Covalent binding of benzo[a]pyrene to rat liver cytosolic proteins and its effect on the binding to microsomal proteins.

Benzo[a]pyrene will bind covalently to rat liver cytosolic proteins when incubated with microsomes and NADPH. The binding is most extensive when microsomes from 3-methylcholanthrene-treated rather than phenobarbital-treated or control rats are used. The binding to cytosolic proteins increases when incubations are performed with increasing concentrations of cytosol. At the same time the covalent binding of benzo[a]pyrene to microsomal proteins decreases. Two cytosolic polypeptides are the main targets for benzo[a]pyrene. These have the same mobility in polyacrylamide gels as the subunits of purified glutathione S-transferase B. These subunits also react covalently with benzo[a]pyrene when the transferase is incubated with microsomes and NADPH.

Interactions of bambuterol with human serum cholinesterase of the genotypes EuEu (normal), EaEa (atypical) and EuEa.

Bambuterol, a carbamate ester prodrug of the bronchodilator terbutaline, was tested as inhibitor and substrate of human serum cholinesterases of the genotypes EuEu (the normal enzyme), EaEa (the atypical enzyme) and EuEa. The IC50 for the normal enzyme was 11 +/- 2.2 nM (mean, SD, N = 10) and for the atypical enzyme 140 +/- 6 nM (N = 13), indicating a much higher affinity of bambuterol to the normal enzyme. The heterozygotes showed a mixed behaviour; the major activity was inhibited like the normal enzyme (IC50 = 9.3 +/- 1.9 nM, N = 9), while a residual activity (10-15%) was inhibited by bambuterol like the atypical enzyme. At a bambuterol concentration of 100 nM each of the three cholinesterase genotypes responded uniquely to bambuterol; the normal enzyme was inhibited to 2.2 +/- 0.9%, the atypical enzyme to 58 +/- 4.6%, and the heterozygote to 10 +/- 1.2% of the basal activity. Bambuterol may therefore be added to the list of inhibitors useful in the genotyping of cholinesterases. Bambuterol was much less efficiently hydrolysed in serum containing the atypical cholinesterase than in serum containing the normal enzyme. The results of the hydrolysis experiments once again illustrate the difference in affinity of bambuterol to the genetic forms of cholinesterase, and also strengthen the evidence that cholinesterase is the major serum enzyme catalysing the hydrolysis of bambuterol.

Hydrolysis of 3H-bambuterol, a carbamate prodrug of terbutaline, in blood from humans and laboratory animals in vitro.

Tritiated bambuterol, a bis-dimethylcarbamate prodrug of terbutaline, was incubated in vitro with blood from both sexes of the following species: man, guinea pig, rat, mouse, dog and rabbit. The rates of hydrolysis of bambuterol to its monocarbamate derivative and further to terbutaline were measured. Large species variations were observed, e.g. blood from two of the human subjects was 15-fold more active than blood from the male rats. The rate of terbutaline formation as a function of initial bambuterol concentration was investigated in human plasma, and was found to describe a bell-shaped curve. Several pieces of evidence indicated that butyrylcholinesterase (EC 3.1.1.8) is the blood enzyme predominantly responsible for hydrolysis of bambuterol, although minor contributions from other esterases cannot be excluded. An exception may be blood from the rabbit, where the kinetics of the hydrolysis was different than in blood from the other species. The kinetics of bambuterol hydrolysis is discussed on basis of the established mechanism of carbamate interactions with cholinesterases, and the high affinity of bambuterol for butyrylcholinesterase.

Metabolism of N-acetyl-L-cysteine. Some structural requirements for the deacetylation and consequences for the oral bioavailability.

Rat liver, lung and intestine homogenates deacetylated N-acetyl-L-cysteine. Nearly stoichiometric amounts of L-cysteine were recovered. In rat liver, the enzyme activity was associated with the cytosolic fraction. Liver cytosol was much less active. N-Acetyl-D-cysteine or the disulphide of N-acetyl-L-cysteine were not deacetylated or in other ways consumed in vitro. Isolated, perfused rat liver did not retain or metabolize N-acetyl-L-cysteine to any measurable extent during single-pass experiments. N-Acetyl-L-cysteine or N-acetyl-D-cysteine were injected into a ligated segment of rat intestine in situ. After 1 hr 2% of the L-isomer and 35% of the D-isomer remained in the intestinal lumen. Systemic plasma levels were less than 3 microM of the L-form and congruent to 40 microM of the D-form. We conclude that deacetylation in the intestinal mucosa and possibly in the intestinal lumen is the major factor determining the low oral bioavailability of N-acetyl-L-cysteine. The deacetylation is discussed on the basis of the subcellular localization and the structural requirement of the reaction.

