Effects of polychlorinated biphenyls and environmental temperature on in vitro formation of benzo[a]pyrene metabolites by liver of trout (Salmo gairdneri)

Rainbow trout (Salmo gairdneri) held at 7° and 16° were given Aroclor 1254 (PCB) (10mg/kg body wt) via intraperitoneal injections. The binding of [³H]benzo[a]pyrene (BaP) to deproteinized salmon sperm DNA was assayed (pmoles BaP equivalents per mg DNA per mg protein) using the post-mitochondrial supernatant (S 10) fractions from livers of fish at 24–168 hr after the PCB exposure. Liver enzymes from the untreated fish acclimated at 7° yielded an average binding value (0.37 ± 0.17) which was significantly greater (P < 0.05) than the value (0.07 ± 0.03) for untreated fish at 16°. Liver supernatant fractions from PCB-induced fish acclimated at 16° and sampled at 24–120 hr showed a substantial increase (P < 0.05) in the binding (average value 2.4 ± 1.8) compared to the value obtained with untreated fish at 16°. At 24, 48 and 120 hr after the PCB treatment of fish held at 7°, there was no significant increase in the binding value or extent of metabolism of BaP compared to that obtained with the untreated fish at 7°. However, at 168 hr, three of four fish at 7° responded to the PCB treatment with significantly (P < 0.05) increased binding values (3.3 ± 1.6). Chromatographic analyses of the ethyl acetate-soluble metabolites revealed that 3-hydroxy BaP and 7,8- and 9,10-dihydrodiols were the major metabolites; K-region metabolites were formed in trace amounts in untreated and PCB-treated fish at both temperatures. No marked qualitative differences were observed in metabolite profiles after the PCB treatment; however, overall metabolism of BaP and production of reactive metabolites by liver enzymes were considerably (P < 0.05) enhanced in the PCB-induced fish at both 7° and 16°.     

Dissociation between ornithine decarboxylase activity and aryl hydrocarbon hydroxylase induction in cell cultures treated with benz[a]anthracene and inhibitors of ornithine decarboxylase

The induction of aryl hydrocarbon hydroxylase and ornithine decarboxylase by benz[a]-anthracene in the presence or absence of ornithine decarboxylase inhibitors was studied in three different cell culture systems. An almost complete abolishment of ornithine decarboxylase activity by 1,3-diamino-2-propanol or α-difluoromethyl ornithine before the addition of the inducer did not affect appreciably the induction of aryl hydrocarbon hydroxylase by benz[a]anthracene in human embryo, HeLa and Reuber H-II-4-E cells in culture. These results suggest that the induction of aryl hydrocarbon hydroxylase does not require ornithine decarboxylase activity per se and can be expressed in the absence of continuous polyamine synthesis.     

On the capacity of human placental enzymes to catalyze the formation of diols from benzo[a]pyrene

Studies were performed on the oxidative biotransformation of benzo[a]pyrene in fortified preparations of human placental microsomes by analysis with high-pressure liquid chromatography. These investigations revealed that the utilization of substrate concentrations (1–2 × 10^?4m) sufficiently high to assure zero-order reaction kinetics (in terms of the generation of phenolic metabolites) produced a marked inhibitory effect on the formation of dihydrodiols in the same reaction mixtures. Relative quantities of dihydrodiols generated increased with decreasing substrate concentrations between 200 and 2.7 μm. Additions of manganese or ferric ions to reaction mixtures altered the ratios of generated phenols to dihydrodiols but did not provide an explanation for the differences observed in the literature. Identical results were obtained with either ¹⁴C- or ³H-labeled benzo[a]pyrene as substrates. The data suggested the possibility that considerable quantities of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, a proximate mutagen/carcinogen, may be generated in vivo by placental tissues of women who smoke.     

Metabolism of 15N-labelled N-nitrosodimethylamine and N-nitroso-N-methylaniline by isolated rat hepatocytes

The N-demethylation of ¹⁵N-labeled N-nitrosodimethylamine (DMN) and N-nitroso-N-methylaniline (NMA) by isolated rat hepatic cells has been investigated. The values obtained in this system for molecular nitrogen formed during metabolism, compared with substrate consumed, were DMN 47%, NMA 23%, and N-nitroso-N-methylurea (NMU) 105%. The results for DMN are roughly halfway between those previously determined with rat liver S-9 fraction in vitro (33%) and in vivo (67%). For NMA, the hepatocyte data are closer to those obtained from S-9 in vitro (19%), rather than the in vivo (52%). No mixed nitrogen (¹⁵N¹⁴N) or labeled nitrogen oxides were found.     

