Preparation of Cells Permeable to Macromolecules by Treatment with Toluene: Studies of Transfer Ribonucleic Acid Nucleotidyltransferase

Transfer ribonucleic acid (tRNA) nucleotidyltransferase was studied after making cells permeable to macromolecules by treatment with toluene. The conditions of toluene treatment necessary for obtaining maximal activity were defined. Toluene treatment was most efficient when carried out for 5 min at 37 C at pH 9.0 on log-phase cells. No activity could be detected if cells were treated at 0 C, or in the presence of MgCl₂, or if the cells were in the stationary phase of growth. However, inclusion of lysozyme and ethylenediaminetetraacetic acid during the toluene treatment did render stationary phase cells permeable. The properties of tRNA nucleotidyltransferase from toluene-treated cells were essentially identical to those of purified enzyme with regard to pH optimum, specificity for nucleoside triphosphates and tRNA, and apparent K_m values for substrates. In addition to tRNA nucleotidyltransferase, a variety of other enzymes which incorporate adenosine 5′-triphosphate into acid-precipitable material could also be detected in toluene-treated cells. Centrifugation of cells treated with toluene revealed that tRNA nucleotidyltransferase leaked out of cells, whereas other activities remained associated with the cell pellets. Chromatography of the material extracted from toluene-treated cells on Sephadex G-100 indicated that toluene treatment selectively extracts lower molecular weight proteins. The usefulness of such a procedure as an initial step in purification of such enzymes, and its application to tRNA nucleotidyltransferase, is discussed.     

Common induction and regulation of biphenyl, xylene/toluene, and salicylate catabolism in Pseudomonas paucimobilis.

A strain of Pseudomonas paucimobilis (strain Q1) capable of utilizing biphenyl was isolated from soil. This strain grew not only on substituted biphenyls, but also on salicylate, xylene or toluene or both (xylene/toluene), and substituted benzoates. Evidence is presented that the catabolism of biphenyl, xylene/toluene, and salicylate is regulated by a common unit in this strain. The catabolism of biphenyl, xylene/toluene, and salicylate is interrelated, since benzoate and toluate are common metabolic intermediates of biphenyl and xylene/toluene, and salicylate is produced from 2-hydroxybiphenyl (o-phenylphenol). All the oxidative enzymes of the biphenyl, xylene/toluene, and salicylate degradative pathways were induced when the cells were grown on either biphenyl, xylene/toluene or salicylate. The P. paucimobilis Q1 cells showed induction of the meta-cleavage enzymes of both 2,3-dihydroxybiphenyl and catechol. Biphenyl-negative derivatives of strain Q1 were simultaneously rendered xylene/toluene and salicylate negative, whereas reversion to the biphenyl-positive character of such derivatives invariably led to a xylene/toluene- and salicylate-positive phenotype. Growth of the P. paucimobilis Q1 cells with benzoate as a sole carbon source allowed the induction of only the ortho pathway enzymes, suggesting that biphenyl, xylene/toluene, or salicylate specifically induced the meta pathway enzymes for the oxidative degradation of these compounds.     

Effects of Aroclor 1254 treatment on the in vitro hepatic metabolism of toluene, aniline and aminopyrine in hybrid sunfish

The in vitro metabolism of the volatile aromatic hydrocarbon toluene by enzymes associated with the 12,000 g supernatant fraction of hybrid sunfish (Lepomis macrochirus X L. cyanellus X L. gibbosus) liver homogenates was studied. Aminopyrine demethylase (APDM) and aniline hydroxylase (AH) activities were measured. Intramuscular injections of Aroclor 1254 (a polychlorinated biphenyl) produced significant increases in APDM and AH activities (P less than or equal to 0.1, ANOVA). There were no significant changes in the metabolism of toluene, liver wet weights, or liver protein concentrations following treatment.     

Measurement of matrix enzyme activity in isolated mitochondria made permeable with toluene.

Mitochondria isolated from rat liver and heart were made permeable to normally nonpentrating substrates and cofactors by treatment with toluene. The optimal conditions for preparing stable, permeable mitochondria were 2% toluene for 2 min at 4 °C in a buffered, isotonic medium containing 8.5% polyethylene glycol (Mr 6000–7500). Without polyethylene glycol, the toluene-treated mitochondria were unstable and released their matrix enzymes. The treated mitochondria were particularly unstable in dilute suspension under normal assay conditions of their enzyme activities. The levels of matrix enzyme activities unmasked by toluene treatment of mitochondria were very close to those of sonicated mitochondria under identical assay conditions. Mitochondria made permeable with toluene lost only small amounts of their protein and retained a major fraction of the nucleotides and coenzymes. Electron microscopic examination of toluenetreated mitochondria indicated that they were relatively intact with swollen and vesiculated cristae membranes. Such preparations will allow the study of mitochondrial enzymes at approximate in vivo concentrations.     

