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°.     

Metabolism of benzo[a]pyrene by guinea pig adrenal and hepatic microsomes

Studies were carried out to compare the metabolism of benzo[a]pyrene (BP) by adrenal and hepatic microsomes obtained from adult male guinea pigs. Adrenal microsomes produced fluorescent metabolites (primarily phenols) approximately three to four times more rapidly than hepatic microsomes, but the differences in the rates were considerably smaller when total BP metabolism was assessed using an isotopic assay. The apparent discrepancy between the two assays is attributable to differences in the profiles of BP metabolites produced by adrenal and liver. Separation of metabolites by high pressure liquid chromatography revealed that adrenal microsomes converted BP to primarily a phenolic metabolite with a retention time identical to that of 3-hydroxy-BP. Liver microsomes, by contrast, produced approximately equal amounts of compounds co-chromatographing with 3-hydroxy-BP and BP-4,5-dihydrodiol. Small amounts of other metabolites were also produced by adrenal and hepatic microsomes. Liver microsomes catalyzed the conversion of BP to metabolites that became covalently bound to exogenous DNA. The amount of binding was dependent upon the duration of incubation and concentration of microsomal protein. Adrenal microsomes, by contrast, did not promote BP binding to DNA. Inhibition of microsomal epoxide hydratase activity with trichloropropene oxide (TCPO) blocked the formation of dihydrodiol metabolites of BP by adrenal and liver microsomes. In the presence of TCPO, liver microsomes produced large amounts of a BP metabolite co-chromatographing with BP-4,5-oxide. TCPO also increased the rate of production of DNA-binding roetabolites by liver microsomes but had no effect on the formation of DNA-binding metabolites by adrenal microsomes. The results demonstrate major differences in the pathways of BP metabolism by guinea pig adrenal and hepatic microsomes. Although adrenal microsomes metabolize BP more rapidly than hepatic microsomes, far greater amounts of reactive metabolites are produced by the liver. Thus, adrenal metabolism of BP may be of little toxicological significance.     

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.     

Lithium-induced increase in the metabolism of benzo[a]pyrene and drugs in rat liver.

Administration of lithium chloride (2.5 mEq/kg/day) to rats for 4 or 12 days increased the rates of hepatic hydroxylation of benzo[a] pyrene, antipyrine and zoxazolamine by 100, 114 and 56 per cent, respectively, and the rate of hepatic conjugation of 4-methylumbelliferone with glucuronic acid by 19 per cent. Liver lithium levels were highly correlated with plasma lithium levels but not with hepatic benzo[a] pyrene hydroxylase activities. In vitro addition of lithium to liver 10,000 g supernatants did not affect the hydroxylation of benzolalpyrene, suggesting that the lithium-induced increase in hepatic drug hydroxylation is not due to enzyme activation and is probably a result of lithium-mediated enzyme induction.     

Pyrrolizidine alkaloid-induced alterations in benzo[alpha]pyrene metabolism and binding of benzo[alpha]pyrene metabolites to deoxyribonucleic acid

Pretreatment of rats by oral administration of jacobine, a pyrrolizidine alkaloid and inducer of epoxide hydrolase, produced a marked shift in hepatic microsomal metabolism in vitro of benzo[alpha]pyrene. The formation of 9-hydroxybenzo[alpha]pyrene and 7,8-dihydroxy-7,8-dihydrobenzo[alpha]pyrene was decreased whereas the formation of 4,5-dihydroxy-4,5-dihydrobenzo[alpha]pyrene was increased following jacobine treatment. This shift in the ratio of benzo[alpha]pyrene metabolites was accompanied by a significant reduction in DNA binding. Addition of purified epoxide hydrolase to control or jacobine microsomes produced a similar decrease in total DNA binding. Chromatography of benzo[alpha]pyrene metabolite-DNA nucleoside adducts showed a marked reduction in four peaks and the elimination of one peak with microsomes from jacobine-treated rats.     

