Morris D. Faiman

Role of taurine in regulation of intracellular calcium level and neuroprotective function in cultured neurons

Glutamate‐induced excitotoxicity has been implicated as an important mechanism underlying a variety of brain injuries and neurodegenerative diseases. Previously we have shown that taurine has protective effects against glutamate‐induced neuronal injury in cultured neurons. Here we propose that the primary underlying mechanism of the neuroprotective function of taurine is due to its action in preventing or reducing glutamate‐induced elevation of intracellular free calcium, [Ca2+]i. This hypothesis is supported by the following findings. First, taurine transport inhibitors, e.g., guanidinoethyl sulfonate and β‐alanine, have no effect on taurines neuroprotective function, suggesting that taurine protects against glutamate‐induced neuronal damage through its action on the extracellular membranes. Second, glutamate‐induced elevation of [Ca2+]i is reduced to the basal level upon addition of taurine. Third, pretreatment of cultured neurons with taurine prevents or greatly suppresses the elevation of [Ca2+]i induced by glutamate. Furthermore, taurine was found to inhibit the influx but not the efflux of 45Ca2+ in cultured neurons. Taurine has little effect on the binding of [3H]glutamate to the agonist binding site and of [3H]MDL 105,519 to the glycine binding site of the N‐methyl‐D‐aspartic acid receptors, suggesting that taurine inhibits 45Ca2+ influx through other mechanisms, including its inhibitory effect on the reverse mode of the Na+/Ca2+ exchangers (Wu et al. [ 2000 ] In: Taurine 4: taurine and excitable tissues. New York: Kluwer Academic/Plenum Publishers. p 35–44) rather than serving as an antagonist to the N‐methyl‐D‐aspartic acid receptors. J. Neurosci. Res. 66:612–619, 2001.

In vitro and in vivo inhibition of rat liver aldehyde dehydrogenase by S-methyl N,N-diethylthiolcarbamate sulfoxide, a new metabolite of disulfiram

In summary, these data provide the first evidence that DETC-MeSO is a natural metabolite of disulfiram, and a potent inhibitor of rat liver mitochondrial low Km ALDH both in vitro and in vivo. It is therefore proposed that, based upon evidence to date, DETC-MeSO appears to be the chemical species to which disulfiram must be bioactivated, and is the metabolite most likely responsible for disulfirams inhibition of rat liver mitochondrial low Km ALDH in vivo. Characterization of the properties of DETC-MeSO as the metabolite responsible for disulfirams action as an ALDH inhibitor is presently in the process of being completed.

Disulfiram metabolism as a requirement for the inhibition of rat liver mitochondrial low Km aldehyde dehydrogenase

In humans and animals, disulfiram produces a disulfiram-ethanol reaction after an ethanol challenge, the basis of which is the inhibition of liver aldehyde dehydrogenase (ALDH). Disulfiram and the metabolites diethyldithiocarbamate (DDTC), diethyldithiocarbamate-methyl ester (DDTC-Me), and S-methyl-N,N-diethylthiolcarbamate (DETC-Me) were studied in order to determine the role of bioactivation in disulfirams action as an inhibitor of rat liver mitochondrial low Km ALDH (RLM low Km ALDH). In in vitro studies, disulfiram and DDTC (0.01 to 2.0 mM) both inhibited RLM low Km ALDH in a concentration-dependent manner. The addition of rat liver microsomes to the mitochondrial incubation did not further increase disulfiram-induced RLM low Km ALDH inhibition. However, DDTC-induced RLM low Km ALDH inhibition was increased further, but only at DDTC concentrations less than 0.05 mM. DDTC-Me and DETC-Me (2.0 mM) similarly exhibited an increased RLM low Km ALDH inhibition after the addition of liver microsomes. In in vivo studies, disulfiram (75 mg/kg), DDTC (114 mg/kg), DDTC-Me (41.2 mg/kg) or DETC-Me (18.6 mg/kg) administered i.p. to female rats inhibited RLM low Km ALDH. Inhibition of drug metabolism by pretreatment of rats with the cytochrome P450 inhibitor N-octylimidazole (NOI) (20 mg/kg, i.p.) prior to either disulfiram, DDTC, DDTC-Me or DETC-Me administration blocked the inhibition of RLM low Km ALDH. The in vitro and in vivo data support the conclusion that bioactivation of disulfiram to a reactive chemical species is required for RLM low Km ALDH inhibition and a disulfiram-ethanol reaction.

