Identification of the heme adduct and an active site peptide modified during mechanism-based inactivation of rat liver cytochrome P450 2B1 by secobarbital.

The olefinic barbiturate secobarbital (SB) is a sedative hypnotic known to be a relatively selective mechanism-based inactivator of rat liver cytochrome P450 2B1. Previous studies have demonstrated that such inactivation results in prosthetic heme destruction and irreversible drug-induced protein modification, events most likely triggered by P450 2B1-dependent oxidative activation of the olefinic pi-bond. However, the precise structure of the SB-modified heme and/or the protein site targeted for attack remained to be elucidated. We have now isolated the SB-heme adduct from P450 2B1 inactivated by [14C]SB in a functionally reconstituted system and structurally characterized it by electronic absorption spectroscopy and tandem collision-induced dissociation (CID), matrix-assisted laser desorption ionization on time of flight (MALDI-TOF), and liquid secondary ion mass spectrometry in the positive mode (+ LSIMS) as the N-(5-(2-hydroxypropyl)-5-(1-methylbutyl)barbituric acid)protoporphyrin IX adduct. The [14C]SB-modified 2B1 protein has also been isolated from similar inactivation systems and subjected to lysyl endopeptidase C (Lys-C) digestion and HPLC-peptide mapping. A [14C]SB-modified 2B1 peptide was thus isolated, purified, electrotransferred onto a poly-(vinylidene) membrane, and identified by micro Edman degradation of its first N-terminal 17 residues (S277NH(H)TEFH(H)ENLMISLL293) as the Lys-C peptide domain comprised of amino acids 277-323. This peptide thus includes the peptide domain corresponding to the distal helix I of P450 101, a region highly conserved through evolution, and which is known not only to flank the heme moiety but also to intimately contact the substrates. This finding thus suggests that SB-induced protein modification of P450 2B1 also occurs at the active site and, together with heme N-alkylation, contributes to the SB-induced mechanism-based inactivation of P450 2B1.

Clotting activity in the rat following acute barbiturate treatment.

Adult male rats were subjected to intraperitoneal (i.p.) saline (controls) or barbiturates (20 mg/rat) as a single injection. Seventeen hours later the rats were sacrificed and plasma collected. Hematocrit (HCT), Platelet count (PLT), prothrombin time (PT), partial thromboplastin time (APTT), and coagulant activity for factors II, V, VII, VIII, X and XII, plus fibrinogen were determined. The data indicates that a single injection of phenobarbital and secobarbital had a greater effect on clotting activity than did barbital or amobarbital. This was primarily reflected in the hepatic synthesized clotting factors, plus the platelets.

Alterations in integrity of goldfish membrane induced by edetate disodium.

The effect of the chelating agent edetate disodium on the integrity of the goldfish membrane was examined. The time to produce death in goldfish exposed to secobarbital sodium was used as a reflection of membrane integrity. Although a minimum edetate disodium concentration was necessary to induce alterations in integrity, no direct relationship between the effect and concentration of the chelating agent was evident. The chelating agent's effect appeared to be an enhancement of the transport of the ionized drug form. The change in membrane integrity existed at least 24 hr after theoretical exposure to edetate disodium, but cyclic alterations in integrity could not be ruled out. The effect on integrity was also demonstrated to be nonpermanent, and the apparent loss in integrity was partially restored by calcium byt not by magnesium.

Twenty-four hour toxicity rhythms of sedative-hypnotic drugs in mice.

Twenty-four hour LD50 values of secobarbital, pentobarbital, phenobarbital, in male Swiss-Webster mice weighing approximately 30 g each. The animals were adapted for three weeks in an environmental room equipped with an automatically-timed photoperiod lasting from 0800 to 2000 h daily. Each mouse was injected intraperitoneally at 6 h time intervals with either phenobarbital or chloral hydrate for toxicity analysis. Secobarbital, pentobarbital and hexobarbital were injected at 3 h time intervals. Peak toxicity was reached at D-0600 with all drugs screened except chloral hydrate which was 180 degrees out of phase. The drugs were least toxic at 1200 h with the exception of chloral hydrate which was least toxic at D-0600 h. These results suggest circadian periodicity in the toxicity of sedative hypnotics. Factors that could be responsible for these variations are discussed.

Dose-response studies on tolerance to multiple doses of secobarbital and methaqualone in a polydrug abuse population.

Patients from a polydrug abuse treatment program were titrated with either secobarbital or methaqualone, their primary drug of abuse, to a state of mild intoxication, consisting of lateral and vertical nystagmus, ataxia, slurred speech, and drownsiness. The mean dose required to produce each sign was compared to that determined in a similarly treated control group. Tolerance to secobarbital was more easily demonstrated than tolerance to methaqualone, and nystagmus was the least sensitive indicator of patient tolerance. The individual signs were also cumulated into a graded rating scale of central nervous system depression which would be related to the dose administered. Tolerence was easily demonstrated at the higher stages of toxicity for secobarbital in the overall patient population, but tolerance to methaqualone was only unequivocal in the subjects indicating a relatively high frequency of abuse. Tolerance to methaqualone occurred at the lower stages of toxicity, suggesting that there is a difference between tolerance to secobarbital and tolerance to methaqualone. There was no indication that patients who also abuse alcohol are more tolerant than their patient counterparts. The patients who also had a history of amphetamine abuse, however, were less tolerant than the nonusers of these drugs.