Studies on the hepatic microsomal metabolism of [14C]phenanthrene

[¹⁴C]Phenanthrene was metabolished in vitro by hepatic microsomes obtained from untreated, sodium phenobarbital (PB), and 3-methylcholanthrene (MC) pretreated Wistar rats, guinea pigs, SW and DBA/2J mouse strains. Metabolite profiles were obtained by comparison of thinlayer radiochromatographs of the products with R_fs of authentic reference compounds, trans-dihydrodi-hydroxyphenanthrenes (dihydrodiols) were the major metabolites in all preparations. In every case, except with microsomes from MC pretreated guinea pigs, trans — 9,10 — dihydro — 9,10 — dihydroxyphenanthrene predominated. With microsomes from MC pretreated guinea pigs, the 1,2-dihydrodiol was the major metabolite. Addition of cyclohexene oxide (5.0 mM) to the incubation mixture inhibited dihydrodiol formation in rat and mouse preparations by 90 per cent, and in guinea pig preparations by 70 per cent. Phenols and phenanthrene — 9,10 — oxide were produced instead.     

Temperature-dependent disposition of [14C]benzo(a)pyrene in the spiny lobster, Panulirus argus

[¹⁴C]Benzo(a) pyrene (BP) (1 mg/kg) was administered by intracardiac injection to groups of spiny lobsters which were killed at various times up to 7 weeks after dosing. Tissues and fluids were evaluated for BP-derived radioactivity. Two studies were conducted in successive summer and winter seasons, when seawater temperatures throughout were 26.5 to 29.0 and 13.5 to 16.5°C, respectively. Highest concentrations of BP-derived radioactivity were found in the hepatopancreas, stomach, intestine, intestinal contents, and the green gland. After an initial distribution phase, the dose was lost from the lobsters in a log linear manner. The elimination half-lives for overall elimination of BP-derived radioactivity were 1.11 weeks in the warmer (summer) and 2.25 weeks in the colder (winter) water. Similarly, for individual organs, elimination was more rapid in the warmer water. For the hepatopancreas, green gland, intestine, and tail muscle, respective $\text{t}\text{1}\text{2}$ values (week) were 1.02, 1.26, 1.71, and 1.42 in the summer and 2.50, 1.50, 5.04, and 2.11 in the winter. There was no suggestion of tissue accumulation of BP-derived radioactivity. HPLC analysis of hepatopancreas samples showed that, in summer, unmetabolized BP concentrations fell rapidly, accounting for only 5% of the total label in the hepatopancreas by 3 days. The fall in unmetabolized BP was accompanied by approximately equal increases in the percentages of both polar metabolites and conjugates. Although the time curve for metabolism of BP in the hepatopancreas was not studied in winter, the metabolic capacity was such that, by 3 days after the dose, only 5% of the ¹⁴C present in hepatopancreas was unmetabolized BP. Thus, it appears that, for this dose of BP, the more rapid elimination of ¹⁴C in summer was due to a more rapid excretion of metabolites, and not to increased metabolism of BP.     

Hydrolysis of permethrin,a pyrethroid insecticide,by rainbow trout and mouse tissues in vitro: A comparative study

Permethrin, a pyrethroid insecticide, is highly toxic to fish relative to its toxicity to mammals and the role of permethrin metabolism in this differential toxicity has not been investigated. A previous study, however, has shown that little hydrolysis of permethrin occurs in vivo in rainbow trout in contrast to mammals where ester hydrolysis is a major detoxification reaction. In the present study, the rates of permethrin hydrolysis in rainbow trout and mouse tissues in vitro were estimated. Mouse liver, kidney, and plasma incubated at 37°C, hydrolyzed trans-[¹⁴C]permethrin approximately 166, 38, and 59 times faster, respectively, than the same rainbow trout tissues incubated at 12°C. At an incubation temperature of 37°C trout liver microsomes hydrolyzed trans-permethrin approximately 45 times slower than mouse liver microsomes. When the total capacity of trout and mouse tissues to hydrolyze trans-permethrin was compared on a whole body basis mice hydrolyzed trans-permethrin 184 times faster than rainbow trout. The results suggest that rainbow trout tissues have a much lower capacity than mouse tissues to hydrolyze permethrin, and this may explain the relative absence of permethrin hydrolysis products in permethrin-exposed raibow trout. It is impossible that the high toxicity of permethrin to rainbow trout is in part related to a low capacity to hydrolyze permethrin.     

