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The interaction of a potent carcinogen, aflatoxin B(1) (AFB(1)), with a probiotic strain of lactic acid bacteria, Lactobacillus rhamnosus strain GG (GG), has been investigated. The binding of AFB(1) to GG in the late exponential-early stationary phase was studied for viable, heat-killed and acid-killed bacteria. In general, viable, heat-killed and acid-killed GG responded in a similar manner. The effects of pronase E, lipase and m-periodate on AFB(1) binding and release were consistent with AFB(1) binding predominantly to carbohydrate components of the bacteria. The effect of urea suggested hydrophobic interactions play a major role in binding. Increasing concentration (0.01-1 M) of NaCl or CaCl(2) had minor effects on AFB(1) binding suggesting some involvement of electrostatic interactions. An increase in pH from 2.5 to 8.5 had no effect on AFB(1) binding but decreased binding of AFB(2a), possibly due to hydrogen bonding interactions.

Aflatoxin M1 (AFM1) is a highly toxic compound found in milk. Its occurrence poses a threat to the health of consumers, especially young children, and leads to economic losses due to contaminated milk. The problem is global but more severe in developing countries. Consequently, there is a great demand for novel strategies to prevent the contamination and adverse effects of AFM1. To develop a safe and practical decontamination method, a preliminary study was carried out with specific lactic acid bacteria strains that were tested for their ability to remove AFM1 from liquid media. All strains, whether viable or heat-killed, could reduce the AFM1 content of a liquid medium. Two most effective strains were also tested using contaminated skim and full cream milk. The results indicate that specific lactic acid bacteria used in dairy products can offer novel means of decontaminating aflatoxin M1 from milk.

The ability of Lactobacillus rhamnosus strains GG and LC-705 to remove AFB1 from the intestinal luminal liquid medium has been tested in vivo using a chicken intestinal loop technique. In this study, the GG strain of L. rhamnosus decreased AFB1 concentration by 54% in the soluble fraction of the luminal fluid within 1 min. This strain was more efficient in binding AFB1 compared with L. rhamnosus strain LC-705 (P < 0.05) that removed 44% of AFBl under similar conditions. Accumulation of AFB1 into the intestinal tissue was also determined. There was a 74% reduction in the uptake of AFB1 by the intestinal tissue, in the presence of L. rhamnosus strain GG compared with 63% and 37% in the case of Propionibacterium freudenreichii ssp. shermanii JS and L. rhamnosus strain LC-705, respectively. The complexes formed in vitro between either L. rhamnosus strain GG or L. rhamnosus strain LC-705 and AFB1 were stable under the luminal conditions for a period of 1 h.

Lactic acid bacteria have been previously reported to possess antimycotoxigenic activities both in vitro and in vivo. The objective of this study was to investigate the effect of aflatoxin B1 on adhesion capability of Lactobacillus rhamnosus strain GG using a Caco-2 adhesion model. Removal of aflatoxin B1 by L. rhamnosus strain GG reduced the adhesion capability of this strain from 30% to 5%. It is therefore concluded that aflatoxins may influence the adhesion properties of probiotics able to sequester them, and subsequently these bacteria may reduce the accumulation of aflatoxins in the intestine via increased excretion of an aflatoxin-bacteria complex.

A stereoselective method of analysis of the antidepressant drug doxepin (DOX, an 85:15% mixture of E-Z stereoisomers), its principal metabolites E- and Z-N-desmethyldoxepin (desDOX) and ring-hydroxylated metabolites in microsomal incubation mixtures is described. DOX and its metabolites were extracted from alkalinised incubation mixtures by either: 9:1 hexane-propan-2-ol (method 1) or 1:1 hexane-dichloromethane (method 2), derivatised with trifluoroacetic anhydride and analysed by GC-MS with selected ion monitoring. Both methods were suitable for the analysis of individual desDOX isomers as indicated by correlation coefficients of > or = 0.999 for calibration curves constructed between 50 and 2500 nM, and good within-day precision at 125 nM (C.V. < or = 14%) and 1000 nM (C.V. < or = 8%). Method 1, however, was unsuitable for the analysis of ring-hydroxylated metabolites of DOX, whereas the hydroxylated metabolites of E-DOX and E-desDOX (generated in situ) were extracted by method 2 with a C.V. of ca. 13%. This is the first assay method that permits the simultaneous measurement of desDOX and hydroxylated metabolites of DOX in microsomal mixtures.

