment for 4 days was followed by an increase up to 125% and a subsequent return to the normal level after 1 week. In rats starved up to 71 h deficiency of exogenous amino acids caused only a minimal or no decrease in cerebral GSH. The specific inhibitor of the gamma-glutamylcysteine synthetase BSO only depleted GSH in the brain of young mice following the repeated s.c. administration of a high dose (890 mg/kg). After cobaltous chloride (20 mg/kg; twice a day for 2 or 4 days) the GSH level in the brain was unchanged. In vivo inhibition of the cerebral glutathione reductase was caused by ammonium metavanadate (12.5 mg/kg; three times a week for 6 weeks). Nitrofurantoin (150 mg/kg) had no effect. After lomustine (10 mg/kg) a minimal increase in glutathione reductase was found, but simultaneously also an increase in GSSG and of the ratio GSSG/total glutathione.

Biphasic effect of pyrazolone derivatives on drug-metabolizing enzyme in rat liver.

The pyrazolone derivatives aminophenazone, phenazone, and propyphenazone known as inducers are capable of inhibiting initially monooxygenase-dependent biotransformation steps. In the dose of 1.5 mmol X kg-1 they prolong the hexobarbital narcosis (max. 1 h after administration) due to a reduced hexobarbital metabolism in the liver. An influence on the N-demethylation (aminophenazone as substrate) ist not substantial. The catalytic binding site of cytochrome P-450 is not influenced. In aminophenazone- and phenazone-treated animals the inhibitory phase is followed by a stimulatory one. After the repeated administration the inhibitory phase continues up to the third measured 1 h after administration. The initially inhibitory effect of inducers seems to be caused by a mutual interference of a complete or partial binding to various forms of cytochrome P-450. However, effects on the CNS cannot be excluded.

Hepatotoxicological studies of pyrazolone derivatives on rats. Part 3: Quantitative immunologic determination of serum concentration of transferrin, IgG, alpha 2-acute phase protein and VLDL.

At present no sufficiently sensitive liver function tests are available to exactly detect small liver impairments caused by drugs. In this respect immunological quantitation of serum proteins could be useful. We have determined quantitative alterations of four serum proteins (IgG, transferrin, alpha 2-AP = alpha 2-acute phase protein, VLDL = very low density lipoprotein) from rats treated with aminophenazone, phenazone and propyphenazone (each 1,55 mmol/kg body mass/d). The serum concentration of the four proteins examined is temporarily elevated immediately after onset of treatment. In chronic treatment transferrin is increased by the influence of all drugs investigated. Propyphenazone treatment increased the amount of VLDL whereas aminophenazone had the opposite effect, phenazone is without influence in the same period. The alpha 2-AP serum level is decreased in long time treatment, IgG remains unchanged. The observed alterations indicate disturbances of liver function in the investigated rats. Among the drugs used, aminophenazone has the strongest, propyphenazone the smallest liver damaging effect. The results reported support the importance of serum protein quantitation in connection with the search for possible liver damage due to drug action.

[Hepatotoxicological studies of pyrazolone derivatives on rats. Part 2: The study of propyphenazone and phenazone (author's transl)]. / Lebertoxikologische Untersuchungen von Pyrazolonderivaten an Ratten. 2. Mitteilung: Untersuchung von Propyphenazon und Phenazon.

In female rats the administration of 1,5 mmol/kg of, respectively, propyphenazone and phenazone over a period of 12 weeks produces a histologically detectable fatty degeneration and reactive-inflammatory changes of the liver, presumably caused by reactive metabolites. These slight morphological alterations do not lead to an increase in plasma enzyme activities. Only in case of phenazone administration, reduced increases in body weight are indicative of a toxic effect. The acceleration of hexobarbital degradation and N-demethylation and the increase in liver weight testify to the inductive activity of both these compounds.

Fluorescent substrate analogue for adenosine deaminase: 3'-O-[5-(dimethylamino)naphthalene-1-sulfonyl]adenosine.