Macromolecular synthesis in permeable liver cells

Macromolecular synthesis was studied in individual liver cells rendered permeable to macromolecules and charged molecules by treatment with toluene. Toluene-treated cells were compared to intact cells with regard to their ability to synthesize protein, RNA, and DNA. The permeable cells catalyzed the incorporation of amino acids into protein in a system which was sensitive to cycloheximide. Maximal incorporation required the addition of tRNA, ATP, GTP, an energy source and various cations. RNA synthesis also took place in these cells and was inhibited by actinomycin D. Maximal incorporation required all four ribonucleoside triphosphates, an energy-generating system, and Mn²⁺, K⁺, and F^?. The toluene-treated cells also were active for DNA synthesis when Ca²⁺ was present to induce endonucleolytic cleavage of the endogenous DNA. For maximal synthesis, all four deoxyribonucleoside triphosphates, ATP, K⁺, Mg²⁺, polyamines, and mercaptoethanol were required. These studies serve to emphasize the potential usefulness of toluene treatment for studying biosynthetic processes in mammalian cells.     

Involvement of thylakoid membranes in supramolecular organisation of Calvin cycle enzymes in Anacystis nidulans

The cells of unicellular photosynthetic cyanobacterium Anacystis nidulans were permeated with lysozyme, toluene, toluene-triton, toluene-triton-lysozyme. Transmission electron microscopy of semi-thin sections (500 nm) using TEM at 160 kV showed that cells permeated with only lysozyme or toluene showed the typical concentric arrangement of thylakoid membranes. However, when toluene-treated cells were further treated with triton and lysozyme the thylakoid membranes were disrupted. Sequential reactions of Calvin cycle were studied in the differentially permeated cells in vivo, using various intermediates such as 3-PGA, GA-3-P, FDP, SDP, R-5-P, RuBP and cofactors like ATP, NADPH depending on the requirement. RuBP and R-5-P + ATP dependent activities could be observed in all types of permeated cells. Sequential reactions of the entire Calvin cycle using 3-PGA could be detected in the cells that had retained the internal organisation of the thylakoid membranes after permeation and were lost on disruption of this organisation. Light dependent CO2 fixation could be detected only in the cells permeated with lysozyme. This activity was abolished in the cells after treatment with toluene. The results suggested that the integrity of thylakoid membranes may be essential for the organisation of sequential enzymes of the Calvin cycle in vivo and facilitate their functioning.     

Cross-linking of mitochondrial matrix proteins in situ

Different cross-linkers (10 mM) of varying specificity and arm length were found to cross-link mitochondrial matrix proteins in situ in 2 min at pH 7.4. As seen by SDS-polyacrylamide electrophoresis, the disappearance of individual protein bands was accompanied by concomitant appearance of polymeric aggregates that failed to enter the 4% spacer gel. The disorganization of the mitochondrial matrix infrastructure either by swelling or sonication of the mitochondria resulted in a decrease in the rate of cross-linking. Leakage of citrate synthase, malate dehydrogenase and fumarase was found to be reduced when cross-linked mitochondria were made permeable with toluene. On lysing the cross-linked mitochondria, a major part of the matrix protein (75%) was found to sediment with the membrane fraction. The activities of citrate synthase, malate dehydrogenase and fumarase in rat liver mitochondria were also found to increase in the precipitates with a concomitant decrease in their activities in the soluble matrix fraction. These results indicate that the cross-linker enters the mitochondria and cross-links matrix proteins including Krebs cycle enzymes either to the mitochondrial membranes, or to themselves resulting in very large molecular weight complexes. These results are interpreted to mean that in liver mitochondria, the Krebs cycle enzymes are preferentially located near the membrane.     