Carbamoylation of Brain Glutamate Receptors by a Disulfiram Metabolite

S-Methyl-N,N-diethylthiolcarbamate sulfoxide (DETC-MeSO), a metabolite of the drug disulfiram, is a selective carbamoylating agent for sulfhydryl groups. Treatment of glutamate receptors isolated from mouse brain with DETC-MeSO blocks glutamate binding. In vivo, carbamoylated glutathione, administered directly to mice or formed by reaction of DETC-MeSO with glutathione in the blood, also blocks brain glutamate receptors. Carbamoyl groups appear to be delivered to brain glutamate receptors or to liver aldehyde dehydrogenase in vivo by a novel glutathione-mediated mechanism. Seizures caused by the glutamate analogs N-methyl-d-aspartate and methionine sulfoximine, or by hyperbaric oxygen, are prevented by DETC-MeSO, indicating that carbamoylation of glutamate receptors gives an antagonist effect. These observations offer an explanation for some of the previously reported neurological effects of disulfiram, such as its ability to prevent O2-induced seizures. Furthermore, some of the physiology of the disulfiram-ethanol reaction, that could not be accounted for based on the known inhibition of aldehyde dehydrogenase alone, may be explained by disulfiram’s effect on glutamate receptors.

Comparative aspects of disulfiram and its metabolites in the disulfiram-ethanol reaction in the rat

Diethyldithiocarbamate-methyl ester (DDTC-Me), a metabolite of disulfiram, has been shown recently to produce a disulfiram-ethanol reaction (DER). Studies were carried out to compare the ethanol-sensitizing properties of DDTC-Me with those of disulfiram and diethyldithiocarbamate (DDTC) in the rat. All three drugs inhibited liver mitochondrial low Km aldehyde dehydrogenase (ALDH) in vivo, with maximal ALDH inhibition occurring 8 hr after drug administration. The onset of ALDH inhibition was most rapid after DDTC-Me administration. ALDH was inhibited approximately 50% 0.5 hr after DDTC-Me, whereas ALDH was inhibited only 5 and 10%, respectively, after disulfiram and DDTC. Not until 8 hr after drug treatment was ALDH inhibition the same for disulfiram, DDTC and DDTC-Me. The degree of ALDH inhibition from 8 to 172 hr after dosing was the same for all three drugs. An ethanol (1 g/kg, 20% v/v) challenge administered to rats treated with disulfiram (75 mg/kg), DDTC (114 mg/kg), or DDTC-Me (41.2 mg/kg) for 8 hr produced similar blood acetaldehyde/ethanol concentration-time profiles. In addition, all three agents produced a DER (hypotension, tachycardia). No DER occurred if ethanol was administered more than 24 hr after drug pretreatment. The hypotension associated with the DER correlated with the increased blood acetaldehyde but not blood ethanol. A threshold blood acetaldehyde of 110 microM appeared to be required for hypotension to occur, and this was related to ALDH inhibition of approximately 40%. The tachycardia associated with the DER correlated more with blood ethanol. After DDTC-Me administration, no disulfiram or DDTC could be detected in the plasma. Furthermore, no DDTC-Me was found in the plasma 8 hr after DDTC-Me administration, suggesting that no correlation exists between the DER and plasma concentration of DDTC-Me and most likely disulfiram. These data suggest that the alcohol-sensitizing properties of DDTC-Me are similar to those observed with disulfiram and DDTC. Since DDTC-Me is an active metabolite and more potent than disulfiram and DDTC in producing a DER, disulfiram metabolism is an important consideration in the disulfiram-ethanol reaction.

Free radical formation and lipid peroxidation in rat and mouse cerebral cortex slices exposed to high oxygen pressure

Abstract Free radical formation and lipid peroxidation were investigated in rat and mouse cerebral cortex slices exposed to O2 at high pressure. Free radical formation as detected by electron paramagnetic resonance spectroscopy, and lipid peroxidation as reflected by malondialdehyde (MDA) formation, increased with increasing O2 pressures. Greater amounts of MDA were formed in mouse than in rat brain slices exposed to O2. It is concluded that O2 exposure initiates free radical formation and subsequently lipid peroxidation, which may lead to cellular damage and seizure onset.

Bioactivation of S-methyl N,N-Diethylthiolcarbamate to S-methyl N,N-diethylthiolcarbamate sulfoxide: Implications for the role of cytochrome P450

Diethyldithiocarbamate (DDTC), diethyldithiocarbamate methyl ester (DDTC-Me), S-methyl N,N-diethylthiolcarbamate (DETC-Me) and S-methyl N,N-diethylthiolcarbamate sulfoxide (DETC-MeSO) are all metabolites of disulfiram. All inhibit rat liver low Km aldehyde dehydrogenase (ALDH) in vivo, with the order of potency being DETC-MeSO > DETC-Me > DDTC-Me > DDTC. Studies were carried out both in vivo and in vitro to further investigate the role of bioactivation as a requirement for the action of disulfiram as a liver ALDH inhibitor. The cytochrome P450 inhibitor 1-benzylimidazole (NBI) was employed as a pharmacological tool to study the metabolism of DETC-Me to DETC-MeSO. Administration of NBI to rats prior to DETC-Me treatment blocked the inhibition of liver mitochondrial low Km ALDH by DETC-Me. This was accompanied by an increase in plasma DETC-ME and a decrease in plasma DETC-MeSO. Pretreatment of rats with NBI prior to DETC-MeSO administration did not block the inhibition of liver mitochondrial low Km ALDH by DETC-MeSO. In in vitro studies, the inclusion of NBI in an incubation containing rat liver microsomes, mitochondria and an NADPH-generating system blocked the formation of DETC-MeSO and inhibition of liver mitochondrial low Km ALDH by DETC-Me. DETC-MeSO was found to be a potent inhibitor of rat liver mitochondrial low Km ALDH both in vivo and in vitro. The data suggest that the metabolism of DETC-Me to DETC-MeSO is mediated by cytochrome P450, and that inhibition of cytochrome P450 by inhibitors such as NBI block the inhibition of low Km ALDH by DETC-Me.