The chronic effects of the chelating agent diethylenetriamine pentaacetic acid (DTPA) on reproduction, condition factor, liver somatic index (LSI), gonad somatic index (GSI), and ethoxyresorufin O-deethylase (EROD) activity of adult Australian crimson-spotted rainbowfish (Melanotaenia fluviatilis) were assessed. Breeding groups of three females and two males were exposed to 0, 1, 10, or 100 mg/liter DTPA (nominal) in a 28-day "static-renewal" experiment. Overall, the toxicity of DTPA to adult crimson-spotted rainbowfish was relatively low. Reproduction was not affected at concentrations up to 100 mg/liter DTPA, although an early effect on hatchability was potentially attributed to direct toxicity to rainbowfish eggs. DTPA also had little effect on the condition of adult rainbowfish, with condition factor and GSI being unaffected at concentrations up to 100 mg/liter, the latter finding supporting the reproduction results. However, LSI in male rainbowfish exposed to 100 mg/liter was significantly lower than in those exposed to 1 mg/liter DTPA (P

Lactobacillus rhamnosus GG and Lactobacillus rhamnosus LC-705, previously shown to effectively bind to aflatoxin B1, were subjected to various chemical and physical treatments to examine the effects of these treatments on the binding affinity of these strains toward aflatoxin B1. Treatment of bacterial pellets of both strains with hydrochloric acid significantly (P < 0.05) enhanced the binding ability to bind aflatoxin B1 was also observed when the bacterial pellets were subjected to heat treatment by either autoclaving or boiling at 100 degrees C in a water bath, put the impact of these two treatments was not as effective as the acid treatment. Ethanol, UV radiation, sonication, alkaline, or pH treatments either had not effect or reduced the binding ability of the bacteria.

Biliary metabolites of 2-chlorosyringaldehyde (2-CSA), the major chlorinated phenol found in chlorine dioxide bleached eucalypt pulp effluent, have been found to be sensitive biomarkers of effluent exposure in the sand flathead (Platycephalus bassensis). Before this method of biomonitoring can be applied in the field, the influences of exposure duration, depuration time, and fish feeding status on the level of this metabolite should be determined. In this study, sand flathead were exposed to a measured concentration of 0.3 microgram/1 of 2-CSA for 1, 2, 4, 8, 12, or 16 days. Fish previously exposed to 2-CSA were then held in sea-water alone for 1, 2, 3, 4, or 6 days. Fish were fed ad libitum throughout the experiment, and the fullness of the fish's stomach at the time of sampling was noted. There were no effects of exposure on biotransformation enzyme activities, either between exposure times or between the exposure and depuration periods. The major metabolite of 2-CSA, 2-chloro-4-hydroxy-3,5-dimethoxybenzylalcohol (2-CB-OH), was first detected in the bile of some fish sampled after 24 h of exposure, and the mean concentration of 2-CB-OH in the bile increased over the exposure period. The mean concentration (+/- SE) of 2-CB-OH in the bile was strongly influenced by fish feeding status, being 94 +/- 18 ng/ml bile in fish with empty stomachs and undetectable in fish with full stomachs. Bile volume was also influenced by fish feeding status, being greatest in fish with empty stomachs at the time of sampling. Results indicate that the feeding status of fish should be taken into consideration when using biliary metabolites as biomarkers of effluent exposure in the field, and methods to establish this are discussed.