Biotransformation of D-limonene to (+) trans-carveol by toluene-grown Rhodococcus opacus PWD4 cells

The toluene-degrading strain Rhodococcus opacus PWD4 was found to hydroxylate D-limonene exclusively in the 6-position, yielding enantiomerically pure (+) trans-carveol and traces of (+) carvone. This biotransformation was studied using cells cultivated in chemostat culture with toluene as a carbon and energy source. The maximal specific activity of (+) trans-carveol formation was 14.7 U (g of cells [dry weight])(-1), and the final yield was 94 to 97%. Toluene was found to be a strong competitive inhibitor of the D-limonene conversion. Glucose-grown cells did not form any trans-carveol from D-limonene. These results suggest that one of the enzymes involved in toluene degradation is responsible for this allylic monohydroxylation. Another toluene degrader (Rhodococcus globerulus PWD8) had a lower specific activity but was found to oxidize most of the formed trans-carveol to (+) carvone, allowing for the biocatalytic production of this flavor compound.     

Stimulation of adenylate cyclase from isolated hepatocytes and Kupffer cells.

Hepatocytes and Kupffer cells were separated from rat liver after prelabeling the Kupffer cells with colloidal iron and perfusion of the liver with digestive enzymes. The activity of several enzymes from Kupffer cells and hepatocytes was compared to validate this method of cell separation. The ratios of hepatocyte to Kupffer cell specific activities of glucose-6-phosphatase, 5'-nucleotidase, adenylate cyclase, and acid phosphatase were 20, 0.39, 0.18, and 0.078, respectively. Adenylate cyclases from hepatocytes and Kupffer cells were stimulated by fluoride ion, GTP, and catecholamines. Hepatocyte adenylate cyclase was also stimulated by glucagon, secretin, vasoactive intestinal polypeptide, and by prostaglandin E1, whereas, the Kupffer cell enzyme was completely insensitive to these hormones. The stimulation of hepatocyte adenylate cyclase by combinations of glucagon plus secretin, or glucagon plus vasoactive intestinal polypeptide, were equivalent to the sum of the individual stimulations. This suggests that the hepatocyte has specific receptors for glucagon and for vasoactive intestinal polypeptide and secretin. Prostaglandin E1 stimulation of hepatocyte adenylate cyclase was not additive to the stimulation caused by polypeptide hormones or catecholamines, nor did prostaglandin E1 decrease stimulation caused by these hormones. Although prostaglandin-sensitive adenylate cyclase was recovered with hepatocytes, 40 to 50% of the total liver prostaglandin-sensitive activity was recovered in a fraction of cell debris mixed with small cells which did not phagocytize colloidal iron.     

Metabolism of Chlorotoluenes by Burkholderia sp. Strain PS12 and Toluene Dioxygenase of Pseudomonas putida F1: Evidence for Monooxygenation by Toluene and Chlorobenzene Dioxygenases

The degradation of toluene by Pseudomonas putida F1 and of chlorobenzenes by Burkholderia sp. strain PS12 is initiated by incorporation of dioxygen into the aromatic nucleus to form cis-dihydrodihydroxybenzenes. Toluene-grown cells of P. putida F1 and 3-chlorobenzoate-grown cells of Burkholderia sp. strain PS12 were found to monooxygenate the side chain of 2- and 3-chlorotoluene to the corresponding chlorobenzyl alcohols. Further metabolism of these products was slow, and the corresponding chlorobenzoates were usually observed as end products, whereas the 3-chlorobenzoate produced from 3-chlorotoluene in Burkholderia sp. strain PS12 was metabolized further. Escherichia coli cells containing the toluene dioxygenase genes from P. putida F1 oxidized 2- and 3-chlorotoluene to the corresponding chlorobenzyl alcohols as major products, demonstrating that this enzyme is responsible for the observed side chain monooxygenation. Two methyl- and chloro-substituted 1,2-dihydroxycyclohexadienes were formed as minor products from 2- and 3-chlorotoluene, whereas a chloro- and methyl-substituted cyclohexadiene was the only product formed from 4-chlorotoluene. The toluene dioxygenase of P. putida F1 and chlorobenzene dioxygenase from Burkholderia sp. strain PS12 are the first enzymes described that efficiently catalyze the oxidation of 2-chlorotoluene.     

