Metabolism of bromobenzene to glutathione adducts in lung slices from mice treated with pneumotoxicants

Recent studies showing that the bronchiolar Clara cell and alveolar Type II cell are major loci of cytochrome P-450 monooxygenases in the lung suggested that measurement of xenobiotic metabolizing enzyme activity might provide a useful and sensitive index of injury to these cell types. Accordingly, an assay has been developed for quantitating the rate of formation of reactive bromobenzene metabolites in lung slices which is based upon measuring the rate of formation of bromobenzene glutathione adducts. To demonstrate that monitoring adduct formation would yield quantitatively similar data to the traditional covalent binding assay for measuring the formation of reactive bromobenzene intermediates, covalent binding and conjugate formation were assayed in incubations of phenobarbital-induced hepatic microsomes conducted in the presence of various cytochrome P-450 monooxygenase inhibitors. Incubation conditions which decreased the rate of covalent binding (incubations done in the absence of glutathione) resulted in similar decreases in conjugate formation (incubations done in the presence of glutathione). In lung slices, the metabolism of bromobenzene to glutathione conjugates was linear for 20 min and continued to increase with time over the entire 160 min of the study. The formation of bromobenzene glutathione adducts in lung slices from piperonyl butoxide-treated animals occurred at a significantly lower rate than control. Likewise, lung slices from animals treated with butylated hydroxytoluene or carbon tetrachloride, agents known to injure alveolar epithelial cells, metabolized bromobenzene to glutathione conjugates at significantly slower rates than control. In contrast, treatment with naphthalene or dichloroethylene, agents which damage the bronchiolar epithelial cells, had little or no effect on conjugate formation. Similarly, there were no significant differences in the rate of bromobenzene glutathione conjugate formation between lungs of air- and ozone-exposed (1.0 ppm × 4 hr) mice killed 2,24,48,72, or 120 hr after exposure. These studies suggest that monitoring the rate of bromobenzene glutathione conjugate formation in lung slices may be a useful and sensitive biochemical index of injury to certain cells of the lung but that severe damage to the nonciliated bronchiolar epithelial cells has little effect on the rate of metabolic activation of this aromatic hydrocarbon.     

Dependence of biliary secretion of inorganic mercury on the biliary transport of glutathione

The interrelation between the biliary transport of glutathione (GSH) and of inorganic mercury was investigated in suckling and adult male and female rats. The 14-day-old rat secreted inorganic mercury into bile at one-seventh the rate of the 28-day-old rat. Development of the ability to secrete mercury paralleled development of the ability to secrete GSH. The inability of the 14-day-old rat to secrete mercury and GSH occurred despite hepatic tissue concentrations of both of these compounds which were similar to those of adult rats. In adult rats, inhibition of GSH secretion by sulfobromophthalein (BSP) administration resulted in a parallel inhibition of mercury secretion. Conversely, the increase in the rate of GSH secretion into bile after cysteine or GSH administration was accompanied by an increase in the rate of mercury secretion into bile. The changes in the biliary secretion of mercury and of GSH after treatment with cysteine or GSH were not closely parallel, probably because of the tissue redistribution of mercury effected by these sulfhydryl-containing compounds. Mercury secretion into bile was independent of the changes in bile flow produced by dehydrocholate (DHC) or hypertonic sucrose, but it was closely related to the rate of GSH secretion. Further, sex differences and individual variability in the biliary secretion of inorganic mercury were correlated with differing abilities to secrete GSH into bile. These results suggest that the biliary secretion of inorganic mercury is in large part dependent on the biliary transport of GSH.     

Role of zinc as an activator of mitochondrial function in rat liver

The effects of zinc on the enzymes of hepatic mitochondria were investigated in rats that had been given zinc sulfate (10 mg Zn2+/100 g body wt) p.o. Administration of zinc caused a marked elevation of succinate dehydrogenase, glutamate dehydrogenase, cytochrome c oxidase and ATPase activities, whereas it did not cause significant changes in pyruvate carboxylase, malate dehydrogenase and isocitrate dehydrogenase activities. The effect of zinc as a function of time was greatest on succinate dehydrogenase. Zinc also produced a marked elevation of ATP concentration in the hepatic cytosol and a corresponding increase in ATPase activity in the hepatic mitochondria. Zinc content of the inner membrane of mitochondria was raised significantly by administration of zinc. The removal of zinc by washing in 10 mM EDTA caused a significant decrease of the increased succinate dehydrogenase activity caused by administration of zinc, while it did not lower ATPase activity. The addition of zinc in amounts of 10-10(3) ng Zn2+ per mg protein produced a significant increase in succinate dehydrogenase activity in the inner membrane of mitochondria, whereas ATPase activity was elevated significantly at 10(3)-10(4) ng Zn2+ per mg protein, indicating that zinc activated succinate dehydrogenase more sensitively than ATPase. The present investigation suggests that zinc taken up by hepatic mitochondria stimulates the electron transport system and oxidative phosphorylation and, as a result, increases the ATP concentration in the hepatic cytosol.     

