Individual and population exposures to gasoline.

Gasoline is a complex mixture of many constituents in varying proportions. Not only does the composition of whole gasoline vary from company to company and season to season, but it changes over time. The composition of gasoline vapors is dominated by volatile compounds, while "gasoline" in groundwater consists mainly of water-soluble constituents. Hydrocarbons, including alkanes, alkenes, and aromatics, make up the large majority of gasoline, but other substances, such as alcohols, ethers, and additives, may also be present. Given this inability to define "gasoline,h' exposures to individual chemicals or groups of chemicals must be defined in a meaningful exposure assessment. An estimated 111 million people are currently exposed to gasoline constituents in the course of refueling at self-service gasoline stations. Refueling requires only a few minutes per week, accruing to about 100 min per year. During that time, concentrations in air of total hydrocarbons typically fall in the range 20-200 parts per million by volume (ppmV). Concentrations of the aromatic compounds benzene, toluene, and xylene rarely exceed 1 ppmV. Some liquid gasoline is also released, generally as drops less than 0.1 g each, but with enough larger spills to raise the average loss per gallon dispensed to 0.23 g for stations with conventional nozzles and 0.14 g per refueling for stations with vapor recovery nozzles (Stage II controls). Some skin exposure may occur from these spills but the exposure has not been quantified. Two major types of vehicular emissions have been studied. Evaporative emissions include emissions while the vehicle is driven (running losses), emissions after the engine has been shut off but is still warm (hot soak), and emissions during other standing periods (diurnal) emissions. These evaporative emissions are dominated by the more volatile gasoline components. Tailpipe emissions include some unreacted gasoline constituents as well as products of combustion (including chemicals identical to some of the original constituents of the gasoline) and a variety of hydrocarbons and related compounds. Running losses are reported to fall in the range of 0.2 to 2.8 g of total hydrocarbons per mile driven, while benzene evaporative emissions range from 0.002 to 0.007 g/mile. Benzene levels inside travelling vehicles have been reported to average about 13 ppbV in Los Angeles. Tailpipe emissions amount to 0.3 to 1.0 g/mile of total hydrocarbons; emissions of benzene, polycylic aromatic hydrocarbons, and 1,3-butadiene have been reported to range from 0.015 to 0.04 g/mile, 0.00025 to 0.00046 g/mile, and 0.001 to 0.005 g/mile, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Catabolism of epoxy fatty esters by the purified epoxide hydrolase from mouse and human liver.

Epoxymethylsterate, 9,10- and 12,13-epoxymethyloleates, and a mixture of isomers of epoxymethylarachidonate and diepoxymethylstearate were synthesized, and their metabolic rates were measured using crude and purified cytosolic epoxide hydrolase. Hepatic epoxide hydrolase was purified from human samples and clofibrate-fed mice by affinity chromatography. The major metabolites under these conditions of all the epoxy fatty esters were their vicinal diols whose structures were confirmed by GC-MS. 12,13-Epoxymethyloleate was metabolized faster than 9,10-epoxymethyloleate and other epoxy fatty esters, but all substrates were turned over rapidly. This rapid turnover suggests that epoxy fatty acids may be endogenous substrates for the cytosolic epoxide hydrolase.

Continuous spectrophotometric assays for cytosolic epoxide hydrolase.

Two convenient and sensitive continuous spectrophotometric assays for cytosolic epoxide hydrolase are described. The assays are based on the differences in the ultraviolet spectra of the epoxide substrates and their diol products. The hydrolysis of 1,2-epoxy-1-(p-nitrophenyl)pentane (ENP5) is accompanied by a decrease in absorbance at 302 nm, while the hydration of 1,2-epoxy-1-(2-quinolyl)pentane (EQU5) produces an increase in absorbance at 315.5 nm. The Km, Vmax values for ENP5 and EQU5 with purified mouse liver cytosolic epoxide hydrolase were 1.7 microM, 11,700 nmol/min/mg and 25 microM, 8300 nmol/min/mg, respectively. Both substrates are hydrolyzed significantly faster than trans-stilbene oxide, which is currently the most commonly used substrate for measuring cytosolic epoxide hydrolase activity. No spontaneous hydrolysis of the substrates is detectable under normal assay conditions. The assays are applicable to whole tissue homogenates as well as purified enzyme preparations. p-Nitrostyrene oxide and p-nitrophenyl glycidyl ether were also examined and found to be very poor substrates for cytosolic epoxide hydrolase from mouse liver.

Cytosolic epoxide hydrolase in human placenta.

