The stereoselectivity of four hepatic glutathione S-transferases purified from a marine elasmobranch (Raja erinacea) with several K-region polycyclic arene oxide substrates.

Product analysis by HPLC demonstrated that three hepatic cytosolic glutathione S-transferases of little skate (E-2, E-3 and E-4) were highly stereoselective, if not stereospecific, for thiol reaction at the R-configured oxirane carbons of four K-region arene oxides; pyrene 4,5-oxide, (+/-)-benz[a]anthracene 5,6-oxide, (+/-)-benzo[a]pyrene 4,5-oxide and phenanthrene 9,10-oxide. A fourth transferase, E-5, exhibited no stereopreference with pyrene 4,5-oxide or phenanthrene 9,10-oxide. Immunological and electrophoretic evidence has shown that the three enzymes exhibiting stereospecificity have a common subunit which is absent from enzyme E-5. The high stereoselectivity of the three enzymes E-2, E-3, and E-4 was accompanied by effective catalysis of the reaction of GSH with these K-region epoxides; for example, the calculated turnover number for E-4 with benzo[a]pyrene 4,5-oxide was 550.

US EPA's IRIS pilot program: establishing IRIS as a centralized, peer-reviewed data base with agency consensus. Integrated Risk Information System.

The US EPA's Integrated Risk Information System (IRIS) contains Agency consensus scientific positions and quantitative values on cancer and noncancer health effects that may result from lifetime oral or inhalation exposure to specific chemical substances in the environment. Combined with specific exposure assessment information, the summary health information in IRIS may be used as a source in evaluating potential public health risks from environmental contaminants. IRIS is available to the public via EPA's Internet server at http://www.epa.gov/iris. Originally developed for internal EPA use, IRIS usage has broadened since being made publicly available in 1988 to include the private and public sectors nationally and internationally. Up to 1995, IRIS summaries were generated from within various EPA Offices and Regions and reviewed by Agency Workgroups, one for cancer and one for noncancer endpoints, before entry onto IRIS. In response to the increasing usage and recognition of IRIS and suggestions for improvement, an IRIS Pilot program was initiated in 1995. The purpose of the Pilot was 3-fold: To improve efficiency in getting information on to IRIS; to improve documentation for the positions reported in IRIS summaries, including applying new methodologies and guidance; and to improve opportunity for public input including external peer review. A new infrastructure was put in place, consisting of a cross-Agency team of 'Chemical Managers', a Pilot Program Manager, and a set of Agency 'Consensus Reviewers'. Cancer and noncancer assessments were prepared in an integrated fashion for Pilot chemical substances, documented in 'Toxicological Reviews' and derivative IRIS summaries. Public input was emphasized via an initial data call and rigorous external peer review. A final step was Agency-wide consensus review by senior staff scientists representing EPA's Offices and Regions. EPA's experience with the Pilot is forming the basis for designing operational aspects of the long-term IRIS program.

The hepatic glutathione transferases of the male little skate, Raja erinacea.

Five cytosolic glutathione transferases were isolated from the liver of the male little skate, Raja erinacea, a marine elasmobranch. They were designated E-1 through E-5 in order of their elution from a DEAE-cellulose column with a 0 to 100 mM KCl gradient in 0.01 M Tris (pH 8.0). Each eluted peak of glutathione transferase activity, after concentration, was applied to an affinity column prepared by reaction of epoxy-activated Sepharose 6B with glutathione (GSH). Elution of the various glutathione transferases from this column with GSH resulted in the further purification of each enzyme; the major glutathione transferase, E-4 and E-1, were purified to apparent homogeneity by this procedure. Skate glutathione transferase E-4 is dimeric and the subunits are either very similar or identical in molecular weight (about 26 000 daltons). Enzymes E-2 through E-5 were acidic proteins (pI less than 7.0) and had high specific glutathione transferase activity (0.3--12 mumol/min/mg protein) with benzo[a]pyrene 4,5-oxide (BPO) as substrate, whereas the other enzyme (E-1) had low activity (0.01 mumol/min/mg) with BPO and a basic pI (greater than 9.5). Bilirubin and hematin, non-substrate ligands, bound tightly to homogeneous E-4, with dissociation constants in the micromolar range.

Epoxide metabolizing enzyme activities in rat testes: postnatal development and relative activity in interstitial and spermatogenic cell compartments.

