Brian R. Smith

[8] Chemical depletion of glutathione in vivo

Publisher Summary The method for lowering tissue glutathione (GSH) levels involves the administration of compounds that react enzymically with GSH to form conjugates. The inhibition of GSH synthesis can also result in the depletion of this tripeptide in the organs with a sufficient turnover rate. The conversion of GSH to its oxidized form, glutathione disulfide (GSSG), has also been used to deplete GSH, mainly in isolated cell preparations; the choice of the depleting agent depends on the type of system under study. Diethyl maleate (DEM) remains one of the most useful compounds for depleting hepatic GSH in vivo. Intraperitoneal administration of DEM reduces the hepatic GSH levels of rats to 6-20% of control values in 30 min and for a period of 2 to 4 hr. Phorone is another α,β-unsaturated carbonyl compound that may be useful as a GSH depleting agent. As in the case of DEM, the conjugation of phorone with GSH is catalyzed by glutathione transferases.

Regiospecificity and stereospecificity in the enzymatic conjugation of glutathione with (±)-benzo(a)pyrene 4,5-oxide

Abstract 13 C-NMR analysis of the glutathione conjugates formed from (±)-benzo(a)-pyrene 4,5-oxide-4,5- 13 C by a purified glytathione transferase from little skate ( Raja erinacea ) liver demonstrated that equivalent amounts of the positional isomers (4,5-dihydro-4-hydroxy-5-glutathionylbenzo(a)pyrene and 4,5-dihydro-4-glutathionyl-5-hydroxybenzo(a)pyrene) were formed. Separation of these conjugates by HPLC and subsequent 13 C-NME studies showed that only one diastereoisomer of each positional isomer was formed by the skate enzyme, each enantiomer of the arene oxide having produced only one of the two possible positional isomers. The non-enzymic reaction of (±)-benzo(a)pyrene 4,5-oxide with glutathione produced the four possible stereoisomers resulting from trans addition to the epoxide ring. This was also true when rat liver cytosol was used as the source of transferase activity. The data demonstrate that skate liver glutathione transferase 4 has high substrate regiospecificity and stereospecificity for (±)-benzo(a)pyrene 4,5-oxide.

[13] Lung perfusion techniques for xenobiotic metabolism and toxicity studies

Publisher Summary The isolated perfused lung preparation is a versatile technique for toxicological, pharmacological, or biochemical studies. This chapter presents a practical perfusion apparatus and describes the surgical procedures to allow investigators, not experienced in animal surgery, to successfully utilize the technique. The perfusion apparatus employed for lung perfusion consists of several discrete parts: a peristaltic (roller) pump for circulating perfusion fluid, a pH meter, an animal ventilator, an artificial thorax with upper reservoir, and a recirculating water bath. Several media have been used for perfusion fluid. Krebs–Ringer bicarbonate buffer fortified with 4.5% bovine serum albumin and 5 mM glucose is a widely used medium. Nembutal is the anesthetic of choice for rats and guinea pigs. Ether or halothane is also appropriate for these species, but none of the aforementioned agents work well with rabbits. The best approach is to anesthetize rabbits with CO2 until respiration has ceased and then to perform the surgery quickly. The short duration of the operation avoids thrombus formation, which is important for successful perfusion.

Evaluation of three metabolic activation systems by a forward mutation assay in Salmonella

The strain SV3 of Salmonella typhimurium was used as the indicator bacterium in the intrasanguineous host-mediated mutagenicity assay. Bacterial distribution and spontaneous mutation frequency were determined after intravenous injection of SV3 into CD1 male mice. Bacteria were cleared at an exponential rate from the blood stream and recovered mainly from the liver and in smaller quantities from the lungs and kidneys. No bactericidal effect was observed during incubation within the animal, and bacterial division occurred in the liver and probably in the kidneys. The significance of an increased mutation frequency of bacteria recovered from untreated animals is discussed. Mutation induction was measured in bacteria recovered from liver, lungs and kidneys of CD1 mice and CD rats treated with dimethylnitrosamine (DMN). The sensitivity of the intrasanguineous host-mediated technique was compared with the sensitivity of the assay in vitro with microsomal preparations from each tissue and host. Activation by isolated perfused liver and lungs from CD rats was included for comparison with the results from experiments in vivo and in vitro.