In vitro benzo[a]pyrene metabolism from lindane-treated rat liver: effect of oral and acute administration, and comparison with phenobarbital and methylcholanthrene pretreatment.

Lindane (γ-hexachlorocyclohexane) was given orally to Sprague-Dawley male rats in their basal diet, at 24, 120, and 240 ppm, for 4 weeks. Acute intoxication was obtained with ip injection, at 20 mg/kg/day for 3 consecutive days, and the inductive effect was compared to phenobarbital (ip, 80 mg/kg/day for 3 days) and methylcholanthrene (ip, 20 mg/kg/day for 3 days) pretreatment. The spectrofluorometric measurement of arylhydrocarbon hydroxylase (AHH) activity did not show any induction after lindane and phenobarbital pretreatment, while the quantitative determination of benzo[a]pyrene (BP) metabolites by thin-layer chromatography indicated an increase of 3-hydroxy-BP (3-OH-BP). Differences in the BP/protein ratio could explain this discrepancy. Consequently, lindane appeared as a weaker inducer of AHH activity than phenobarbital. The in vitro binding of [¹⁴C]BP metabolites to DNA was largely enhanced by the oral administration of lindane from as low as 24 ppm in the diet and seemed dose related until 120 ppm, reaching two or three times the control value. The acute intoxication by lindane resulted in an increase in BP metabolite binding to DNA similar to that obtained with 120 ppm in the diet. From the chromatographic pattern of BP metabolites, it is concluded that lindane induced a large formation of 4,5-BP-dihydrodiol, which was related to a two-fold increase in epoxide-hydratase activity. From these results, lindane could be considered as a phenobarbital-like inducer.     

Development of a radioimmunoassay for [DES-ARG9]-bradykinin

Antisera to [des-Arg9]-bradykinin were elicited in rabbits immunized with the peptide conjugated to thyroglobulin and/or ovalbumin. Sera were screened for the presence of antibody with three radioactive antigens, mono-125I-labeled derivatives of [Tyr1, des-Arg]-kallidin, [Tyr, des-Arg]-bradykinin, and [Tyr, des-Arg]-bradykinin that were prepared by treating mono-125I-labeled [Tyr]-kallidin, [Tyr]-bradykinin, and [Tyr]-bradykinin with carboxypeptidase B. Of the six animals immunized, five produced antibodies to [des-Arg]-bradykinin as evidenced by the ability of their sera to bind at least 33% of the added radioactivity at a final dilution of 1:500. Sensitivity and specificity studies were performed with each labeled antigen and a dilution of antiserum that bound 30-50% of the radioactivity. The best labeled antigen-antibody combination, with respect to titer, sensitivity, and specificity was obtained with [mono-125I-Tyr, des Arg]-bradykinin and serum from a rabbit immunized with [des-Arg]-bradykinin conjugated to ovalbumin with toluene diisocyanate. The lowest concentration of [des-Arg]-bradykinin inhibiting 50% of this radioactive antigen binding was 0.23 ng/ml and the lowest concentration which could be distinguished from no [des-Arg]-bradykinin added was 67 pg/ml. This antiserum cross-reacts with bradykinin and lysyl-bradykinin about 9% but not with methionyl-lysyl-bradykinin.     

