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J R Gillette

Publications and source records attributed to J R Gillette.

178 records · Page 10Linked to original sources
PubMedDate not supplied

The formation of glutathione conjugate derived from propranolol.

Mixed function oxidases in liver microsomes from 3-methylcholanthrene-pretreated guinea pigs converted S-propranolol to 4-hydroxypropranolol, 5-hydroxypropranolol, and desisopropyl-propranolol. The addition of glutathione and cytosol from rat liver to the system resulted in the formation of a water-soluble metabolite. Evidence that the metabolite was a glutathione adduct was obtained by showing that treatment with gamma-glutamyltranspeptidase changed its HPLC retention time. The formation of the glutathione adduct required glutathione S-transferases in cytosol and was accompanied by a decrease in the formation of 5-hydroxypropranolol, but not of 4-hydroxypropranolol or desisopropylpropranolol. The formation of the propranolol-glutathione adduct was confirmed by two independent approaches. When [14C]glutathione and [3H]S-propranolol were used, the relationship between 14C and 3H in the metabolite indicated that it contained equal amounts of glutathione and propranolol. When a pseudoracemic mixture of S-propranolol and [2,4-2H2]R-propranolol was used, thermospray LC/MS analysis of the glutathione adduct revealed two peaks having different retention times. The first peak, representing a [2,4-2H2]R-propranolol-GSH adduct, gave fragment ions at m/z 294, 278, 262, while the second, which represented the S-propranolol-GSH adduct, gave fragment ions having 2 mass units less than those obtained with [2,4-2H2]R-propranolol. There was no evidence of any loss of deuterium during the formation of these fragment ions. The material in both peaks gave ions of m/z 308, 147, and 130, which is consistent with the presence of a glutathione group.

Animals↗
PubMedDate not supplied

Pharmacokinetic procedures for the estimation of organ clearances for the formation of short-lived metabolites. Acetaminophen-induced glutathione depletion in hamster liver.

Several approaches have been developed to estimate in vivo the intrinsic clearances of enzymes that catalyze the formation of chemically reactive metabolites that do not escape the organs in which they are formed. Two basic models are considered. Model 1 is a general model in which the chemically reactive metabolite is inactivated by a combination of a pseudo-first order reaction, such as a reaction with large pools of protein, and a second order reaction with a depletable endogenous substance, such as glutathione or an enzyme. Model 2 is a special case, in which at low doses of the parent compound the reactive metabolite preferentially reacts with a depletable endogenous substance, such as glutathione. In developing both models we have assumed that the rate of formation of reactive metabolite follows first order kinetics and that the concentration of reactive metabolite in liver reaches a steady state almost instantaneously. In developing Model 2 we also have assumed that the depletion of hepatic reduced glutathione is due solely to the formation of glutathione conjugates. The uses of the approaches based on Model 2 were illustrated by studying the effects of a marginally toxic dose of acetaminophen on the depletion and subsequent repletion of hepatic glutathione in hamsters. From the calculated rate of synthesis of glutathione in liver, the fraction of the dose of acetaminophen converted to the glutathione conjugate in liver, and the clearance for the formation of glutathione conjugate in vivo was estimated and was found to be similar to that obtained with hepatic 9,000 g supernatant preparations. Other uses of the models are described.

Acetaminophen↗
PubMedDate not supplied

Complications in the estimation of hepatic blood flow in vivo by pharmacokinetic parameters. The area under the curve after the concomitant intravenous and intraperitoneal (or intraportal) administration of acetaminophen in the rat.

Hepatic blood flow can be estimated from the area under the blood or plasma concentration-time curve of a drug following an intravenous dose and either an oral or intraperitoneal dose. The validity of the method depends on several factors which alter the area under the curve, that is, hemodynamic interactions due to the drug or its metabolites, the use of plasma concentration as opposed to blood concentration data, incomplete absorption on oral or intraperitoneal administration, enterohepatic recycling of the drug or its metabolite, and the presence of extrahepatic metabolism. The validity of the method was tested in rats with tracer doses of acetaminophen. After the simultaneous administration of an iv dose of 14C-acetaminophen and an intraportal dose of 3H-acetaminophen to rats, the availability after the first-pass hepatic extraction of acetaminophen in rats was 0.56 +/- 0.05. Hepatic blood flow estimated by the area under the curve for the respective routes of administration was 78.1 +/- 16.1 ml/min/kg. However, after the simultaneous administration of an iv tracer dose of 14C-acetaminophen and an ip dose of 3H-acetaminophen, the apparent availabilities calculated from the areas under the curve were highly variable and tended to be greater (0.73 +/- 0.11). Thus the estimates of the hepatic blood flow also tended to be higher (159 +/- 65 ml/min/kg).

Acetaminophen↗
PubMedDate not supplied

Production of a dimer of 2-acetylaminofluorene during the sulfation of N-hydroxy-2-acetylaminofluorene in vitro.

During the sulfation of N-hydroxy-2-acetylaminofluorene (NOH-2AAF) by rat liver 100,000 g supernatant fraction in vitro, an unidentified metabolite is produced which accounts for 22% of the N-OH-2AAF metabolized. This product has been characterized as the 2AAF dimer, 1-(N-2'-fluorenylacetamido-2-acetylaminofluorene) by comparing its TLC, HPLC, UV, and mass spectral properties with a synthetic standard which was prepared from the reaction of N-acetoxy-2-acetylaminofluorene (N-AcO-2AAF) with 2AAF. Increasing amounts of 2AAF added to the incubation mixture of N-OH-[acetyl-14C]2AAF and rat liver 100,000 g supernatant fraction decreased the irreversible binding of 14C to protein, and increased the formation of 2AAF dimer proportionately. This suggests that the 2AAF dimer is formed from the reaction of 2AAF and the electrophilic species produced from the sulfated N-OH-2AAF. In the presence of the 9,000 g fraction of rat liver, the dimer of 2AAF was aroximately 1/25 as active as 2AAF in producing mutations in the Salmonella mutagenesis test system.