Trichloroethylene cometabolic degradation by Rhodococcus sp. L4 induced with plant essential oils

Cometabolic degradation of TCE by toluene-degrading bacteria has the potential for being a cost-effective bioremediation technology. However, the application of toluene may pose environmental problems. In this study, several plant essential oils and their components were examined as alternative inducer for TCE cometabolic degradation in a toluene-degrading bacterium, Rhodococcus sp. L4. Using the initial TCE concentration of 80 muM, lemon and lemongrass oil-grown cells were capable of 20 +/- 6% and 27 +/- 8% TCE degradation, which were lower than that of toluene-grown cells (57 +/- 5%). The ability of TCE degradation increased to 36 +/- 6% when the bacterium was induced with cumin oil. The induction of TCE-degrading enzymes was suggested to be due to the presence of citral, cumin aldehyde, cumene, and limonene in these essential oils. In particular, the efficiency of cumin aldehyde and cumene as inducers for TCE cometabolic degradation was similar to toluene. TCE transformation capacities (T (c)) for these induced cells were between 9.4 and 15.1 mug of TCE mg cells(-1), which were similar to the known toluene, phenol, propane or ammonia degraders. Since these plant essential oils are abundant and considered non-toxic to humans, they may be applied to stimulate TCE degradation in the environment.     

Regiospecificity of two multicomponent monooxygenases from Pseudomonas stutzeri OX1: molecular basis for catabolic adaptation of this microorganism to methylated aromatic compounds

The pathways for degradation of aromatic hydrocarbons are constantly modified by a variety of genetic mechanisms. Genetic studies carried out with Pseudomonas stutzeri OX1 suggested that the tou operon coding for toluene o-xylene monooxygenase (ToMO) was recently recruited into a preexisting pathway that already possessed the ph operon coding for phenol hydroxylase (PH). This apparently resulted in a redundancy of enzymatic activities, because both enzymes are able to hydroxylate (methyl)benzenes to (methyl)catechols via the intermediate production of (methyl)phenols. We investigated the kinetics and regioselectivity of toluene and o-xylene oxidation using Escherichia coli cells expressing ToMO and PH complexes. Our data indicate that in the recombinant system the enzymes act sequentially and that their catalytic efficiency and regioselectivity optimize the degradation of toluene and o-xylene, both of which are growth substrates. The main product of toluene oxidation by ToMO is p-cresol, the best substrate for PH, which catalyzes its transformation to 4-methylcatechol. The sequential action of the two enzymes on o-xylene leads, via the intermediate 3,4-dimethylphenol, to the exclusive production of 3,4-dimethylcatechol, the only dimethylcatechol isomer that can serve as a carbon and energy source after further metabolic processing. Moreover, our data strongly support a metabolic explanation for the acquisition of the ToMO operon by P. stutzeri OX1. It is possible that using the two enzymes in a concerted fashion confers on the strain a selective advantage based on the ability of the microorganism to optimize the efficiency of the use of nonhydroxylated aromatic hydrocarbons, such as benzene, toluene, and o-xylene.     

Comparative transcriptome analysis of Methylibium petroleiphilum PM1 exposed to the fuel oxygenates methyl tert-butyl ether and ethanol

High-density whole-genome cDNA microarrays were used to investigate substrate-dependent gene expression of Methylibium petroleiphilum PM1, one of the best-characterized aerobic methyl tert-butyl ether (MTBE)-degrading bacteria. Differential gene expression profiling was conducted with PM1 grown on MTBE and ethanol as sole carbon sources. Based on microarray high scores and protein similarity analysis, an MTBE regulon located on the megaplasmid was identified for further investigation. Putative functions for enzymes encoded in this regulon are described with relevance to the predicted MTBE degradation pathway. A new unique dioxygenase enzyme system that carries out the hydroxylation of tert-butyl alcohol to 2-methyl-2-hydroxy-1-propanol in M. petroleiphilum PM1 was discovered. Hypotheses regarding the acquisition and evolution of MTBE genes as well as the involvement of IS elements in these complex processes were formulated. The pathways for toluene, phenol, and alkane oxidation via toluene monooxygenase, phenol hydroxylase, and propane monooxygenase, respectively, were upregulated in MTBE-grown cells compared to ethanol-grown cells. Four out of nine putative cyclohexanone monooxygenases were also upregulated in MTBE-grown cells. The expression data allowed prediction of several hitherto-unknown enzymes of the upper MTBE degradation pathway in M. petroleiphilum PM1 and aided our understanding of the regulation of metabolic processes that may occur in response to pollutant mixtures and perturbations in the environment.     

Effect of glutaraldehyde treatment on enzyme-loaded erythrocytes.

In principle, enzyme-loaded erythrocytes can be used as a vehicle for enzyme replacement therapy in lysosomal storage diseases. Glutaraldehyde treatment renders these erythrocytes more resistant to lysis without inactivating the enzymes that have been entrapped inside them. Glutaraldehyde treatment does not prevent ingestion of enzyme-loaded erythrocytes by macrophages in vitro so that these cells can be used to deliver enzymes to lysosomes. In vivo, the glutaraldehyde-treated cells are quickly removed from the circulation by the spleen or liver. The degree of glutaraldehyde treatment allows the erythrocytes to be targeted either to the spleen (low glutaraldehyde concentrations) or to the liver (higher glutaraldehyde concentrations).     