Metabolism, disposition and excretion of [C]melamine in male Fischer 344 rats

The metabolism, excretion and disposition of melamine were determined after administration of a single oral dose of 0.025 mCi (0.38 mg) [¹⁴C]melamine to adult male Fischer 344 rats. Within the first 24 hr, 90% of the administered dose was excreted in the urine. Negligible radioactivity appeared in breath and faeces. There was little difference in blood, liver or plasma concentrations of ¹⁴C, suggesting that melamine distributes in body water. The only organs showing radioactivity levels much higher than plasma were the kidney and bladder. The bladder level was by far the highest, a finding probably due either to back diffusion from urine or to contamination of bladder tissue with urine. Virtually no residual radioactivity was observed in tissues examined at 24 hr or later. The elimination-phase half-life calculated from plasma data, 2.7 hr, was in good agreement with the urinary-excretion half-life of 3.0 hr. The renal clearance of melamine was 2.5 ml/min. Radioactivity in plasma or urine co-chromatographed with that of the dosing solution, indicating that melamine is not metabolized in the male Fischer 344 rat.     

Inhibition by suloctidil of [3H] nitrendipine binding to cerebral cortex membranes

[3H] Nitrendipine binds specifically with high affinity and high capacity (KA congruent to 0.20 +/- 0.01 nM, Bmax = 4.4 +/- 0.3 pM/g tissue, means +/- S.E.M.; N = 4) to guinea-pig cerebral cortex membranes. Suloctidil fully inhibits [3H] nitrendipine binding with a Ki value of 0.45 microM. The interaction between suloctidil and the putative Ca2+ channels is allosteric as shown by competition experiments performed in the presence of D 600 or diltiazem. Comparison of the activity of some close analogs of suloctidil provides evidence for the importance of the amino group and of the hydrophobic amino substituent in the interaction of suloctidil and the putative Ca2+ channel. It is suggested that part of the previously reported blockade of Ca2+ entry induced by suloctidil is due to a blockade of the Ca2+ channels.     

Metabolism of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in chicken aortas: Monooxygenation,bioactivation to mutagens,and covalent binding to DNA in vitro

The capacity of enzymes present in chicken aortas to catalyze the conversion of polynuclear aromatic hydrocarbons to metabolites that covalently bind to DNA and produce mutations in Salmonella typhimurium auxotrophs is described. Chicken aortic homogenates contained active monooxygenases capable of catalyzing the conversion of benzo[a]pyrene (BaP) and 7,12-dimethylbenz[a]anthracene (DMBA) to oxidized products as measured by a highly sensitive radiometric assay and high-pressure liquid chromatography. BaP was converted primarily to phenols, while DMBA was converted to hydroxymethyl, phenolic, and diol metabolites. Pretreatment of chickens with Aroclor 1254, 3-methylcholanthrene (3-MC), or 5,6-benzoflavone (5,6-BF) resulted in significant (p < 0.05) increases in the aortic oxidative metabolism of both BaP and DMBA; phenobarbital (Pb) and pregnenolone 16α-carbonitrile (PCN) were ineffective as inducers of chicken aortic monooxygenation. Chicken aortic S-9 homogenates catalyzed the bioactivation of BaP and DMBA to mutagens for S. typhimurium strains TA-98 and TA-1538. Aroclor 1254, 3-MC, 5,6-BF, Pb, or PCN pretreatments did not significantly enhance detectable mutagenesis. When aortic homogenates from untreated chickens were utilized, low specific activities of bound products of BaP, DMBA, or 3-MC to DNA were observed. Pretreatment of chickens with either Aroclor 1254 or 3-MC resulted in 7- to 12-fold increases in specific binding activity. Inhibition of DMBA and BaP metabolism as well as DNA binding was observed when 7,8-benzoflavone (130 μm) or β-estradiol (100 μm) were added to incubation flasks. 1,2-Epoxy-3,3,3-trichloropropane (13 μm) enhanced binding of [³H]BaP and [³H]DMBA to DNA. Additions of glutathione to reaction flasks inhibited ³H binding to DNA, suggesting an important role of aortic nonoxidative metabolism of polycyclic aromatic hydrocarbons.     