It has been shown that cytosol from human term placenta contains cytosolic epoxide hydrolase activity. This cytosolic epoxide hydrolase was enriched more than 700-fold by affinity chromatography and appears similar to the enzyme from mouse and human liver in terms of molecular mass (Mr 59,000) and antigenic reactivity.

Epoxide-metabolizing enzymes in mammary gland and liver from BALB/c mice and effects of inducers on enzyme activity.

Epoxide hydrolases (EC 3.3.2.3) (EH) are hydrolytic enzymes which may play an important role in the activation and detoxification of mammary carcinogens. In this study, microsomal, cytosolic, and cholesterol epoxide hydrolases along with glutathione S-transferase were characterized in liver and mammary gland from nulliparous and lactating BALB/c mice and from mice transplanted with preneoplastic hyperplastic outgrowths. Clofibrate, butylated hydroxyanisole, and beta-naphthoflavone were used to induce EH. Significant epoxide hydrolysis was observed in microsomal and cytosolic subcellular fractions assayed with cis- and trans-stilbene oxide, benzo(a)pyrene-4,5-oxide, and cholesterol epoxide. The hydrolysis rates were significantly different for nulliparous and lactating animals, in both mammary gland and liver. Clofibrate increased the activity of all forms of EH in liver, but not mammary gland. Butylated hydroxyanisole and beta-naphthoflavone appeared to induce cytosolic glutathione S-transferase as well as some, but not all, forms of EH in liver and mammary gland regardless of hormonal stimuli. The inducers produced different effects in mammary gland as compared with liver. This may be due to either differing amounts of inducer reaching the target site or different regulation of the enzymes in mammary gland and liver. Hyperplastic outgrowths and liver from hyperplastic outgrowth-transplanted animals demonstrated significantly different EH and cytosolic glutathione S-transferase activities from those of nulliparous and lactating animals. This observation offers preliminary evidence that levels of epoxide-metabolizing enzymes are altered when mammary tissue is transformed. Mammary gland cytosolic EH was purified by affinity chromatography and compared to that from liver by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, enzyme-linked immunosorbent assay, isoelectric focusing, and enzyme inhibition by 4-phenylchalcone oxide. Cytosolic EH from the mammary gland appears to be identical to the liver enzyme by all the above mentioned biochemical and biophysical parameters.

Affinity purification of cytosolic epoxide hydrolase using derivatized epoxy-activated Sepharose gels.

Improved affinity chromatography procedures for the purification of cytosolic epoxide hydrolase are described. An earlier affinity purification method using immobilized 7-methoxycitronellyl thiol (MCT) sporadically produced final enzyme preparations containing major impurities. To eliminate these impurities, we tested alternate ligands, spacer arms, and ligand concentrations. A series of alkyl and aryl thiols coupled to epoxy-activated Sepharose were found to exhibit markedly different binding characteristics as compared with commercially available alkyl- and aryl-Sepharose gels. Using one of these new matrices, benzylthio-Sepharose, cytosolic epoxide hydrolase from mouse liver was purified over 100-fold, appeared homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was obtained with 60-90% recovery of enzyme activity. The impurities previously observed with the MCT-Sepharose procedure were reduced or eliminated by using an MCT ligand concentration of 5 microequivalents per gram or less. MCT-Sepharose and benzylthio-Sepharose provide rapid and convenient one-step procedures for obtaining purified cytosolic epoxide hydrolase from numerous species and tissues.

Comparison of crude and affinity purified cytosolic epoxide hydrolases from hepatic tissue of control and clofibrate-fed mice.

An affinity purification procedure was developed for the cytosolic epoxide hydrolase based upon the selective binding of the enzyme to immobilized methoxycitronellyl thiol. Several elution systems were examined, but the most successful system employed selective elution with a chalcone oxide. This affinity system allowed the purification of the cytosolic epoxide hydrolase activity from livers of both control and clofibrate-fed mice. A variety of biochemical techniques including pH dependence, substrate preference, kinetics, inhibition, amino acid analysis, peptide mapping, Western blotting, analytical isoelectric focusing, and gel permeation chromatography failed to distinguish between the enzymes purified from control and clofibrate-fed animals. The quantitative removal of the cytosolic epoxide hydrolase acting on trans-stilbene oxide from 100,000g supernatants, allowed analysis of remaining activities acting differentially on cis-stilbene oxide and benzo[a]pyrene 4,5-oxide. Such analysis indicated the existence of a novel epoxide hydrolase activity in the cytosol of mouse liver preparations.