Quantification of biochemical compounds in Bauhinia Variegata Linn flower extract and its hepatoprotective effect

Liver disorders may occur as a result of exposure to chemical compounds capable of inducing the oxidative stress and hepatic injuries. The aim of present study was to investigate the effects of flower extracts of B. Variegata for the treatment of liver injury induced by the CCl₄. About 1 ml/kg body weight (b.w) of CCl4 was induced to experimental mice by intraperitoneal way for 14 days. The methanol and chloroform extracts (100, 200 and 300 mg/kg b.w) were administered to experimental animals for 14 days along with standard drug Silymarine (100 mg/kg b.w). The extracts alone showed no evidence of hepatic toxicity but animals exposed to CCl₄ without the treatment with B. Variegata presented variations in levels of liver enzymes, antioxidant enzymes, proteins and blood cells as well as injuries in liver cells were also observed during histopathological study. However, after the treatments especially with 300 mg/kg b.w of methanol flower extracts levels of liver markers (ALT, AST and ALP), antioxidant enzymes and blood cells decreases and turned towards normal levels. Whereas level of total proteins and bilirubin was improved and damaged liver cells were repaired. The curative activity of flower extracts can be correlated to the higher potential of antioxidants and occurrence of Quercetin and some other organic compounds those were investigated from flower extracts of B. Variegata during HPLC and GC-MS analysis. The finding of this study supports the use of B. Variegata flower formulation in folk medicines.     

Effect of low flow ischemia-reperfusion injury on liver function

The release of liver enzymes is typically used to assess tissue damage following ischemia-reperfusion. The present study was designed to determine the impact of ischemia-reperfusion on liver function and compare these findings with enzyme release. Isolated, perfused rat livers were subjected to low flow ischemia followed by reperfusion. Alterations in liver function were determined by comparing rates of oxygen consumption, gluconeogenesis, ureagenesis, and ketogenesis before and after ischemia. Lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP) activities in effluent perfusate were used as markers of parenchymal and endothelial cell injury, respectively. Trypan blue staining was used to localize necrosis. Total glutathione (GSH + GSSG) and oxidized glutathione (GSSG) were measured in the perfusate as indicators of intracellular oxidative stress. LDH activity was increased 2-fold during reperfusion compared to livers kept normoxic for the same time period whereas PNP activity was elevated 5-fold under comparable conditions. Rates of oxygen consumption, gluconeogenesis, and ureagenesis were unchanged after ischemia, but ketogenesis was decreased 40% following 90 min ischemia. During reperfusion, the efflux rates of total glutathione and GSSG were unchanged from pre-ischemic values. Significant midzonal staining of hepatocyte nuclei was observed following ischemia-reperfusion, whereas normoxic livers had only scattered staining of individual cells. Reperfusion of ischemic liver caused release of hepatic enzymes and midzonal cell death, however, several major liver functions were unaffected under these experimental conditions. These data indicate that there were negligible changes in liver function in this model of ischemia and reperfusion despite substantial enzyme release from the liver and midzonal cell death.     

Use of 3-hydroxyphenylacetylene for activity-dependent, fluorescent labeling of bacteria that degrade toluene via 3-methylcatechol

3-hydroxyphenylacetylene (3-HPA) served as a novel, activity-dependent, fluorogenic and chromogenic probe for bacterial enzymes known to degrade toluene via meta ring fission of the intermediate, 3-methylcatechol. By this direct physiological analysis, cells grown with an aromatic substrate to induce the synthesis of toluene-degrading enzymes were fluorescently labeled.     