Glutathione in human melanoma cells. Effects of cysteine, cysteine esters and glutathione isopropyl ester.

Thiols are of great importance for the regulation of many cellular functions including metabolism, transport and cell protection. In this study the usefulness of L-cysteine methyl and octyl esters, of N,S-diacetyl-L-cysteine methyl ester and glutathione isopropyl ester as cellular cysteine and GSH delivery systems was investigated in the human IGR 1 melanoma cell line. The L-cysteine methyl and octyl esters proved to be highly toxic to the cells. Treatment of the cultures with 1 mM N,S-diacetyl-L-cysteine methyl ester or 3 mM glutathione isopropyl ester for 24 h resulted in marked elevation of the cellular glutathione level without apparent or with slight cell loss, respectively. Thus the administration of the latter two compounds seems to be suitable for inducing GSH elevation in the cultured melanoma cells.

Microsomal target proteins of metabolically activated aromatic hydrocarbons.

The specificity of binding to microsomal proteins of metabolically activated hydrocarbons has been studied. Radioactively labelled benzene, phenol, chlorobenzene, BP and MC were incubated with liver microsomes from control, phenobarbital- and MC-treated rats in the presence of an NADPH-generating system. The patterns of metabolite binding to microsomal proteins were examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and fluorography. Benzene, phenol and chlorobenzene metabolites showed one type of binding pattern dominated by a band at 72 000 Mr. This band was strong both in control and induced microsomes. Additional radioactive bands were seen in the 50 000--60 000 Mr region particularly in MC-induced microsomes. BP and MC metabolites showed a different type of binding pattern with incorporation of radioactivity into several fractions in the 50 000--60 000 Mr region of MC-induced microsomes. Two other strongly labelled fractions occurred at 68 000 and 72 000 Mr. The incorporation was low into control and phenobarbital-induced microsomes. Two labelled bands (Mr 56 000 and 72 000) were common for all hydrocarbons in MC-induced microsomes. The 56 000 Mr band had the same mobility in the gel as an MC-induced protein likely to be cytochrome P-448. The NADPH-generating system was essential for metabolite binding and GSH and UDPGA greatly reduced binding. We suggest that differences in metabolite binding patterns reflect differences in the routes of metabolite formation and that activated hydrocarbons are likely to bind to proteins close to their site of formation.

Mechanism of benzene toxicity. Effects of benzene and benzene metabolites on bone marrow cellularity, number of granulopoietic stem cells and frequency of micronuclei in mice.

The effects of benzene and benzene metabolites *hydroquinone and catechol) on bone marrow cellularity, number of granulopoietic stem cells and on the frequency of micronuclei in polychromatic erythrocytes were investigated in mice. The dose-effect curve for benzene revealed that there was a threshold dose (approx. 100 mg benzene/kg body wt./day injected subcutaneously on 6 consecutive days) above which severe toxicity occurred in all three parameters. Also hydroquinone gave rise to adverse effects in the parameters studied, but the sequence of occurrence was different from that observed with benzene. These data are interpreted to indicate that hydroquinone is a hemotoxic metabolite of benzene in mice in vivo, but that other metabolites, or benzene itself, also probably contribute to the toxicity. Catechol gave no effects. However, due to acute effects like tremor and convulsions only rather low doses could be tested. Simultaneous administration of toluene dramatically reduced the toxicity of benzene, but gave only a small reduction of the hydroquinone-induced effects.

A rapid and sensitive method for determination of covalent binding of benzo[a]pyrene to proteins.

A method is presented for the quantitative determination of covalent binding of metabolically activated benzo[a]pyrene to microsomal proteins. After incubation of radiolabelled benzo[a]pyrene with microsomes and NADPH, the mixture is applied to filter paper discs. These are immersed in ethanol to precipitate the proteins. Unbound radiolabel is removed by repeated washes of the filters in organic solvents before scintillation counting. The method is simple, rapid, sensitive and accurate, and works both with 14C- and 3H-labelled compounds. The method is suitable for measuring the incorporation of other radiolabelled xenobiotics to proteins of both microsomes and other subcellular fractions and for the analysis of binding to isolated proteins.

Catalytic effects of glutathione peroxidase mimetics on the thiol reduction of cytochrome c.