The synthesis of the fluorescent derivative of adenosine, by reaction with 5-(dimethylamino)naphthalene-1-sulfonyl chloride in dry pyridine at low temperature, yielding 3'-O-[5-(dimethylamino)naphthalene-1-sulfonyl]adenosine (3'-O-dansyladenosine), is here described. 3'-O-Dansyladenosine is partially soluble in water (approximately 10(-4) M) and upon excitation at 325 nm exhibits maximum fluorescence emission at 516 +/- 22 nm (corrected) in buffered aqueous solution at pH 7.6 with a quantum yield of 0.21 and a lifetime of 11.8 +/- 0.2 ns. The fluorescence of 3'-O-dansyladenosine is sensitive to the polarity of its solvent: in pyridine, a quantum yield of 0.61 at the emission maximum of 435 nm was observed. 3'-O-Dansyladenosine is a reversible competitive inhibitor of adenosine deaminase with a moderate inhibitive dissociation constant K1 = (1.54 +/- 0.13) X 10(-5) M. The enzyme-substrate analogue association constant was determined by equilibrium dialysis to be K = (0.69 +/- 0.05) X 10(5) M-1, very close to KI-1. The hydrophobic nature of its binding site in adenosine deaminase is evident from the strong blue shift of the fluorescence emission maximum to 440 nm, the 4-fold increase in fluorescence quantum yield, and the longer lifetime of 15.8 +/- 0.2 ns; the tight, rigid nature of the complex is evident from its high fluorescence polarization value, 0.23. An 85% decrease in the fluorescence emission intensity of the adenosine deaminase-3'-O-dansyladenosine complex in the presence of adenosine indicates the selective binding to the enzyme active site. Correlation between the conformation of the probe, either when free in various solvents or when bound to the enzyme, and its fluorescence quantum yield is noted. 3'-O-Dansyladenosine is suitable for fluorescent labeling of adenosine deaminase in cell systems.

[Hepatotoxicological studies of pyrazolone derivatives on rats. Part 1: The study of aminophenazone (author's transl)]. / Lebertoxikologische Untersuchunen von Pyrazolonderivaten an Ratten. 1. Mitteilung: Untersuchung von Aminophenazon.

After oral application of aminophenazone to animals (170 and 340 mg/kg daily, over up to 17 weeks), the authors investigated the effect of this drug on some liver functions and performed also a morphological study. They found that aminophenazone produces an increase of the smooth ER, peripheral fatty degradation and reactive-inflammatory responses of the liver, the intensity of these phenomena being dependent upon the dose and time of application. The increase of the smooth ER is the expression of the inductive effect which persists throughout the whole experimental period and manifests itself by accelerated degradation of hexobarbital, increased N-demethylation, increased ascorbic acid synthesis and increased liver weight. The increase in body weight is reduced in the animals treated. The enzyme activities in the plasma lie within the range observed with control animals; they are no indicator of the slight live changes stated. Reactive metabolites and/or the impairment of other metabolic reactions may be considered to be the cause of the hepatotoxic effect of aminophenazone.

The excretion of ascorbic acid and hippuric acid in the urine of rats with liver injury.

The excretion of ascorbic acid and hippuric acid in the urine as liver function tests has no marked advantages when compared with other tests (activities of serum enzymes glutamate dehydrogenase and L-alanine: 2-oxoglutarate aminotransferase, hexobarbital sleeping time).

Evaluation of liver function tests after administration of aminophenazone.

Known liver function tests (activities of ASAT = L-Aspartate: 2-oxoglutarate aminotransferase, ALAT = L-Alanine: 2-oxoglutarate aminotransferase, GLDH = Glutamate-dehydrogenase, LDH = Lactate-dehydrogenase in serum) are not qualified for detecting minimal histopathological liver alterations induced by aminophenazon.

[Bromosulfan and indocyanine green test in liver-damaged rats]. / Bromsulfan- und Indozyaningrün-Test bei lebergeschädigten Ratten.

The susceptibility and valuation of bromsulphthalein- and indocyanine green-test in liver damaged rats was investigated. The removal of BSP from the blood after the administration of carbon tetrachloride (0,1 ml/kg) showed already a delay after two weeks, while the content of ICG was only slightly altered. Both impaired uptake or transport mechanisms into liver cells and impaired metabolism or elimination processes can be indicated by these findings. After application of aminophenazone (340 mg/kg), resulting in histological detectable lesions of liver, an accelerated elimination of the two dyes from the blood was observed, which was significant for BSP and may be caused by an induction effect.

[The excretion of hippuric acid and ascorbic acid in the urine of liver-damaged rats (author's transl]. / Die Ausscheidung von Hippursäure und Ascorbinsäure im Urin bei lebergeschädigten Ratten.

The authors investigated the effects of the administration of thioacetamide, carbon tetrachloride and aminophenazone on the excretion of ascorbic acid and hippuric acid in adult male and female Wistar rats. After a single application of thioacetamide and aminophenazone, the ascorbic acid content in the urine showed a dose-dependent increase, whereas that in the liver had decreased. This increase in the urinary ascorbic acid might be due to a release of stored ascorbic acid from the liver cells. When thioacetamide was given for a prolonged period, the ascorbic acid content in the urine increased at the beginning; later one, at the end of three weeks, it was slightly inferior to the control value. Both single and repeated applications of thioacetamide led to a decrease in the excretion of hippuric acid in the urine, which is attributed to an impairment of the mitochondrial hippuric acid synthesis. Long-term treatment with aminophenazone resulted in an increase of ascorbic acid in the urine, which is indicative of an induction effect, whereas the ascorbic acid content in the liver remained unchanged. There was no effect on the excretion of hippuric acid. In regard to their use in the toxicological evaluation of drugs, these two metabolic effects offer no decisive advantage over current liver function tests.