Endocytosis of lysosomal enzymes by non-parenchymal rat liver cells. Comparative study of lysosomal-enzyme uptake by hepatocytes and non-parenchymal liver cells

Cultured non-parenchymal rat liver cells internalize human urine alpha-N-acetylglucosaminidase, human skin beta-N-acetylglucosaminidase and pig kidney alpha-mannosidase. Different heat-stabilities of endocytosed and endogenous alpha-mannosidase activity provided indirect evidence that the increase in intracellular activity resulted from uptake. The high efficiency and the saturation kinetics of uptake indicated that these enzymes become internalized by adsorptive endocytosis. Competition experiments with glycoproteins bearing known carbohydrates at their non-reducing terminals, with mannans, methyl glycosides and monosaccharides, established that the uptake of these three lysosomal enzymes is mediated by the binding to cell-surface receptors that recognize mannose and N-acetylglucosamine residues. The decreased uptake after treatment of these enzymes with either beta-N-acetylglucosaminidase or alpha-mannosidase was in accordance with the results of the inhibition experiments. Removal of oligosaccharides of the high-mannose type by treatment with endoglucosaminidase H inhibited uptake almost completely, suggesting that the sugars recognized by cell-surface receptors of non-parenchymal liver cells are located in the outer core of these oligosaccharides. A comparison of the uptake of these three lysosomal enzymes by parenchymal and non-parenchymal rat liver cells indicates that infused alpha-N-acetylglucosaminidase is taken up preferentially by hepatocytes, whereas alpha-mannosidase and beta-N-acetylglucosaminidase are localized predominantly in non-parenchymal rat liver cells.     

Arhodomonas sp. Strain Seminole and Its Genetic Potential To Degrade Aromatic Compounds under High-Salinity Conditions

Arhodomonas sp. strain Seminole was isolated from a crude oil-impacted brine soil and shown to degrade benzene, toluene, phenol, 4-hydroxybenzoic acid (4-HBA), protocatechuic acid (PCA), and phenylacetic acid (PAA) as the sole sources of carbon at high salinity. Seminole is a member of the genus Arhodomonas in the class Gammaproteobacteria, sharing 96% 16S rRNA gene sequence similarity with Arhodomonas aquaeolei HA-1. Analysis of the genome predicted a number of catabolic genes for the metabolism of benzene, toluene, 4-HBA, and PAA. The predicted pathways were corroborated by identification of enzymes present in the cytosolic proteomes of cells grown on aromatic compounds using liquid chromatography-mass spectrometry. Genome analysis predicted a cluster of 19 genes necessary for the breakdown of benzene or toluene to acetyl coenzyme A (acetyl-CoA) and pyruvate. Of these, 12 enzymes were identified in the proteome of toluene-grown cells compared to lactate-grown cells. Genomic analysis predicted 11 genes required for 4-HBA degradation to form the tricarboxylic acid (TCA) cycle intermediates. Of these, proteomic analysis of 4-HBA-grown cells identified 6 key enzymes involved in the 4-HBA degradation pathway. Similarly, 15 genes needed for the degradation of PAA to the TCA cycle intermediates were predicted. Of these, 9 enzymes of the PAA degradation pathway were identified only in PAA-grown cells and not in lactate-grown cells. Overall, we were able to reconstruct catabolic steps for the breakdown of a variety of aromatic compounds in an extreme halophile, strain Seminole. Such knowledge is important for understanding the role of Arhodomonas spp. in the natural attenuation of hydrocarbon-impacted hypersaline environments.     

Identification of CYP2C9 as a human liver microsomal linoleic acid epoxygenase

Leukotoxin (9,10-epoxy-12-octadecanoate) and isoleukotoxin (12, 13-epoxy-9-octadecenoate) are monoepoxides of linoleic acid, synthesized by a cytochrome P450 monooxygenase and possibly by an oxidative burst of inflammatory cells. Recent experiments in this laboratory have indicated that the toxicity of leukotoxin and isoleukotoxin is not due to these epoxides, but to the 9,10- and 12, 13-diol metabolites. Leukotoxin and isoleukotoxin are metabolized primarily by the soluble epoxide hydrolase to form leukotoxin diol. Investigations with recombinant cytochrome P450 enzymes have demonstrated that leukotoxin and isoleukotoxin can be formed by these enzymes. This study used a combination of experimental approaches to identify the major cytochrome P450 enzyme in human liver involved in linoleic acid epoxidation. The kinetic paramenters were determined; the K(m) of linoleic acid epoxidation by pooled human liver microsomes was 170 microM and the V(max) was 58 pmol/mg/min. Correlation analysis was performed using individual samples of human liver microsomes, and the best correlation of linoleic acid epoxidation activity was with tolbutamide hydroxylase activity, CYP2C9. Recombinant CYP2C9 was the most active in linoleic acid epoxygenation, and antibody and chemical inhibition also indicated the importance of CYP2C9. This enzyme, therefore, may serve as a therapeutic target in the treatment of inflammation in order to reduce the amount of circulating leukotoxin/isoleukotoxin and their related diols.