Nonenzymatic glucosylation of human serum albumin and its influence on binding capacity of sulfonylureas

To estimate the functional change occurring in human serum albumin by nonenzymatic glucosylation, glucosylated human serum albumin was prepared by in vitro incubation with glucose. The rate of glucosylation proceeded as a first-order reaction. The binding of sulfonylureas to serum albumin was determined by equilibrium gel filtration. Through this method, it was possible to estimate the binding capacity of a low water solubility drug with a high affinity to protein. The amounts of the sulfonylureas bound to glucosylated HSA decreased by 44% with tolazamide and acetohexamide, 50% with glibenclamide, and 52% with tolbutamide, compared to human serum albumin (HSA). This suggests that a high concentration of glucosylated HSA in diabetic patients may possibly cause an increase in free drug concentration exceeding normal levels. This study shows that the decrease in the binding capacity of sulfonylureas with protein is due to the modification of albumin molecules by the covalent binding of glucose.     

Dietary modification of metabolism and toxicity of chemical substances—with special reference to carbohydrate

Rats were fed various test diets only on the day before sacrifice or every day for 3 weeks prior to sacrifice in order to assess the effects of protein (casein), fat (a mixture of olive and corn oils) and carbohydrate (sucrose) on the liver mixed-function oxidase activity. The activity was determined by measuring metabolic rates of 8 volatile hydrocarbons, i.e., benzene, toluene, styrene, chloroform, carbon tetrachloride, 1,2-dichloroethane, 1,1-dichloroethylene, and trichloroethylene. Contrary to the general belief, it was found that carbohydrate, not protein or fat, regulates the metabolism of these hydrocarbons: a diet which was deficient in carbohydrate remarkably enhanced the metabolism irrespective of protein and fat contents in the diet. This conclusion was confirmed by employing two types of diet, one in which the carbohydrate was replaced by an isocaloric amount of protein or fat (thus keeping total calories of each diet constant) and the other in which the carbohydrate content was varied with protein and fat contents fixed (total calories of each diet differed from others according to the carbohydrate content). In accordance with this, dietary carbohydrate intake also exerted a remarkable influence on the hepatotoxicity of carbon tetrachloride which needs to be metabolically activated to become cytotoxic: the smaller the intake, the more severe the liver injury.     

Effects of several antimalarials and phenothiazine compounds on the formation of coat structure from clathrin

We have evaluated the effects of two phenothiazine and several antimalarial drugs on the rates of polymerization of 8S clathrin molecules to 300S coat structures. Most of the drugs investigated have been shown in other studies to inhibit receptor-mediated endocytosis through the coated pit regions of plasma membranes. The two types of drugs were found to accelerate the polymerization rate without having much effect on the size distribution of the polymer species. The activities of the drugs appear to depend on a dibasic moiety and a large, hydrophobic aromatic ring in their structures.     

Dissociation of pharmacological and enzymatic activities of snake venom phospholipases A2 by modification of carboxylate groups

The carboxylate groups in an acidic and in a basic phospholipase A2 (PLA2) enzyme, purified, respectively, from Naja naja atra and Naja nigricollis snake venoms, were modified with carbodiimide and semicarbazide. The derivatives modified at pH 3.5 and pH 5.5 had less than 1% (N. nigricollis) or 2% (N. n. atra) residual enzymatic activity, whereas 12-16% enzymatic activity remained following modification at pH 5.5 in the presence of Ca2+. In marked contrast, these derivatives retained variable, but significantly greater, levels of lethal potency, hemolytic and anticoagulant activities, and abilities to block indirectly and directly induced contractions of the diaphragm. By this modification of aspartic and glutamic acid residues we have, for the first time, obtained derivatives of PLA2 which selectively retain greater pharmacological activity relative to enzymatic activity. Previously, we had found that modification of lysine and arginine residues produced derivatives which retain enzymatic activity but show a loss of pharmacological properties. These findings suggest that some pharmacological effects of snake venom PLA2 enzymes are due to a non-enzymatic action, suggesting two distinct but perhaps overlapping active sites.     