A partial cytochrome P450 sequence was RT/PCR amplified from total RNA isolated from the whole body of worker class termites (Mastotermes darwiniensis). The degenerate primers used were designed from conserved regions from 4 different species: rat, human, cockroach and drosophila. The sequence was defined by the presence of the typical P450 heme-binding region and invariant residues found in all P450 proteins. The deduced amino acid sequence is 67% identical to cockroach (Blaberus discoidalis) CYP4C1, with only 39% and 42% identity to either CYP4A1 or CYP4B1, respectively, and has been named CYP4C8. Similar low sequence homology was observed between the termite sequence and the mouse CYP3a16 (39%) and black-swallow butterfly CYP6B3 (41%) P450 proteins. The CYP4C8 sequence contains variations in the 13-residue sequence characteristic of family 4 members, distinct from those seen for CYP4D1 and CYP4F family members. M. darwiniensis has been proposed as the "missing link between cockroaches and termites," with the genus Mastotermes dating back some 20-40 million years. The phylogenetic distance between B. discoidalis and M. darwiniensis would suggest that CYP4C8 represents the most ancient of the CYP4 family members.

Peroxisome proliferators are known to modulate the activity of xenobiotic-metabolising enzymes, including glutathione S-transferase (GST) and cytochrome P-450 (CYP). In this study the effect of peroxisome proliferators silvex and di(2-ethylhexyl)phthalate (DEHP) on the formation of (+)-anti-benzo(a)pyrene -7,8-dihydrodiol-9,10-epoxide (BPDE)-DNA adducts from a proximate mutagen and carcinogen (-)-transbenzo(a)pyrene-7,8-dihydrodiol (BPDD) has been investigated. Rat CYP1A1 metabolises BPDD to mutagenic BPDE, which may form DNA adducts or, alternatively, be detoxified by hydrolysis or glutathione conjugation. In this experiment the formation of BPDE-DNA adducts was significantly increased in hepatocytes isolated from all silvex treated rats and two out of four DEHP treated rats (14 day treatment). The activity of CYP1A1 was increased whereas GST was reduced by the peroxisome proliferator silvex. These changes were more significant than those induced by DEHP. We have hypothesised that the formation of BPDE-DNA adducts was primarily due to the increased BPDD activation to BPDE versus reduced detoxication of BPDE. Other hepatic changes induced by the peroxisome proliferators, e.g. peroxisome proliferation per se and increased mitotic activity of the liver could have an effect on the outcome of BPDD exposure.

Total RNA isolated from a rainbow trout (Oncorhynchus mykiss) liver was subjected to RT/PCR using degenerate primers designed from homologous regions amongst cytochrome P450 CYP4 proteins. PCR amplification resulted in a single electrophoretic band which was excised, purified and sequenced directly, using cycle sequencing. The deduced protein sequence demonstrated the closest amino acid identity to rabbit CYP4B1 (54.6%) and rat CYP4B2 (55.4%). Phylogenic analysis of this sequence was found to be significantly different to any other CYP4 sequence and has been named CYP4T1. This represents the first CYP4 family member to be identified in an aquatic vertebrate.

Peroxisome proliferators are ubiquitous rodent hepatocarcinogens, known to modulate the activities of xenobiotic-metabolising enzymes such as glutathione S-transferases (GST) and mixed-function oxidase (cytochrome P-450). In addition these compounds induce pleiotropic changes in the liver of rodents even after a short-term treatment. It has been hypothesised that the enzymatic and cellular changes induced by peroxisome proliferators may alter the toxicity of other compounds activated by cytochrome P-450 and detoxified by GST isoenzymes. The effect of nafenopin-induced changes in the liver of rats on the toxicity of an anti-cancer drug cyclophosphamide was studied using cyto- and geno-toxicity parameters in the liver and bone marrow cells. The administration of cyclophosphamide (10 or 20 mg/kg bw) to the rats pre-treated with 80 mg/kg bw of nafenopin for 2 days resulted in significantly increased cytotoxic response in bone marrow cells. However, genotoxicity of cyclophosphamide was increased only in the liver of nafenopin pre-treated rats. Low level of genotoxicity in bone marrow could be accounted for potentiated cytotoxicity of cyclophosphamide. These events coincided with a significant, up to 5-fold, increase in indirect activation-detoxication index for cyclophosphamide, determined as a ratio of ECOD and GST activities, in nafenopin treated rats. This resulted from the induction of ECOD responsible for the formation of reactive metabolites of cyclophosphamide and reduced activity of GST responsible for their detoxication. In addition, mitotic activity of hepatocytes was increased in nafenopin treated rats that might also have an impact on the genotoxicity of cyclophosphamide in this organ.