2-Acetylaminofluorene↗
PubMedDate not supplied

3-Hydroxyacetaminophen: a microsomal metabolite of acetaminophen. Evidence against an epoxide as the reactive metabolite of acetaminophen.

3-Hydroxyacetaminophen has been isolated and identified as a microsomal metabolite of acetaminophen. Analysis of the metabolite by gas chromatography-mass spectrometry revealed that the metabolite had a molecular ion and fragmentation pattern identical to that of authentic 3-hydroxyacetaminophen. Glutathione and ascorbic acid blocked covalent binding of reactive metabolite(s) to protein but did not block the formation of 3-hydroxyacetaminophen. Moreover, epoxide hydrolase did not block covalent binding of the reactive metabolite(s) to protein. Thus, the reactive metabolite apparently is not an epoxide substrate of the hydrolase, nor are 3-hydroxyacetaminophen and the reactive metabolite(s) formed from a common intermediate.

Acetaminophen↗
PubMedDate not supplied

The mechanism of formation of o-bromophenol from bromobenzene.

Both o-bromophenol and p-bromophenol are formed from bromobenzene in rat liver microsomes. It has been established that p-bromophenol is formed via bromobenzene-3,4-oxide, but o-bromophenol could conceivably arise via either the 2,3-epoxide or the 1,2-epoxide or by direct insertion of oxygen. As described in the present article, we have isolated and identified bromobenzene 2,3-dihydrodiol as a microsomal metabolite of bromobenzene. Identification of the dihydrodiol therefore indicates the formation of its obligatory precursor, bromobenzene-2,3-oxide. Moreover, using bromo(2,4,6-2H3)benzene, we have clarified the mechanism of formation of o-bromophenol from bromobenzene. The rate of formation of o-bromophenol from bromobenzene and bromo(2,4,6-2H3)benzene in liver microsomes from 3-methylcholanthrene-treated rats was 0.72 +/- 0.02 and 0.74 +/- 0.06 nmol/mg/min (kH/kD = 0.99), respectively. The lack of a significant isotope effect indicates that the hydroxylation of bromobenzene to o-bromophenol is not by a direct insertion mechanism. Furthermore, the mass spectrum of o-bromophenol isolated from a microsomal incubation with bromo(2,4,6-2H3)benzene indicated that 70% of the product retained all three deuterium atoms. These results are consistent with the view that o-bromophenol is formed from the 2,3-epoxide intermediate but do not preclude formation by the addition of oxygen to the 2-position carbons followed by an NIH shift and rearrangement before an epoxide is formed.

Animals↗
PubMedDate not supplied

Multiple reactive metabolites derived from bromobenzene.

It has recently been shown that bromobenzene is converted to multiple reactive metabolites. However, the nature of these reactive metabolites is unclear. In the present manuscript, we have demonstrated that the same cytochromes P-450 activate both bromobenzene and p-bromophenol, and each substrate competitively inhibits the metabolism of the other. Moreover, the covalent binding of p-bromophenol to rat liver microsomes was inhibited by epoxide hydrolase, catechol-O-methyltransferase, superoxide dismutase, glutathione, and ascorbic acid but not by catalase. In contrast, the amount of 4- bromocatechol isolated from microsomal incubations containing p-bromophenol was decreased by glutathione and increased by ascorbic acid and superoxide dismutase. It is thus likely that p-bromophenol is converted to an epoxide that decomposes to 4- bromocatechol and that both the epoxide and the quinone formed from oxidation of 4- bromocatechol may become covalently bound to tissue proteins. However, these chemically reactive metabolites are apparently nontoxic because treatments which increase the covalent binding of p-bromophenol in vitro do not cause toxicity in vivo.

Animals↗
PubMedDate not supplied

Theoretical kinetics of sequential metabolism in vitro. Study of the formation of 16 alpha-hydroxyandrostenedione from testosterone by purified rat P450 2C11.

P450 2C11 from rat liver is known to metabolize testosterone to 2 alpha-, 16 alpha-, and 6 beta-hydroxytestosterone, and to androstenedione and 16 alpha-hydroxyandrostenedione. Because Waxman (J. Biol. Chem. 259, 15481-15490) has reported that the enzyme converts androstenedione to 16 alpha-hydroxyandrostenedione, it seemed likely that the metabolite was formed from testosterone by way of androstenedione. Indeed, we have found that P450 2C11 does not convert 16 alpha-hydroxytestosterone to 16 alpha-hydroxyandrostenedione to any significant extent and, therefore, that the metabolite is formed from testosterone almost solely by way of androstenedione. To determine whether some of the 16 alpha-hydroxyandrostenedione might be formed directly from the androstenedione-enzyme complex, we developed an approach by which it is possible to calculate the amount of the androstenedione, released into the medium, relative to the amount of the androstenedione-enzyme complex that is converted directly to 16 alpha-hydroxyandrostenedione under initial conditions when the concentration of released androstenedione will be negligible. The approach uses two factors: factor A is the androstenedione/(androstenedione + 16 alpha-hydroxyandrostenedione) present at the end of the incubation, and factor B corrects for the amount of released androstenedione that recombines with the enzyme and is converted to 16 alpha-hydroxyandrostenedione. Although the values of both factors A and B will vary with the concentrations of testosterone and preformed androstenedione present in the incubation mixtures and with the duration of incubation, the value of A*B will be independent of these parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Androstenedione↗