Influence of delta 9-tetrahydrocannabinol on expression of histone and ribosomal genes in normal and transformed human cells

The influence of delta 9-tetrahydrocannabinol (delta 9-THC) on the cellular levels of histone mRNAs and ribosomal RNAs was examined in several normal and transformed human cell lines--HeLa S3 cells, WI-38 human diploid fibroblasts, SV40-transformed WI-38 cells, and A549 lung carcinoma cells. RNA sequences were quantitatively assayed by electrophoretic fractionation, transfer to nitrocellulose, and hybridization with cloned genomic human histone or ribosomal DNA sequences. Treatment with delta 9-THC (10-40 microM) for 10 hr resulted in a concentration-dependent decrease in the representation of H2A, H2B, H3 and H4 histone mRNAs without a significant inhibitory effect on the levels of ribosomal RNAs. The cannabinoid-mediated inhibitory effect on histone gene expression was less evident in cells with active drug-metabolizing systems.     

Interaction of phencyclidine with mouse neuroblastoma cells

Growth of mouse neuroblastoma (Nb) cells (clone Ml) was not affected by phencyclidine (PCP) concentrations of 10^?6M up to 2 × 10^?4M, whereas 10^?3 PCP caused a 100% inhibition of cell growth. Several PCP analogs, including the quaternary PCP methiodide, exerted effects similar to those of PCP. The uptake of [piperidyl-3,4-³H]PCP ([³H]PCP) by the Nb cells was studied using cell monolayers in Petri dishes. Non-specific entry of PCP into the cells was linear with added substrate but specific uptake exhibited saturation kinetics. The concentration for half-maximum specific uptake was 2 × 10^?5 M, and the capacity of the cells at saturation was 2–3 nmoles [³H]PCP/mg protein, at 22°. The uptake rate constant was 0.2 ± 0.05 × 10⁵ (M^?1min^?1) and the dissociation constant was 0.25 ± 0.05 (min^?1). Uptake was temperature dependent and was inhibited by 2,4-dinitrophenol (DNP). This may indicate that this binding represents (at least in part) an active uptake process of PCP into the cells.     

Induction of cadmium-thionein in mouse tumor cells

Copper and zinc concentrations (per milligram of protein) in the liver of control mice were 18 and 57% lower, respectively, than in liver of animals bearing subcutaneous tumors originally derived from a hepatoma. Zinc in both tumor and liver was associated with a fraction identified as zinc-thionein by gel filtration. Tumor-bearing mice treated with cadmium (1 μg of CdCl₂/g) accumulated the metal mostly in liver, and more than 90% of the liver cadmium was bound to cadmium-thionein after 24 hr. Some cadmium was present in the tumors as well. Although the metal content of the tumors was dependent on the route of administration (ip or sc), a constant fraction (approximately 50%) was associated with cadmiun-thionein. When primary cultures of tumor cells were exposed to cadmium (0.25 μg/ml), the cells incorporated the metal into a fraction identified as cadmiumthionein by gel filtration and ion-exchange chromatography. The appearance of cadmiumthionein in tumor cells was accompanied by the incorporation of radioactive cysteine into this protein indicating de novo synthesis of thionein in tumor cells upon cadmium exposure.     

Mechanism of depression of brain phospholipid levels by an epileptogenic drug

Treatment of the rat with U18666A [3 beta-(2-diethylaminoethoxy) androst-5-en-17-one HCl] resulted in development of a chronic seizure state and 20-40% reductions in the concentration of all major phospholipid in whole brain. The mechanism of the phospholipid changes was explored in the present study. Incorporation of intracerebrally injected [1,3-3H]glycerol and [32P]orthophosphate into glycerolipids was decreased by 30-40% in treated rats. U18666A added in vitro to brain slices totally blocked glycerolipid synthesis at a high drug level (10(-3) M) but stimulated incorporation into diacylglycerol, phosphatidic acid and phosphatidylinositol at a lower level (10(-4) M). When added in vitro to cell fractions from liver or brain, U18666A readily inhibited phosphatidate phosphohydrolase and the acyltransferase enzymes which convert glycerolphosphate to phosphatidic acid and which convert diacylglycerol to triacylglycerol. Fifty percent inhibition of all three enzymes occurred at drug concentrations of between 0.4 and 1.0 mM. Phosphatidate cytidylyltransferase, an enzyme important to formation of phosphatidylinositol, was comparatively resistant to inhibition. Taken together, the results indicate that the marked reduction in the concentration of brain phospholipids caused by treatment of the young rat with U18666A is likely due to decreased phospholipid synthesis secondary to inhibition of several key enzymes in glycerolipid synthesis and, particularly, to inhibition of glycerolphosphate acyltransferase and phosphatidate phosphohydrolase.     