Microsomal epoxide hydrase (EH) and 176 000 g supernatant fraction glutathione-S-transferase (GSH-S-T) activities were determined with styrene oxide as substrate in rat testes during postnatal development. The development of these enzymes was also followed in liver for comparison. Testes of 6-day-old rats had high GSH-S-T activities (66 nmol/min/mg protein), which were about 50% of the adult levels. Transferase activity then developed slowly and reached a maximum by 165 days of age. Specific testicular GSH-S-T activities of 6-day-old rats were 3--4 times those of hepatic GSH-S-T activities in the same animals. In contrast, EH activities of both liver and testes were very low in prepubertal rats, but they increased dramatically at the onset of puberty and reached maximum activities by 45 days of age. Microsomal and microsomal supernatant fractions prepared from adult rat spermatogenic cells had about twice the EH and GSH-S-T specific activities (with styrene oxide or benzo[a]pyrene 4,5-oxide as substrates) of similar fractions prepared from interstitial cells. On the other hand, benzo[a]pyrene hydroxylase (AHH) activity and cytochrome P-450 content were at least 2-fold greater in microsomes from interstitial cells than in those from spermatogenic cells.

1-14C-n-hexadecane disposition in the spiny lobster, Panulirus argus and the American lobster, Homarus americanus.

1-14C-n-hexadecane, a model compound for the non-volatile aliphatic hydrocarbon components of crude oil, was administered by intrapericardial injection to the spiny lobster, Panulirus argus, and the clawed or American lobster, Homarus americanus. Experiments were conducted in Florida (spiny lobster) and Maine (American lobster). The animals were sacrificed at various times from 0.5 hr to 8 wks after administration of the dose. The tissues and fluids were analyzed for 14C content by digestion or catalytic oxidation and liquid scintillation counting. Selected tissues (hepatopancreas, tail muscle and hemolymph) were extracted with ethyl acetate to allow quantitation of the unmetabolized n-hexadecane by thin layer chromatography. n-Hexadecane-derived radioactivity was very persistent in both the spiny lobster (t1/2 = 4.6 wk) and the American lobster (t1/2 = 11.2 wk). In both lobsters, the hepatopancreas (HP) acquired the highest specific activity and the tail muscle had the longest half life for elimination from an individual tissue. Although hexadecane was metabolized more rapidly in the HP of the spiny lobster than in the HP of the American lobster, unmetabolized hexadecane persisted in the HPs of both species for at least 8 weeks after the dose (the longest time studied).

Formation of S-[2-(N7-guanyl)ethyl] adducts by the postulated S-(2-chloroethyl)cysteinyl and S-(2-chloroethyl)glutathionyl conjugates of 1,2-dichloroethane.

The formation of S-[2-(N7-guanyl)ethyl]glutathione (GEG) from dihaloethanes is postulated to occur through two intermediates: the S-(2-haloethyl)glutathione conjugate and the corresponding episulfonium ion. We report the formation of GEG when deoxyguanosine (dG) was incubated with chemically synthesized S-(2-chloroethyl)glutathione (CEG). The depurination of GEG was shown to be first order with a half-life of 7.4 +/- 0.4 h at 27 degrees C. Evidence is also presented for the formation of S-[2-(N7-guanyl)ethyl]-L-cysteine (GEC) in incubation mixtures containing dG and S-(2-chloroethyl)-L-cysteine (CEC), the corresponding cysteine conjugate of CEG. This finding demonstrates that this (haloethyl)cysteine conjugate does not require activation by enzymatic action of cysteine conjugate beta-lyase but, instead, can directly alkylate DNA. The half-life of the depurination of GEC was 6.5 +/- 0.9 h, which is no different from that of GEG. Of the two conjugates, CEC is a somewhat more active alkylating agent toward dG than CEG as N7-guanylic adduct was detected in reaction mixtures with lower concentrations of CEC than with CEG.

Peroxidase-mediated formation of glutathione conjugates from polycyclic aromatic dihydrodiols and insecticides.

Using two peroxidative systems (prostaglandin H synthase/arachidonic acid and horseradish peroxidase/H2O2) we observed GSH conjugate formation with a number of compounds including polycyclic aromatic hydrocarbon-diols (PAH-diols), insecticides, and steroids. Several of the conjugates were characterized by chromatography, uv-vis spectrophotometry, and FAB mass spectroscopy. Conjugate formation is dependent upon a functioning peroxidase, GSH, and is markedly enhanced (3- to 10-fold) by the inclusion of a number of reducing cosubstrates including phenol, uric acid, phenylbutazone, and acetaminophen. The mechanism of conjugate formation appears to involve addition of thiyl radical to alkene bonds conjugated to an electron releasing group probably by resonance stabilization of the carbon-centered radical intermediate. Thiyl radicals are formed either directly by GSH reduction of the peroxidase or indirectly by GSH reduction of radicals formed from reducing cosubstrates. The nitrone spin trap, 5,5-dimethyl-1-pyrroline N-oxide, which traps thiyl radicals, totally inhibits production of GSH conjugates in both peroxidative systems. Conjugation of PAH-diols, some of which are penultimate carcinogens, would prevent their metabolism to the diol-epoxides, an ultimate carcinogenic species of PAH. Conjugation by peroxidases appears to be a general pathway for glutathione conjugate formation that may lead to potential detoxification of chemicals.