Lung retention and metabolic fate of inhaled benzo(a)pyrene associated with diesel exhaust particles

Polycyclic aromatic hydrocarbons (PAHs) are a class of compounds considered to have human carcinogenic potential and have been found associated with many respirable, environmental particle pollutants. The effect of these ultrafine, insoluble, carrier particles on the lung retention and metabolic fate of inhaled PAHs was investigated with a radiolabeled model PAH, [3H]benzo(a)pyrene (3H-BaP). Fischer-344 rats were exposed (30 min) by nose-only inhalation to 3H-BaP adsorbed (approximately 0.1% by mass) onto diesel engine exhaust particles. These aerosols were generated in a dynamic aerosol generation system by vapor condensation methods. The total mass concentration of these aerosols was 4-6 micrograms/liter of air with a mass median diameter of 0.14 micron. Lung clearance of the inhaled particle-associated 3H radioactivity occurred in two phases. The initially rapid clearance of this inhaled radiolabel had a half-time of less than 1 hr. The second, long-term component of lung clearance had a half-time of 18 +/- 2 days and represented 50 +/- 2% of the 3H radioactivity that had initially deposited in lungs. In contrast, previous inhalation studies with a pure 3H-BaP aerosol showed that greater than 99% of the 3H radioactivity deposited in lungs was cleared within 2 hr after exposure (Sun et al., Toxicol. Appl. Pharmacol. 65, 231-244, 1982). By HPLC analysis, the majority of diesel soot-associated 3H radioactivity retained in lungs was BaP (65-76%) with smaller amounts of BaP-phenol (13-17%) and BaP-quinone (5-18%) metabolites also being detected. No other metabolites of BaP were detected in lungs of exposed rats. Tissue distribution and excretion patterns of 3H radioactivity were qualitatively similar to previous inhalation studies with 3H-BaP coated Ga2O3 aerosols (Sun et al., 1982). These findings suggest that inhaled PAHs may be retained in lungs for a greater period of time when these compounds are associated with diesel engine exhaust particles. In addition, these compounds retained in lungs can be metabolized in lungs. These results may have significant implications for the health risks that may be involved with human exposure to particle-associated organic pollutants.     

Localisation of [3H]clonidine binding to membranes from guinea pig renal tubules

The selective radioligand [3H]clonidine has been used to localise alhpa 2 adrenoceptors in guinea pig kidney. Chemical sympathectomy with 6-hydroxydopamine produced no significant change in the number of sites labeled by [3H]clonidine indicating that the majority of binding sites were not located on sympathetic nerve terminals. Binding was enhanced in membranes prepared from renal tubules and considerably reduced in preparations from glomeruli. Subcellular fractions of renal cortex revealed that binding was to plasma membranes and that the greatest binding capacity was present in the fraction rich in basal lateral membranes. It is concluded that the major concentration of renal alpha 2 adrenoceptors are present on renal tubules and that they may be localised to a particular pole of the renal tubule cell.     

Some characteristics of hamster liver and lung microsomal aryl hydrocarbon (biphenyl and benzo(a)pyrene) hydroxylation reactions