Potentiation of the biochemical effects of β-phenylethylhydrazine by deuterium substitution

The concentrations of dopamine (DA), m-tyramine (mTA), p-tyramine (pTA) and serotonin (5-HT) in the striata of rats 18 hr after the administration of three different doses (5, 50, or 100 mg/kg) of β-phenylethylhydrazine (phenelzine, PEH) were measured. These concentrations were compared to those following the administration of the same doses of 1, 1, 2, 2-tetradeutero-PEH (d₄PEH). In general, PEH and d₄PEH caused dose-dependent increases in the levels of mTA, pTA and 5-HT. The lowest dose of d₄PEH caused greater increases than PEH in the levels of all four monoamines. The concentration of 5-HT was increased more by d₄PEH than PEH at all three doses. The inhibition of mitochondrial MAO obtained from rat striatum by PEH or d₄PEH in vitro revealed no differences. However, the inhibition of striatal MAO obtained from rats injected with d₄PEH was found to be greater than that from rats injected with PEH. It was concluded that deuteration of PEH potentiates its ability to inhibit MAO following its administration to the rat by slowing its degradation in vivo.     

Tight binding inhibitors—IX: Kinetic parameters of dihydrofolate reductase inhibited by methotrexate,an example of equilibrium study

Equilibrium studies in the presence of methotrexate (MTX), based on the new theories of tight-binding inhibitors and on classical initial velocity analysis, indicated that the reaction mechanism of dihydrofolate reductase Lactobacillus casei MTX/R is consistent with a rapid equilibrium random bi-bi and that MTX inhibits the enzyme competitively with respect to dihydrofolate but noncompetitively with respect to NADPH. The kinetic parameters determined at pH 7.3 and 23° were: K_m for DHF, 9.8 ± 1.3 μM; K_m for NADPH, 6.0 ± 1.2 μM; K_d for E·DHF, 5.7 ± 0.7 μM; K_d for E·NADPH, 0.037 ± 0.028 μM; K_d for E·MTX, 1.20 ± 0.15 nM; K_d for E·NADPH·MTX → E·NADPH + MTX, 0.19 ± 0.04 nM; and K_d for E·NADPH·MTX → E·MTX + NADPH, 7.6 ± 5.9 nM; the molar equivalency factor was 3.33 ± 0.44 nM per unit/liter of the enzyme, and the catalytic number was 300 min^?.     

Pharmacological modulation of chemotactic factor-elicited release of granule-associated enzymes from human neutrophils: Effects of prostaglandins,nonsteroid anti-inflammatory agents and corticosteroids

Human neutrophils demonstrated a selective release of granule-associated β-glucuronidase and lysozyme but not of cytoplasmic lactate dehydrogenase during cell contact with N-formyl-methionyl-leucyl-phenylalanine (FMLP). Enzyme discharge was dependent upon treatment of neutrophils with cytochalasin B prior to exposure to FMLP. Prostaglandins (PG) D₂, E₂ and I₂ inhibited enzyme release from cytochalasin B-treated neutrophils incubated with FMLP in phosphate buffered saline, pH 7.4, at 37°. Flurbiprofen, ibuprofen, indomethacin, ketoprofen and benoxaprofen reduced the extrusion of β-glucuronidase and lysozyme from FMLP-stimulated neutrophils; acetylsalicylic acid was inactive. Methylprednisolone sodium succinate, hydrocortisone sodium succinate, prednisolone sodium succinate and triamcinolone acetonide hemisuccinate also demonstrated the capacity to inhibit the selective release of granule-associated enzymes from human neutrophils. Aldosterone hemisuccinate and deoxycorticosterone hemisuccinate were inactive. These studies indicate that certain pharmacological and therapeutic agents may function to alleviate various inflammatory conditions by curtailing the extrusion of degradative enzymes from neutrophils.     

Identification of the metabolites of an antitumor tricyclic nucleoside (NSC-154020)

The metabolism of 3-amino-1,5-dihydro-5-methyl-1-β-d-ribofuranosyl-1,4,5,6,8-pentaazaacenaphthylene (TCN, NSC-154020) was studied in human erythrocytes. With use of high performance liquid chromotography, three metabolites in addition to the major metabolite, TCN 5′-monophosphate, were detected. After isolation and purification of the metabolites, their structures were elucidated by spectrometric methods and incubations with cultured cells and catabolic enzymes. All resulted from the oxidation of TCN. Two are proposed to be α- and β-anomers of a d-ribofurasonyl nucleoside with a pyrimidol[4,5-c]pyridazine-4-one base structure. The third metabolite is proposed to be the 5′-monophosphate of the β-anomer. A mechanism for the oxidation of TCN is presented, but the exact biological oxidant involved remains unclear.