A specific and useful method was developed for the determination of dexfenfluramine metabolism by microsomal systems utilising GC-MS. The synthesis of two metabolites 1-(3-trifluoromethylphenyl)propan-2-ol ('alcohol') and 1-(3-trifluoromethylphenyl)-1,2-propanediol ('diol') via straightforward routes, were confirmed by MS and NMR spectra. The conditions for extraction from alkalinised microsomal mixtures of the metabolites nordexfenfluramine, 1-(3-trifluoromethylphenyl)propan-2-one ('ketone'), alcohol and diol, their conversion to trifluoroacetate derivatives and analysis by GC-MS-SIM are described. Calibration curves were constructed between 48 and 9662 nM and fitted to quadratic equations (r2>0.999). The method precision was good over low (121 nM) medium (2415 nM) and above medium (9662 nM) concentrations for all metabolites; the within- and day-to-day coefficients of variation ranged between 2.5-12.4% and 6.7-17.5%, respectively. The accuracy, measured as bias, was very good both within- and day-to-day (range: -0.4-12.6%, 0.8-18.9%). For most metabolites, the C.V. for the assay and bias increased at 121 nM. Dexfenfluramine metabolism by rat liver microsomes was investigated using the assay method and showed a concentration dependent increase in nordexfenfluramine and ketone metabolites over the substrate range of 5-200 microM.

We have investigated the effects of peroxisome proliferators silvex, nafenopin and diethylhexylphthalate (DEHP) on rat liver glutathione S-transferase (GST) isoenzyme activities and patterns. Silvex was a more potent in vitro GST inhibitor than nafenopin and DEHP. After 14 days oral administration to rats a reduction in total GST activity was observed, doses of compounds were chosen so that peroxisome proliferation was equivalent between compounds, nevertheless total GST activity was altered to different extents: nafenopin approximately silvex > DEHP approximately control. GST isoenzyme profiles were also altered, the proportion of GST 2-2 increased and 4-4 decreased compared to control levels. The results indicated that: (i) the peroxisome proliferators studied had similar effects on GST isoenzyme profile: (ii) modulation of the GST activity was apparently independent of peroxisome proliferation per se.

Metabolites of chlorinated phenolic compounds in fish bile have been found to be sensitive biomarkers of bleached pulp mill effluent exposure. Chlorinated syringaldehydes are largely unstudied chlorophenolics found in bleached hardwood effluent. Sand flathead (Platycephalus bassensis), Australian marine fish, were exposed to 100% chlorine dioxide-bleached eucalypt pulp effluent at concentrations of 0.5, 2, and 8% (v/v) for 4 days. Metabolites of 2-chlorosyringaldehyde (2-CSA), the predominant chlorophenolic in this effluent, were measured in the bile. The major metabolite was the conjugate of 2-chloro-4-hydroxy-3,5-dimethoxy-benzylalcohol (2-CB-OH), the reduced product of 2-CSA. 2-CB-OH was found in all fish exposed to diluted effluent and was concentrated in the bile over 1000 times above 2-CSA levels in the effluent. A separate experiment examined the metabolic fate of 2,6-dichlorosyringaldehyde (2,6-DCSA), which is one of the major chlorophenolics in chlorine-bleached eucalypt pulp effluent. Sand flathead were exposed to 2,6-DCSA by intraperitoneal injection at 15 mg/kg or through the water to 0.5, 2, or 8 micrograms/liter for 4 days. Analysis of the bile revealed the major metabolite of 2,6-DCSA to be the conjugate of 2,6-dichloro-4-hydroxy-3,5-dimethoxybenzylalcohol, which was found in all exposed fish and was concentrated in the bile over 20,000 times above 2,6-DCSA exposure levels. Results reveal that the analysis of metabolites of chlorinated syringaldehydes in fish bile can provide a biomarker of bleached hardwood effluent exposure that is sensitive to low levels of exposure, specific to certain bleaching sequences, and correlates well with exposure concentrations.