Metabolism and toxicity of benzo(a)pyrene-4,5-oxide in the isolated perfused rat liver.

Abstract Metabolically generated arene oxides are believed to be responsible for much of the biological activity of polynuclear aromatic hydrocarbons. Epoxides are further metabolized in vitro by epoxide hydrase (EC 4.2.1.63) and by glutathione S-transferases (EC2.5.1.18). This is a report of the metabolism and toxicity of benzo(a)pyrene 4,5-oxide (BP-4,5-oxide), a metabolite of benzo(a)pyrene, in the isolated perfused rat liver and also in ether-anesthetized rats. [G-3H]BP-4,5-oxide was metabolized predominantly to conjugates (mainly thioether derivatives) and to a lesser extent to the dihydrodiol. These results reflect the activities of the two major epoxide-metabolizing pathways described in in vitro studies. The metabolites were released from the livers into the vascular circulation, but biliary excretion was the major route of removal of the metabolic products from the liver. Metabolism of BP-4,5-oxide in rats and in the isolated perfused liver was quantitatively and qualitatively similar, but recovery of applied materials was reduced in rats due to redistribution of the radioactivity within the animal. BP-4,5-oxide caused periportal damage in isolated perfused livers and was found to be covalently bound to tissue DNA, RNA, and protein. The covalent binding may be a causative factor leading to the tissue damage.

The intrasanguineous host mediated assay procedure using Saccharomyces cerevisiae: Comparison with two other metabolic activation systems

3 metabolic activation systems--organ homogenates, perfused liver, and the intrasanguineous host mediated assay (IHMA)--were compared in their abilities to activate demethylnitrosamine (DMN) and induce gene conversion in Saccharomyces cerevisiae D4. Both rats and mice were used for the organ homogenates and IHMA studies. All activation systems were able to activate DMN; where the different organs were compared, liver was more active than kidney, followed by lung. The IHMA was the most sensitive of the systems examined.

PULMONARY METABOLISM AND EXCRETION OF ARENE OXIDES

Publisher Summary This chapter discusses the pulmonary metabolism and excretion of arene oxides. Lung is a target organ for toxic environmental agents. This tissue is exposed to airborne materials and to substances carried in the bloodstream as the entire cardiac output passes through the pulmonary capillary bed. Measurement of pulmonary arene oxide metabolism, in vivo, is difficult because of the high rate of blood flow through the lung and the relatively low activity of this tissue. Results from the in vitro studies are difficult to interpret in terms of what happens in the whole organ because of the structural and organizational losses suffered during the preparation of sub-cellular fractions from the intact tissue. The chapter describes a study that investigates the causes of discrepancies in arene oxide metabolism seen when comparing results from perfused lungs with in vitro data.

BILIARY METABOLITES OF BENZO(a)PYRENE 4,5-OXIDE IN THE RAT

Publisher Summary This chapter describes the biliary metabolites of benzo(a)pyrene (BP). The initial step in the metabolic activation of BP is the conversion to arene oxides, which may be further metabolized to ultimate carcinogens, such as diol epoxides, or to non-carcinogenic metabolites. The biotransformation pathways for intermediate arene oxides are, therefore, important in determining whether activation or detoxication occurs. The interest in the fate of reactive metabolites and the role of the glutathione 5-transferases in their detoxication led to investigate the biliary metabolites of benzo(a)pyrene 4, 5-oxide (BPO) in the rat. Previous studies of BP metabolism have focused on the solvent-extractable metabolites such as phenols, quinones, and dihydrodiols. However, the water-soluble metabolites have been less extensively investigated. As thioether conjugates arising from the reaction of arene oxides with glutathione are indicative of detoxication, more attention should be paid to the compounds of this type.