The reduction of ferric cytochrome c by various thiols was studied. It was found that L-cysteine, L-cysteine methyl ester and D-penicillamine were very efficient reductants for cytochrome c, whereas N-acetylated amino acids (N-acetyl-L-cysteine and N-acetyl-D-cysteine) reacted considerably slower. A series of glutathione peroxidase mimetics and related compounds were studied as catalysts for the N-acetyl-L-cysteine reduction of ferric cytochrome c. Diphenyl diselenide, t-butylthio phenyl selenide, S-(phenylseleno)-glutathione, N-(phenylseleno)-phthalimide and alpha-(phenylselenenyl)-acetophenone were all efficient reduction catalysts. Diphenyl disulfide, Ebselen and several derivatives thereof were less potent catalysts whereas diaryl selenides and diphenyltelluride did not affect the rate of reduction when present in catalytic amounts. The catalysis of diphenyl diselenide, selenosulfides, alpha-(phenylselenenyl)acetophenone, N-(phenylseleno)-phthalimide and Ebselen and derived compounds was suggested to involve the formation of areneselenolate ions as redox-active species capable of transferring one electron to the ferric cytochrome c. The resulting selenium centered arylseleno radicals would then dimerize to regenerate the catalyst in the diselenide form. In the presence of diaryl ditellurides and N-acetyl-L-cysteine, ferric cytochrome c was also rapidly reduced. This reaction was stoichiometric with respect to the ditelluride reagent.

Effects of N-acetylcysteine stereoisomers on oxygen-induced lung injury in rats.

The effects of the stereoisomers of N-acetylcysteine (L-NAC and D-NAC) on oxygen-induced lung oedema have been studied in rats. The NAC-isomers were given by an osmotic minipump in order to attain continuous administration, either intravenously or intragastrically. In some experiments, plasma concentrations of NAC, cysteine and glutathione (total concentrations, i.e., concentrations obtained after reduction of the samples with dithiothreitol) were recorded. Exposure to oxygen induced an almost two-fold increase of the lung wet weight. When L-NAC or D-NAC were given intravenously, in dose of 1.1 mmol/day/kg body weight, the increase of lung wet weight was prevented by 40-50%. The plasma concentrations were approximately 40 microM (L-NAC) and approximately 90 microM (D-NAC). Following intragastrical administration of the same doses, plasma concentrations of L-NAC and D-NAC reached approximately 3 and approximately 60 microM, respectively. Using this method of administration, only D-NAC significantly diminished the increase of the lung wet weight. The difference in plasma concentrations of the NAC isomers, particularly after intragastric administration, most likely reflects the fact that L-NAC is effectively hydrolysed in most tissues, while D-NAC is resistant to enzymatic hydrolysis, thus penetrating largely intact into the systemic circulation. The data presented shows that NAC, regardless of stereoconfiguration, will protect the lung against oxygen toxicity, provided sufficient systemic levels are obtained. Since D-NAC is not a precursor of L-cysteine, formation of glutathione cannot explain the protective effects of this isomer. L- and D-NAC may therefore act via direct antioxidant/radical scavenging mechanisms and not necessarily as precursors of glutathione in this model.

Glutathione in bronchoalveolar lavage fluid from smokers is related to humoral markers of inflammatory cell activity.

Cigarette smoking results in variable degrees of inflammation in the lower respiratory tract. Furthermore, smoking produces oxidant-mediated changes in the lung, important to the pathogenesis of emphysema. Since glutathione can neutralize reactive oxygen species and prevent peroxidation of unsaturated lipids, it may constitute an important component of the lung's defense against oxidant and inflammatory injury. In the present study, broncholaveolar lavage (BAL) was performed in 27 smokers, and the concentrations of total glutathione as well as the cellular and humoral markers of inflammatory activity were studied. There were significant correlations between total glutathione and neutrophils; two neutrophil granule components, myeloperoxidase and elastase; and chemotactic activity for neutrophils. Moreover, the total glutathione correlated with the eosinophil cationic protein (ECP), a granule constituent of the eosinophil, with two locally produced antiproteases, secretory leukocyte protease inhibitor (SLPI) and antichymotrypsin (ACHY), but not with an alpha 1-protease inhibitor and albumin. These data suggest that the total glutathione levels in BAL fluid may reflect a degree of oxidative and inflammatory stress caused by cigarette smoke, and they are therefore likely to contribute to the protection against this stress.

Breath concentration as an index of the health risk from benzene. Studies on the accumulation and clearance of inhaled benzene.