The effects of algal concentration (Selanastrum capricornatum) on the toxicity of the organochlorine pesticide endosulfan and the synthetic pyrethroid esfenvalerate to Daphnia carinata was investigated. The study progressed through four stages: (1) A dose-response experiment on the effects of endosulfan and esfenvalerate on the survival, growth, and reproduction of D. carinata at a single nonlimiting food level. (2) An experiment to investigate the effects of five different food concentrations on survival, growth, and reproduction of D. carinata at sublethal concentrations of endosulfan and esfenvalerate compared with nonexposed controls. (3) An experiment to investigate the effects of route of exposure (water, food-borne, or water+food-borne exposure) on the toxicity of endosulfan to D. carinata. (4) An experiment to investigate the effects of algal concentration on persistence of endosulfan in the water column and on the relative toxicity of the alpha and beta isomers and of endosulfan sulfate to D. carinata. In the first experiment all daphnids exposed to 500 ng/liter esfenvalerate died within 3 days. There was a significant effect of esfenvalerate on reproduction at 50 ng/liter by the second brood. Endosulfan did not cause significant mortality to daphnids but brood size was reduced at 320 micrograms/liter. In the second experiment the toxicity of esfenvalerate increased significantly with decreasing food concentration. In contrast, the toxicity of endosulfan to D. carinata was greatest at the higher food concentrations. Direct water-borne exposure to endosulfan was the most toxic route of exposure and the presence of algae decreased toxicity of this pesticide. The total amount of endosulfan (alpha + beta + endosulfan sulfate) persisting in the water column after 24 hr was greater at high food levels, suggesting that this may be one mechanism for increased toxicity at high food concentrations. The 48-hr LC50s of technical endosulfan, endosulfan sulfate, alpha-endosulfan, beta-endosulfan, and a 50:50 mixture of alpha, and beta endosulfan were 478, 756, 249, 205, and 234 micrograms/liter, respectively.

The inhibition of ethoxy coumarin O-deethylase (ECOD) activity by aucubin and its aglycone was examined in a microsomal system and in freshly isolated hepatocytes. Aucubin was found to be inactive but the aglycone was found to be a potent time-dependent inhibitor of ECOD activity in both systems. The close structural similarity between the aglycone of aucubin and glutaraldehyde suggests a similar mechanism of enzyme inhibition through protein cross-linking by Schiff reactions. The similarity between the 2 compounds was demonstrated through their closely similar binding spectra to bovine serum albumin. The biological activities reported for the aglycone are suggested to be due to this similarity to glutaraldehyde.

First-instar Daphnia carinata were exposed to one of four or five sublethal concentrations of the industrial chelating agent diethylenetriamine pentaacetic acid (DTPA) either alone, or in conjunction with, high (90-100%) or low (10-25%) oxygen saturation and high (2 x 10(5) cells/ml) or low (2 x 10(4) cells/ml) food conditions for 6 to 7 days, in a series of three experiments. Survival, growth, reproduction, and hemoglobin (Hb) content were assessed. Mortality increased significantly from 6.5 +/- 4.2 to 38.9 +/- 5.2%, and mean length was significantly reduced from 2.73 +/- 0.02 to 1.37 +/- 0.01 mm at 100 mg/liter DTPA in experiment 1. Mean length was also significantly reduced from 2.64 +/- 0.12 to 1.9 +/- 0.1 mm at 50 mg/liter DTPA in experiment 3. This was attributed to an indirect effect via the food supply in the third experiment. There was a significant decrease in the mean number of first-brood eggs at 10 mg/liter DTPA in all three experiments. Hemoglobin concentration was significantly increased under low oxygen conditions from 27.6 +/- 1.7 to 65.5 +/- 4.6 mg Hb/g Daphnia dry wt, and 23.0 +/- 1.8 to 49.4 +/- 3.5 mg Hb/g Daphnia dry wt in experiments 2 and 3, respectively. However, DTPA had no effect on hemoglobin concentration in any experiment. DTPA toxicity to D. carinata was not significantly altered by oxygen stress or food limitation and could not be attributed to an inhibition of Hb synthesis. Increased exposure times may result in further reproductive effects and also an indirect effect on hemoglobin concentration via the gradual depletion of iron stores. The no-observed effect concentration and the lowest observed effect concentration for D. carinata in this study were 1.0 and 10 mg/liter DTPA, respectively, based on reproduction, giving an estimated threshold concentration of 3.2 mg/liter DTPA.