Effects of dietary cabbage,brussels sprouts, Illicium verum,Schizandra chinensis and alfalfa on the benzo[a]pyrene metabolic system in mouse liver

Male C57B16 mice were fed on diets containing either 20% cabbage, 20% Brussels sprouts, 20% alfalfa, 5% Schizandra chinensis or 5% Illicium verum (two Chinese medicinal herbs) or on a chow or purified basal diet for 14 days after a 1-wk equilibration period on the basal diet. Liver microsomal fractions were assayed for cytochrome P-450 content, aryl hydrocarbon hydroxylase (AHH) and epoxide hydrolase (EH). Liver microsome-mediated benzo[a]pyrene (BP) metabolism (with and without an EH inhibitor, 1,2-epoxy-3,3,3-trichloropropane) was analysed by HPLC. Liver weights of the animals fed on Brussels sprouts and I. verum were significantly increased compared with those of the animals fed on basal diets. S. chinensis induced a 3-fold increase in cytochrome P-450 (P < 0.05). Although P-450 induction in the other groups was as high as 1.8-fold (for chow), statistical significance was not established. Chow induced AHH activity 2.2-fold (P < 0.05), while S. chinensis and alfalfa induced 1.6-fold and 1.7-fold increases, respectively, in AHH activity, although neither increase was statistically significant. EH was stimulated significantly in the following order: I. verum (2.1-fold) > chow (1.7-fold) >S. chinensis (1.6-fold) > Brussels sprouts (1.4-fold). Total levels of BP metabolism and phenol II (primarily 3-hydroxybenzo[a]pyrene) formation were closely associated for each dietary treatment. Total BP metabolism was significantly increased (2.1-fold) in the chow-fed group and increased 1.6-fold in the S. chinensis group (P > 0.05). No increase was seen with the other diets. Phenol II formation relative to total metabolites was significantly increased for the S. chinensis and I. verum groups compared to the basal group. Diet-related variations in phenol production relative to total metabolism were eliminated by addition of the EH inhibitor to the incubation media.     

Conversion of N-hydroxyamphetamine to phenylacetone oxime by rat liver microsomes

These in vitro studies indicate that N-oxidation of N-hydroxyamphetamine (NOHA) by rat liver homogenates yields phenylacetone oxime (PAOx) as the major metabolite. This oxidation was NADPH and oxygen dependent but was not appreciably increased in microsomes from phenobarbital-pretreated animals. The addition to microsomal incubations of Superoxide dismutase (SOD), catalase (CAT), azide or mannitol did not alter the rate of oxidation, suggesting that O₂su?. H₂O₂, or OH^. are not involved in this process. The reaction was minimally inhibited by a 2:1 ratio of CO/O₂, and there was no significant reduction in the formation of product by the presence of diethylaminoethyl diphenylvalerate (SKF-525A) or 2,4-dichloro-6-phenylphenoxyethylamine (DPEA) in micromolar concentrations. Thus, although this NADPH-dependent N-oxidation pathway was catalyzed by rat hepatic microsomes, the data suggest that it was not a cytochrome P-450 mediated monooxygenase reaction.     

Fate of [3H]amezinium in sympathetically innervated rabbit tissues

Perfused rabbit hearts accumulated intra-aortically infused [³H]amezinium. At concentrations of 1 or 10 nM, the accumulation proceeded at a constant rate for at least 60 min. After 60 min, tissue medium ratios were between 6 (1 μM) and approx. 100 (1 or 10 nM [³H]amezinium). Cocaine or pretreatment with 6-hydroxydopamine abolished, and pretreatment with reserpine reduced the accumulation of [³H]amezinium (1nM). Kinetic analysis yielded a K_m value of 0.9 μM and a V_max of 1.2 nmole g^?1 min^?1. When hearts had been labelled with 1 or 10 nM [³H]amezinium, the fractional rate of the subsequent efflux was very low (0.001 min^?1). It was greatly increased when the animals had been pretreated with reserpine. Electrical stimulation of the sympathetic nerves released [³H] amezinium from pre-labelled rabbit pulmonary artery strips. The electrically evoked overflow was abolished by tetrodotoxin or omission of Ca²⁺; it was enhanced by cocaine, desipramine and yohimbine and decreased by clonidine. The results show that amezinium, at least at low concentrations, is selectively taken up into postganglionic sympathetic neurones, that it is partly sequestered in the vesicles, and that it is released by action potentials. Amezinium is a structurally novel substrate of both the noradrenaline transport mechanism of the axolemma and the transport mechanism of the noradrenaline-storing synaptic vesicles.     