Human subjects were exposed to known concentrations of benzene in air for single and repeated daily periods. The breath concentrations measured repeated exposures approached a maximum after 3 d, and this phenomenon indicated that the tissues were approaching saturation under the experimental conditions. The breath concentrations measured after exposure indicated an initial rapid clearance of benzene with a half-time of 2.6 h, followed by a slower phase with a half-time of 24 h. The decay in breath concentration after prolonged occupational exposure appeared to be slower; the difference between the laboratory and industrial studies was, however, not significant. The hygienic significance of these results was discussed, and it was recommended that control measures be employed when a morning breath concentration exceeds 10 ppb.

Bambuterol, a carbamate ester prodrug of terbutaline, as inhibitor of cholinesterases in human blood.

Bambuterol, the bis-dimethyl carbamate prodrug of terbutaline, was tested for its potency in inhibiting cholinesterases in human blood. Preincubation of blood with bambuterol in the absence of thiocholine ester substrate was essential for obtaining maximal inhibition. The inhibition exerted by bambuterol after such preincubation was reversible and noncompetitive. Bambuterol was an extremely effective inhibitor of cholinesterase when butyrylthiocholine was used as substrate (I50 = 1.7 +/- 0.3 x 10(-8) M, N = 10) whereas it was 2400-fold less efficient in inhibiting cholinesterase with acetylthiocholine as substrate (I50 = 4.1 +/- 0.5 x 10(-5) M, N = 10). Because butyrylthiocholine is the preferred substrate for cholinesterase (EC 3.1.1.8) and acetylthiocholine for acetylcholinesterase (EC 3.1.1.7), these results indicate that bambuterol is a remarkably selective and potent inhibitor of cholinesterase.

Reversible formation of fatty acid esters of budesonide, an antiasthma glucocorticoid, in human lung and liver microsomes.

Microsomes from human lung and liver catalyze the formation of fatty acid esters of budesonide, a glucocorticoid used for inhalation treatment of asthma. The conjugation was dependent on coenzyme A and ATP. Addition of free fatty acids to the incubations affected the pattern of metabolites, but ester formation was observed also without such addition. Budesonide oleate, palmitate, linoleate, palmitoleate, and arachidonate were identified as metabolites. The fatty acid conjugates of budesonide were shown to be substrates for lipase in vitro, thus budesonide is regainable from the conjugates. The data suggest that an equilibrium between budesonide and these pharmacologically inactive lipoidal conjugates will be established in tissues at repeated exposure to budesonide. Since the fatty acid conjugates most likely will be retained intracellularly for a longer time than unchanged budesonide, the duration of tissue exposure to budesonide will depend partly on the rate of lipase-catalyzed hydrolysis of the conjugates. The findings in this study provide a possible explanation for the efficacy of budesonide in mild asthmatics also when inhaled once daily.

Synthesis and enzymatic hydrolysis of esters, constituting simple models of soft drugs.

One way to minimise systemic side effects of drugs is to design molecules, soft drugs, in such a way that they are metabolically inactivated rapidly after having acted on their pharmacological target. Hydrolases (esterases, peptidases, lipases, glycosidases, etc.) are enzymes well suited to use for drug inactivation since they are ubiquitously distributed. Insertion of ester bonds susceptible to enzymatic cleavage may represent one approach to make the action of a drug more restricted to the site of application. The present study describes the chemical synthesis of fourteen model compounds comprising a bicyclic aromatic unit connected by an ester-containing bridge to another aromatic ring. Initial attempts to define a) the tissue selectivity of the hydrolytic metabolism and b) the molecular structural factors affecting the rate of enzymatic ester cleavage are presented. The data show that human and rat liver fractions were more active than human duodenal mucosa and human blood leukocytes at hydrolysing the compounds. The rank order of the compounds was, however, very similar in the different biological systems. Commercially available pig liver carboxyl esterase and cholesterol esterase both reasonably well predict the rank order in the tissue fractions.

Biological threshold limits for benzene based on pharmacokinetics of inhaled benzene in man.

Volunteers were exposed to benzene, 2--10 ppm, under controlled conditions up to 6 h a day during five consecutive days. The accumulation and elimination of benzene was measured by determination of benzene concentration -- down to 0.001 ppm -- in exhaled breath. From these observations, a multicompartment model, which approximately describes the kinetics of benzene elimination and accumulation has been designed. On the basis of this model, benzene concentrations in breath, corresponding to exposure levels of benzene, have been estimated. Thus, at a daily exposure to 10 ppm the exhaled benzene concentration in the morning after a day of exposure will not exceed 0.1 ppm.