trans-7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE] is rapidly inactivated in aqueous solvents due to hydrolysis to tetraols. No significant effect on the rate of hydrolysis is observed in the presence of glutathione (GSH)-depleted cytosol. However, when the cytosolic fraction is replaced by a mixture of glutathione (GST)-isoenzymes (corresponding to about 10% of the cytosolic protein) a significant inhibition of the rate of hydrolysis is observed, indicating a physical interaction between the diol epoxide and GST. This is compatible with the proposed role of certain GST-isoenzymes as intracellular carriers for lipophilic compounds. Studies on the accessibility of (+)-anti-BPDE to hydrolysis and GST-catalysed conjugation with GSH reveal that the presence of rat liver microsomes or liposomes, in particular those composed of the neutral phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), very effectively protect the diol epoxide from hydrolysis. Addition of cytosolic fraction depleted of GST to a mixture of GST-isoenzymes and microsomes or liposomes do not significantly increase the rate of GSH-conjugation. This implies the absence of high molecular factors in the cytosol that may increase the accessibility of (+)-anti-BPDE and thus promote conjugation. In contrast to liposomes of PC and PE, those composed of the negatively charged phospholipids phosphatidylserine (PS) and phosphatidylinositol (PI) are considerably less efficient in protecting (+)-anti-BPDE. In fact, these lipids seem to promote hydrolysis, an effect which is lost when PS and/or PI are present together with PC and PE. Taken together, the results presented here suggest that (+)-anti-BPDE and most probably other diol epoxides, are not accessible for GSH-conjugation by direct interaction between GST and the membrane-bound compound. Moreover, there is little support for the existence of cytosolic components that increase the accessibility of (+)-anti-BPDE for conjugation. In agreement with previous results using other compounds, the results indicate that only the fraction of diol epoxide that is free in solution is accessible for conjugation with GSH.

Ammonium 4-chloro-7-sulfobenzofurazan (Sbf-Cl) is a water-soluble fluorescent reagent which is highly specific for thiol groups. We describe here a simple and sensitive fluorescence assay which is highly specific for subunit 3 of the rat glutathione S-transferases. Specific activities of isoenzymes 3-3 and 3-4 were an order of magnitude greater than those of isoenzymes 1-1, 1-2, 2-2, and 4-4, with glutathione and Sbf-Cl concentrations of 1 and 2 mM, respectively. The catalytic specificity constant, kcat/Km, was in the range of 10(4)-10(6) M-1 s-1, indicating that Sbf-Cl is a very good substrate for rat hepatic glutathione S-transferases. The specificity constants measured for the heterodimers 1-2 and 3-4 were greater than the values predicted when dimeric independence is assumed. This suggests that binding of Sbf-Cl to the glutathione S-transferases may result in steric alterations and subsequent elevation in enzymatic activity. Sbf-Cl was shown to be at least 10-fold more sensitive for the detection of rat GST isoenzyme 3-3 than DCNB. Consequently, Sbf-Cl will have an important future role in the investigation of the active site topology of glutathione S-transferases, in addition to its obvious potential as a substrate for the detection and quantitation of rat glutathione S-transferase subunit 3.