3H]Dihydroergocryptine binding to alpha-adrenergic receptors of human platelets. A reassessment using the selective radioligands [3H]prazosin, [3H]yohimbine, and [3H]rauwolscine

Which subtype(s) of the alpha-adrenergic receptor occurs on human platelets? Studies of platelet responsiveness to adrenergic compounds and indirect radioligand binding studies addressing this question have yielded contradictory conclusions. These binding studies employed the ligand [³H]dihydroergocryptine ([³H]DHE), an alpha-adrenergic antagonist that does not select between alpha₁- and alpha₂-adrenergic receptors and that also binds to other receptor types in some tissues. To determine the subtype of the platelet alpha-adrenergic receptor, we have examined the binding to intact human platelets of [³H]prazosin (alpha₁-selective), [³H]yohimbine (alpha₂-selective), and [³H]rauwolscine (alpha₂-selective), and we have compared the binding of these selective radioligands with that of [³H]DHE. [³H]Yohimbine and [³H]rauwolscine both bound with high affinity (K_D = 2.7 and 4.6 nM, respectively) to an equal number and a single class (Hill coefficient ～1.0) of sites (～300 per platelet), but [³H]yohimbine yielded lower nonspecific binding than did [³H]rauwolscine. In paired experiments, [³H]DHE bound to 1.5 times as many (phentolamine-displaceable) sites as did [³H]yohimibine or [³H]rauwolscine. Unlabeled vohimbine and epinephrine competed for fewer [³H]DHE binding sites than did phentolamine. Thus, in addition to binding to the alpha₂-adrenergic receptors identified by [³H]yohimbine and [³H]rauwolscine, [³H]DHE seems to bind to other sites on human platelets. The nature of these sites is not clear. We found that [³H]prazosin did not identify alpha₁-adrenergic receptors on platelets, and that phenoxybenzamine only inhibited [³H]yohimbine and [³H]DHE binding at higher concentrations than usually observed for alpha₁-adrenergic receptors. We conclude that (1) all alpha-adrenergic sites on human platelets are of the alpha₂ subtype, (2) [³H]DHE may bind to additional, as yet ill-defined, sites in addition to those sites identified by [³H]yohimbine and [³H]rauwolscine, and (3) [³H]yohimbine is the preferred antagonist radioligand for studying the alpha₂-adrenergic receptors on human platelets.     

Effect of pyrrolizidine alkaloids from tansy ragwort (Senecio jacobaea) on hepatic drug-metabolizing enzymes in male rats

Ad lib. consumption of diets containing 5% tansy ragwort (Senecio jacobaea) for 1–4 weeks produced a 5- to 6-fold increase in hepatic microsomal epoxide hydrase and significant increases in cytosolic glutathione S-transferase activities in male Long-Evans rats. An enhancement of these enzyme activities was also observed when a diet containing 1% tansy ragwort was fed for a period of 3 weeks. Feeding a diet containing 0.5% pyrrolizidine alkaloid (PA) mixture extracted from tansy ragwort for 1 week produced a 5-fold increase in hepatic epoxide hydrase and a 73 per cent increase in glutathione S-transferase activities. In contrast, hepatic microsomal aryl hydrocarbon hydroxylase activity (AHH) was reduced significantly by feeding diets containing 5% tansy ragwort or a 0.5% alkaloid mixture. Hepatic microsomal cytochrome P-450 content was lowered following consumption of the 0.5% alkaloid mixture but not by feeding a 5% tansy ragwort diet, the difference presumably being a result of the lowered PA intake by the latter animals. Exposure to the pyrrolizidine alkaloids, therefore, may influence significantly the capacity of animals to metabolize endogenous or foreign compounds and possibly also affect the subsequent biotransformation and toxicity of these plant constituents.     

Selective inhibition of rat pulmonary monooxygenase by O, O, S-trimethyl phosphorothioate treatment

The effects of oral administration of O,O,S-trimethyl phosphorothioate (OOS), an impurity present in widely used organophosphorus insecticides, were studied using pulmonary and hepatic microsomal enzymes of rats. The animals were treated with OOS at 10, 20 and 40 mg/kg, and were killed on day 3 after treatment. Their relative lung weights increased markedly at 20 and 40 mg/kg, increasing 94% at the highest dose, whereas the weight of liver decreased. At 20 mg/kg OOS, the cytochrome P-450 content of the lung and liver decreased to 83 and 80% of the control levels respectively. Pulmonary microsomal 7-ethoxycoumarin (7-Ec) O-deethylase decreased in a dose-dependent manner; activities were less than 10% of control at the 40 mg/kg dose. The activity of pulmonary coumarin hydroxylase also decreased following OOS treatment, but the decrease was not dose-dependent since no activity was detectable at doses over 10 mg/kg. In contrast, the effect of OOS treatment on hepatic monooxygenase activity was moderate. 7-Ec deethylase activity was not affected by OOS treatment at any dose level, while p-nitroanisole (p-NA) demethylase activity was decreased only at the 40 mg/kg dose of OOS. Pulmonary malathion carboxylesterase activity was not affected by OOS treatment. In contrast, a dose-dependent decrease was observed in the liver carboxylesterase. Time course effects of OOS treatment on these parameters were examined by treating rats at 20 mg/kg. The animals were killed 0.5, 1, 3 and 7 days after the treatment. The 7-Ec deethylase activity of pulmonary microsomes was decreased on days 0.5, 1 and 3 after treatment, the maximum decrease being observed on day 1. Significant decreases were not observed in hepatic microsomal activities of 7-Ec deethylase or p-nitroanisole demethylase throughout the experimental period; rather, these activities were higher on day 7. Hepatic microsomal malathion carboxylesterase was lower on days 0.5, 1 and 3 after OOS treatment.     

