Cytochrome c oxidase inhibition in the rice weevil Sitophilus oryzae (L.) by formate, the toxic metabolite of volatile alkyl formates.

Volatile alkyl formates are potential replacements for the ozone-depleting fumigant, methyl bromide, as postharvest insecticides and here we have investigated their mode of insecticidal action. Firstly, a range of alkyl esters, ethanol and formic acid were tested in mortality bioassays with adults of the rice weevil, Sitophilus oryzae (L.) and the grain borer, Rhyzopertha dominica (F.) to determine whether the intact ester or one of its components was the toxic moiety. Volatile alkyl formates and formic acid caused similar levels of mortality (LC(50) 131-165 micromol l(-1)) to S. oryzae and were more potent than non-formate containing alkyl esters and ethanol (LC(50)>275 micromol l(-1)). The order of potency was the same in R. dominica. Ethyl formate was rapidly metabolised in vitro to formic acid when incubated with insect homogenates, presumably through the action of esterases. S. oryzae and R. dominica fumigated with a lethal dose of ethyl formate had eight and 17-fold higher concentrations of formic acid, respectively, in their bodies than untreated controls. When tested against isolated mitochondria from S. oryzae, alkyl esters, alcohols, acetate and propionate salts were not inhibitory towards cytochrome c oxidase (EC 1.9.3.1), but sodium cyanide and sodium formate were inhibitory with IC(50) values of 0.0015 mM and 59 mM, respectively. Volatile formate esters were more toxic than other alkyl esters, and this was found to be due, at least in part, to their hydrolysis to formic acid and its inhibition of cytochrome c oxidase.

Role of cytochrome P450 2D6 (CYP2D6) in the stereospecific metabolism of E- and Z-doxepin.

The tricyclic antidepressant, doxepin, is formulated as an irrational mixture of E (trans) and Z (cis) stereoisomers (85%: 15%). We examined the stereoselective metabolism of doxepin in vitro, with the use of human liver microsomes, recombinant CYP2D6 and gas chromatography-mass spectrometry. In human liver microsomes over the concentration range 5-1500 microM, the rate of Z-doxepin N-demethylation exceeded that of E-doxepin above 100 microM in two of three livers. Eadie-Hofstee plots were curvilinear indicating the involvement of several enzymes in N-demethylation. Coincubation of doxepin with 7,8-naphthoflavone and ketoconazole reduced the rates of N-demethylation of E- and Z-doxepin by 30-50% and 40-60%, respectively, suggesting the involvement of CYP1A and CYP3A4, whilst quinidine had little effect on N-demethylation. In contrast, doxepin hydroxylation was exclusively stereo-specific; E-doxepin and E-N-desmethyldoxepin were hydroxylated with high affinity in liver microsomes and by recombinant CYP2D6 (Km in the range of 5-8 microM), but there was no evidence of Z-doxepin hydroxylation. In 'metabolic consumption' experiments with liver microsomes (having measurable CYP2D6 activity) and initial substrate concentration of 1 microM, the consumption of E-doxepin was greater (P < 0.05, n = 5) than that of Z-doxepin. Quinidine inhibited the consumption of E-doxepin but did not affect the consumption of Z-doxepin. With N-desmethyldoxepin, quinidine inhibited the consumption of E-N-desmethyl-doxepin whereas Z-N-desmethyldoxepin appeared to be a terminal oxidative metabolite. In summary, CYP2D6 is a major oxidative enzyme in doxepin metabolism; predominantly catalysing hydroxylation with an exclusive preference for the E-isomers. The relatively more rapid metabolism of E-isomeric forms, and the limited metabolic pathways for the Z-isomers may explain the apparent enrichment of Z-N-desmethyldoxepin that is observed in vivo.

Stereoselective measurement of E- and Z-doxepin and its N-desmethyl and hydroxylated metabolites by gas chromatography-mass spectrometry.

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.

Metabolism of dexfenfluramine in human liver microsomes and by recombinant enzymes: role of CYP2D6 and 1A2.