Role of flavin-dependent monooxygenases and cytochrome P450 enzymes in the sulfoxidation of S-methyl N,N-diethylthiolcarbamate

Disulfiram is bioactivated to S-methyl N,N-diethylthiolcarbamate sulfoxide (DETC-MeSO), the metabolite proposed to be responsible for the action of disulfiram as an aldehyde dehydrogenase inhibitor. This bioactivation process includes a reduction, an S-methylation, and two successive oxidations. Sulfur-containing functional groups are substrates for cytochrome P450 enzymes or flavin-containing monooxygenases (FMO). In the present study, we investigated the contribution of these monooxygenases to the formation of DETC-MeSO from its immediate precursor S-methyl N,N-diethylthiolcarbamate (DETC-Me). Liver microsomes obtained from mature male rats were incubated with DETC-Me. The formation of DETC-MeSO was blocked completely by solubilization of the microsomes with the detergent Emulgen 911, or by the presence of the cytochrome P450 inhibitor 1-benzylimidazole. However, thermal-inactivation of FMO resulted in only a partial loss in DETC-MeSO formation. Liver microsomes from phenobarbital-treated rats showed a 4- to 5-fold increase in the rate of formation of DETC-MeSO, compared with controls. Liver microsomes from pyrazole-treated rats showed a 50% decrease in the sulfoxidation of DETC-Me compared with controls. In a purified reconstituted system, cytochrome P450 2B1 (CYP2B1) catalyzed the formation of DETC-MeSO at a rate of 51 nmol DETC-MeSO formed/min/nmol cytochrome P450. Antibodies to CYP2B1 caused a 60% inhibition of DETC-MeSO formation by liver microsomes from phenobarbital-treated rats. These results suggest that in male rat liver microsomes, cytochrome P450 plays a major role in catalyzing the sulfoxidation of DETC-Me, whereas FMO plays a minor role (< 10%). Also, in liver microsomes from phenobarbital-treated rats, CYP2B1 is the major catalyst for the sulfoxidation of DETC-Me.

S-methyl-N,N-diethylthiolcarbamate: a disulfiram metabolite and potent rat liver mitochondrial low Km aldehyde dehydrogenase inhibitor.

S-methyl-N,N-diethylthiolcarbamate-methyl ester (DETC-Me), a proposed disulfiram metabolite, was investigated both in vivo and in vitro for its effectiveness as a liver mitochondrial low Km aldehyde dehydrogenase (L Km ALDH) inhibitor. Male Sprague-Dawley rats were treated intraperitoneally with DETC-Me, killed at various times and L Km ALDH determined. DETC-Me was found to be a more potent in vivo inhibitor of L Km ALDH than either disulfiram, diethyldithiocarbamate (DDTC) or diethyldithiocarbamate-methyl ester (DDTC-Me). The ID50 for DETC-Me, DDTC-Me and disulfiram was 6.5, 15.5 and 56.2 mg/kg, respectively. The ID50 for DDTC was similar to DDTC-Me. Maximal inhibition of L Km ALDH occurred 30 minutes after DETC-Me administration. DETC-Me was ineffective as an in vitro inhibitor. DETC-Me produced a marked disulfiram-ethanol reaction (DER) at one-quarter of the dose of disulfiram or DDTC. Plasma DETC-Me in rats was greater after DETC-Me administration than after DDTC-Me, DDTC or disulfiram. In conclusion, DETC-Me is proposed to be a metabolite of disulfiram, and may be the immediate precursor of the chemical species responsible for L Km ALDH inhibition.

The role of lipid, free radical initiator, and oxygen on the kinetics of lipid peroxidation

Abstract The relationship between the concentration of unsaturated lipid, free radical initiator, and oxygen concentration on the kinetics of lipid peroxidation was determined. The rate of lipid peroxidation was studied with the thiobarbituric acid (TBA), diene conjugation (DC), and ferrithiocyanate (Fe-SCN) methods. The rate of peroxidation was half-order with respect to unsaturated lipid, initiator, and oxygen. The half-order relationship could be expressed as: rate = ( fk 1 k 2 k 3 k 6 1 2 ( azobisisobutyronitrile ) 1 2 ( RH 1 2 ( O 2 ) 1 2 . The half-order relationship was found with linoleic (18:2), linolenic (18:3), and arachidonic (20:4) acids. A linear relationship existed between the logarithm of unsaturation and the rate of peroxidation. No peroxidation of linolenic acid was indicated when the DC method was employed, but was when the TBA and Fe-SCN methods were used.