α-monofluoromethyl and α-difluoromethyl putrescine as ornithine decarboxylase inhibitors: In vitro and in vivo biochemical properties

In vitro, 5-fluoropentane-l,4-diamine and 5,5-difluoropentane-l,4-diamine are potent enzyme-activated inhibitors of rat liver ornithine decarboxylase (EC 4.1.1.17). The two α-fluoromethyl derivatives of putrescine activate to different degrees 5-adenosyl-l-methionine decarboxylase (EC 4.1.1.50). The difluoromethyl derivative differs from the monofluoromethyl derivative in that it is not a substrate of diamine oxidase (EC 1.4.3.6), but is a better substrate of mitochondrial monoamine oxidase (EC 1.4.3.4) than the monofluoromethyl derivative. In vivo, a single i.p. injection of 200 mg/kg of 5-fluoropentane-l,4-diamine to rats causes a marked decrease of the ornithine decarboxylase activity in the ventral prostate and to a lesser extent in the thymus, whereas 5,5-difluoropentane-l, 4-diamine causes only a slight decrease of this enzyme activity in the prostate and does not affect it in the thymus. Both compounds produce a decrease of 4-aminobutyrate: 2-oxoglutarate aminotransferase (EC 2.6.1.19) activity in the brain. The differences observed between the biochemical properties of the two α-fluoromethyl derivatives of putrescine are discussed in relation to the pK_a value of the α-amino group which decreases from 7.75 for 5-fluoropentane-l,4-diamine to 6.4 for 5,5-difluoropentane-l, 4-diamine.     

Serotonin-releasing effects of substituted piperazines in vitro

The effects of various piperazine-containing compounds on the release of endogenous serotonin (5-HT) from rat hypothalamic slices were evaluated. Incubation of hypothalamic slices with m-chlorophenylpiperazine (mCPP) or m-trifluoromethylphenylpiperazine (mTFMPP) evoked a potent, dose-dependent release of endogeous 5-HT that was similar in magnitude to that seen with tryptamine, p-chloroamphetamine, or fenfluramine. In the presence of the 5-HT uptake blockers fluoxetine or chlorimipramine, this release was reduced dramatically. Furthermore, removal of calcium from the incubation medium had little effect on the drug-induced release, suggesting that the release mechanism involved displacement of 5-HT stores and not depolarization-induced exocytosis. Trazodone, MK-212, and quipazine had only small effects on release. These studies show that several piperazine-containing compounds can evoke a potent release of endogenous stores of hypothalamic 5-HT in vitro, actions which should be considered together with their direct agonist activity when interpreting the CNS effects in vivo     

Binding of adrenergic ligands ([3H]clonidine and [3H]WB-4101) to multiple sites in human brain

In order to identify alpha-adrenoceptors in post-mortem human brain and to detect the possible existence of multiple types of binding sites for adrenergic [³H]ligands, we studied the binding of [³H]clonidine and [³H]WB-4101 to human brain cerebral cortex, hippocampus, hypothalamus and striatum. Frontal cortex revealed two binding sites for [³H]clonidine (with K_D values of approximately 1 and 8 nM), as indicated by the biphasic Scatchard plot, the biphasic pattern of dissociation kinetics, and the biphasic inhibition by phentolamine on the binding of [³H]clonidine; the high-affinity site was heat-labile. Two high-affinity binding sites for [³H]WB-4101 were also detected in the human frontal cortex (with K_D values of about 0.09 and 1.5 nM), as revealed by a biphasic pattern of dissociation. A third site with low affinity binding for [³H]WB-4101 was detected by the biphasic inhibition by phentolamine (as well as by WB-4101 and prazosin) on the binding of [³H]WB-4101. The three other brain regions revealed very similar patterns exhibited by the frontal cortex, except that the density of the [³H]clonidine sites (of either high or low affinity) was highest in the hypothalamus, whereas the density of [³H]WB-4101 sites was highest in the hippocampus.