Dexfenfluramine has been widely used as an appetite suppressant in the treatment of obesity. It was recently shown that the apparent non-renal clearance of dexfenfluramine was significantly lower in poor metabolizers than in extensive metabolisers of debrisoquine which suggested the involvement of the polymorphically expressed enzyme, CYP2D6, in dexfenfluramine metabolism. In this study, human liver microsomes and yeast-expressed recombinant enzymes were used to examine dexfenfluramine metabolism in vitro. In human liver microsomes, the major product of dexfenfluramine was nordexfenfluramine with lesser amounts of a novel metabolite, N-hydroxynordexfenfluramine, and ketone and alcohol derivatives being formed. Eadie-Hofstee plots (v against v/[s]) of nordexfenfluramine formation between 1 and 1000 microM substrate concentration were biphasic in three of four liver microsome samples examined, with mean Km values of 3 and 569 microM for the high and low affinity enzymes, respectively. At a substrate concentration (0.5 microM) around the known therapeutic plasma concentration, there was negligible inhibition of microsomal dexfenfluramine N-dealkylation by sulphaphenazole and ketoconazole, but between 33 and 100% inhibition by quinidine, and 0-58% inhibition by 7,8-naphthoflavone in seven liver samples. In human liver microsomes, there was also a significant correlation (rs= 0.79, n = 10, P < 0.01) between dextromethorphan O-demethylation and dexfenfluramine (at 1 microM) N-dealkylation activities. Dexfenfluramine was a specific inhibitor (IC50 46 microM) of CYP2D6-mediated dextromethorphan O-demethylation in human liver microsomes but did not appreciably inhibit six other cytochrome P450 isoform-selective activities for CYP1A2, 2A6, 2C9, 2C19, 2E1 and 3A activities in human liver microsomes. Yeast-expressed recombinant human CYP2D6 metabolized dexfenfluramine with high affinity (Km 1.6 microM, Vmax 0.18 nmol min(-1) nmol P450(-1)) to nordexfenfluramine which was the sole product observed. Recombinant CYP1A2 was a lower affinity enzyme (Km 301 microM, Vmax 1.12 nmol min(-1) nmol P450(-1)) and produced nordexfenfluramine with small amounts of N-hydroxynordexfenfluramine. This is the first detailed study to examine the in-vitro metabolism of dexfenfluramine in human liver microsomes and by recombinant human P450s. We were able to identify CYP2D6 (high affinity) and CYP1A2 (low affinity) as the major enzymes catalysing the N-dealkylation of dexfenfluramine in human liver microsomes.

The effects of exposure duration and feeding status on fish bile metabolites: implications for biomonitoring.

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.

Measurement of dexfenfluramine metabolism in rat liver microsomes by gas chromatography-mass spectrometry.

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.

Isolation and identification of a cytochrome P450 sequence in an Australian termite, Mastotermes darwiniensis.

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.

Glutathione transferases and glutathione-binding proteins of termites: purification and characterisation.

Termites have an important role in the cycling of carbon and trace elements in the biosphere through their degradation of wood, grasses and humus. Glutathione transferases (GSTs) are detoxication enzymes found in all organisms; GSTs are known to protect insects from the toxic effects of plant chemicals and pesticides. The activities and characteristics of termite GSTs were investigated in three Australian termite families. Multiple GST isozymes were purified from whole body preparations of the three termite species by affinity chromatography and subsequent chromatofocusing. Termite GSTs exhibited a broad range of activities toward model substrates but were most active with 1-chloro-2,4-dinitrobenzene. The pI values of termite GSTs ranged between 7.4 and 5.8, while the apparent molecular weights of subunits ranged between 25.9 and 27.7 kDa. The N-terminal sequence of a glutathione-binding protein that was purified from Mastotermes darwiniensis was similar to the N-terminal sequence of a streptococcal protein of unknown function previously implicated in glomerulonephritis in humans.

Metabolites of chlorinated syringaldehydes in fish bile as biomarkers of exposure to bleached eucalypt pulp effluents.

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.

Metabolites of 2-chlorosyringaldehyde in fish bile: indicator of exposure to bleached hardwood effluent.

1. 2-Chlorosyringaldehyde (2-CSA) is the major chlorinated phenol produced by the 100% chlorine dioxide bleaching of eucalypt pulp and is found in other bleached hardwood effluents. Almost nothing is known of the environmental or metabolic fates of this chemical. 2. Sand flathead (Platycephalus bassensis) was given 2-CSA by intraperitoneal injection at 0.15, 1.5, 15 and 75 mg/kg doses and, 4 days later, bile was collected and solvent extracted before and after enzymatic cleavage of conjugates. The acetate derivatives of bile extracts were analysed by gas chromatography/mass spectrometry. 3. The major metabolite 4 days after administration was the glucuronide or sulphate conjugate of 2-chloro-4-hydroxy-3,5-dimethoxy-benzylalcohol (2-CB-alcohol). The identity of 2-CB-alcohol was confirmed by chemical synthesis. 4. The quantity of 2-CB-alcohol in the bile was linearly related to dose of 2-CSA and was detected at all dose levels. Minor metabolites identified were conjugated 2-CSA, unchanged 2-CSA and 2-chloro-4-hydroxy-3,5-dimethoxy-benzoic acid. 5. The amount of 2-CB-alcohol in bile has the potential to be a sensitive and specific indicator of fish exposure or bleached hardwood effluent.

Ammonium 4-chloro-7-sulfobenzofurazan: a fluorescent substrate highly specific for rat glutathione S-transferase subunit 3.

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.