Aryl hydrocarbon hydroxylation (AHH) reactions were compared using liver and lung microsomes of corn oil- and 3-methylcholanthrene (3-MC)-treated hamsters, employing benzo(a)pyrene (BAP) and biphenyl as substrates. The predominant AHH activity of liver and lung microsomes from corn oil- or 3-MC-treated hamsters was biphenyl 4-hydroxylase. Biphenyl 2-hydroxylase and BAP-hydroxylase activities were approximately 50 per cent as active as biphenyl 4-hydroxylase in liver and approximately 1–3 per cent as active as biphenyl 4-hydroxylase in lung microsomes. Biphenyl 4-hydroxylase activity was 70–80 per cent as active in lung as in liver microsomes. Treatment with 3-MC in vivo induced the biphenyl 4-hydroxylation reaction in liver but not in lung microsomes, the biphenyl 2-hydroxylation reaction both in lung and liver microsomes, and the BAP hydroxylation reaction in lung but not in liver microsomes. Biphenyl 2- and 4-hydroxylase activities of liver microsomes displayed similar sensitivities to inhibition by a number of chemical inhibitors in vitro. Inhibition of biphenyl hydroxylation reactions by metyrapone or carbon monoxide did not distinguish between lung or liver microsomal mono-oxygenases of corn oil- or 3-MC-treated hamsters. While small differences were expressed by inhibition with ethylmorphine, large differences became apparent through inhibition studies with BAP or α-naphthoflavone. It is concluded that the major aromatic hydroxylase activity of lung microsomes from corn oil- or 3-MC-treated hamsters resembles the constitutive (uninduced) AHH of the liver microsomes and that the minor aromatic hydroxylase activity of lung microsomes from corn oil- or 3-MC-treated hamsters resembles the induced AHH of the liver microsomes.     

Effects of dietary Schizandra chinensis, brussels sprouts and Illicium verum extracts on carcinogen metabolism systems in mouse liver

Ethanol extracts of Brussels sprouts (BRX), Schizandra chinensis (SZX) or Illicium verum (IVX) were added to a semi-purified basal diet and fed to adult male and female C57B1/6 mice for 14 and 10 days, respectively. Other groups received the unsupplemented basal diet or a mouse chow. Liver fractions were prepared from these mice to investigate the effects of the diets on the enzyme systems involved in benzo[a]pyrene (BP) and aflatoxin B1 (AFB1) metabolism. The effects of the microsomal or cytosolic liver fractions on the in vitro mutagenicity of BP and AFB1 and on the DNA binding of AFB1 were also studied. There were several apparently sex-related differences in the responses of the monooxygenase system components measured. In males, cytochrome P-450 levels were significantly increased only in the chow group, while significant increases in both 7-ethoxycoumarin O-deethylase (ECD) and aryl hydrocarbon hydroxylase (AHH) activities were seen only in the SZX group. In females, cytochrome P-450 levels were significantly increased in both the BRX and SZX groups, whereas AHH activity was significantly increased only in the chow and BRX groups and ECD activity was increased in the SZX and IVX groups. Microsomal epoxide hydratase (EH) was induced in males in the SZX and IVX groups and in females only in the SZX group, while cytosolic EH was significantly increased only in IVX males. Diet-induced changes in monooxygenase activities were found to be the best indicators of changes in microsome-mediated BP mutagenesis and AFB1 mutagenesis and binding to DNA in vitro, with a direct correlation between high AHH and/or ECD activities and the levels of mutagenic response to BP or AFB1 in the Ames assay and of DNA binding of AFB1.     

Effects of anticonvulsants on the in vivo and in vitro release of gaba

The actions of various anticonvulsant compounds on GABA release in vivo and in vitro were studied. An in vivo, superfusion of sensorimotor cerebral cortex was employed and drugs were administered either by intraperitoneal injection, or in superfusion fluid and release of endogenous amino acids was measured. The in vitro method involved superfusion of synaptosomes, with drugs dissolved in superfusate, with monitoring of the release of pre-loaded [U¹⁴C]-GABA. Two alkyl-GABA analogues, γ-acetylenic GABA and γ-vinyl GABA caused enhanced release of GABA to superfusate both in vivo and in vitro. However, phenobarbitone, diphenyl hydantoin, sodium n-dipropyl acetate and carbamazepine were without effect on GABA release in either test system. Taurine caused no detectable GABA release in vivo, or from purified synaptosomes in vitro, but did stimulate release in vitro, from crude synaptosome preparations containing mitochondria in large quantities, though histidine and leucine were equally effective.