The reaction of ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) with thiols was investigated with particular attention to the formation of an ebselen selenol intermediate. The selenol intermediate could be trapped in a mixture of ebselen and thiols with 1-chloro-2,4-dinitrobenzene and the resulting product displayed unique spectral characteristics. The reaction of authentic, synthesised ebselen selenol with 1-chloro-2,4-dinitrobenzene (CDNB) was shown to give rise to the same compound (2,4-dinitrophenyl (N-phenyl-2-carboxamido phenyl) selenide as characterized by light spectroscopy, NMR, IR and elemental analysis. The determination of the absorbtion coefficient at 400 nm (E = 7.5 mM-1 cm-1) and the initial rate constant of the reaction (1.4 +/- 0.3 mM-1 min-1) allows for the convenient quantification of ebselen selenol concentrations by initial rate measurements after addition of CDNB. The choice of 400 nm to monitor the reaction excludes the interference of other intermediates in the reaction of ebselen with thiols as well as the reaction of the thiols with CDNB. When the assay is applied to typical incubation conditions used for investigating the glutathione peroxidase-like activity of ebselen it was shown that as much as 10-20% of ebselen is in the selenol form. If a stronger reductant (dithiothreitol) is used 60% is in the selenol form. These data could also be confirmed by the direct determination of ebselen selenol by UV spectroscopy, due to its peak absorption at 370 nm (E = 2 mM-1 cm-1). In conclusion, this investigation demonstrates, for the first time, the identity and quantity of ebselen selenol in the reaction of ebselen with thiols and also describes a convenient assay for its quantification. These observations allow further possibilities for investigation of the molecular species responsible for the antioxidant and peroxidase activities of ebselen.

Tienilic acid, a phenoxyacetic acid diuretic, reduces the amount of total sulphobromophthalein (BSP) excretion in the isolated perfused rat liver (IPRL). This reduction was primarily by reduction in excretion of conjugated BSP, with excretion of unchanged BSP being relatively unaffected. TA also reduces the amount of conjugated BSP formed in vitro, indicating that its effect in the IPRL may be by means of inhibiting the glutathione S-transferase enzymes involved in the formation of the conjugate. It would appear that a reduction in the biliary excretion of BSP cannot be taken to be an indication of reduced liver function in a general sense.

A modification of equilibrium dialysis in which alpha 1-acid glycoprotein and plasma compete directly for disopyramide has been used in conjunction with binding curves to measure the extent of the alpha 1-acid glycoprotein-disopyramide interaction. At concentrations in the therapeutic range, 80-90% of disopyramide was bound to alpha 1-acid glycoprotein for plasma from each of six healthy adults. Also, equilibrium dialysis data are presented, indicating that pH does not influence the binding of disopyramide within the therapeutic range.

Soluble rat liver glutathione S-transferases have been purified and a previously undescribed peak was observed. This peak contained glutathione S-transferase activity which was extensively inhibited by indomethacin. Glutathione conjugation of 1-chloro-2,4-dinitrobenzene by this isozyme, designated glutathione S-transferase VII, was inhibited 44 and 68% at indomethacin concentrations of 0.20 and 1.00 microM, respectively. The other six basic glutathione S-transferase isozymes were relatively unaffected by low concentrations of indomethacin. The pharmacological significance of this inhibition by indomethacin is largely dependent on the role of the glutathione S-transferase VII in leukotriene synthesis.

A new reversed-phase high-performance liquid chromatographic method allowing simultaneous measurement of plasma concentrations of disopyramide and quinidine is described. Disopyramide and quinidine were separated on a reversed-phase column using 0.05 M phosphate buffer (pH 3.0)--acetonitrile (73:27, v/v), as mobile phase and the peaks were monitored by UV absorbance at the wavelengths of 254 and 325 nm. The drugs were extracted from alkaline plasma with chloroform containing the internal standard. The organic phase was evaporated to dryness and the residue was redissolved in a small volume of the mobile phase before analysis by high-performance liquid chromatography. The method is convenient and reliable in routine monitoring of both drugs.