Peroxidase-mediated glutathione conjugation of benzo[a]pyrene-7,8-dihydrodiol is enhanced by benzo[a]pyrene phenols in vitro.

We reported previously that glutathione (GSH) is oxidized by peroxidases to a thiyl radical that can react with a number of chemicals, including the penultimate carcinogenic metabolite benzo[a]pyrene-7,8-dihydrodiol (7,8-B[a]PD), to give GSH conjugates. Here, we report that phenolic metabolites of benzo[a]pyrene (B[a]P) enhance the peroxidase-mediated formation of glutathione conjugates of 7,8-B[a]PD. The GSH conjugation of 7,8-B[a]PD in a horseradish peroxidase/peroxide system was increased over control values as follows: 9-OH-B[a]P by 4-fold, 7-OH-B[a]P by 3-fold, 1-OH-B[a]P by 2-fold. In contrast 3-OH-B[a]P was ineffective. A phenolic derivative of another polycyclic aromatic hydrocarbon (PAH), benz[a]anthracene, also enhanced GSH conjugation of 7,8-B[a]PD. The enhancement was dependent upon the presence of the phenol, horseradish peroxidase and peroxide. The phenolic compounds, including 3-OH-B[a]P, were also efficient reducing cofactors for the peroxidase. With the exception of 3-OH-B[a]P, the phenolic metabolites of PAH enhanced peroxidase-mediated formation of thiyl radical as detected by electron spin resonance spectrometry. Since both phenols and dihydrodiols are metabolites of B[a]P catalyzed by the cytochromes P450 system, enhancement of peroxidase-dependent 7,8-B[a]PD-GSH conjugation by phenols suggests a possible interaction between peroxidases and cytochromes P450 systems. This interaction may contribute to the detoxication of the penultimate carcinogenic PAH-dihydrodiols and other chemicals.

Detection of glutathione thiyl free radical catalyzed by prostaglandin H synthase present in keratinocytes. Study of co-oxidation in a cellular system.

We report here the application of the electron spin resonance technique to detect free radicals formed by the hydroperoxidase activity of prostaglandin H synthase in cells. Studies were done using keratinocytes obtained from hairless mice. These cells can be prepared in large number and possess significant prostaglandin H synthase activity. Initial attempts to directly detect free radical metabolites of several amines in cells were unsuccessful. A technique was developed based on the ability of some free radicals formed by prostaglandin hydroperoxidase to oxidize reduced glutathione (GSH) to a thiyl radical, which was trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Phenol and aminopyrine are excellent hydroperoxidase substrates for this purpose and thus were used for all further experiments. Using this approach we detected the DMPO/GS.thiyl radical adduct catalyzed by cellular prostaglandin hydroperoxidase. The formation of the radical was dependent on the addition of substrate, inhibited by indomethacin, and supported by either exogenous arachidonic acid or endogenous arachidonic acid released from phospholipid stores by Ca2+ ionophore A-23187. The addition of GSH significantly increased the intracellular GSH concentration and concomitantly stimulated the formation of the DMPO/GS.thiyl radical adduct. Phenol, but not aminopyrine, enhanced thiyl radical adduct formation and prostaglandin formation with keratinocytes while both cofactors were equally effective in incubations containing microsomes prepared from keratinocytes. These results suggest that prostaglandin hydroperoxidase-dependent co-oxidation of chemicals can result in the intracellular formation of free radical metabolites.

Stereoselectivity of styrene oxidation in microsomes and in purified cytochrome P-450 enzymes from rat liver.

The stereochemistry of the cytochrome P-450(P-450)-dependent oxidation of styrene to styrene 7,8-oxide (SO) enantiomers was evaluated with rat hepatic microsomes and with individual rat liver P-450 enzymes in reconstituted monooxygenase systems. The stereoselectivity of the monooxygenase reaction with styrene was determined by high-performance liquid chromatographic analysis of the glutathione conjugates formed quantitatively from SO, the product of the monooxygenase reaction. Hepatic microsomes from control rats oxidized styrene at a rate of 13.1 +/- 4.5 nmol/min/nmol of P-450 and with a ratio of the amount of the (R)-styrene 7,8-oxide enantiomer to the amount of the (S)-styrene 7,8-oxide enantiomer (R/S) SO ratio of 0.65 +/- 0.1. These values were determined under incubation conditions in which epoxide hydrolase activity was inhibited by cyclohexene oxide, and at least 95% of the SO formed was converted enzymatically to glutathione conjugates. Treatment of rats i.p. with phenobarbital (PB) or beta-naphthoflavone (beta NF) caused changes in both parameters. Whereas the rates of oxidation in hepatic microsomes prepared from PB-treated rats was unchanged at 15.4 +/- 7.5 nmol/min/nmol of P-450 and decreased in hepatic microsomes from beta NF-treated rats to 9.4 +/- 2.8 nmol/min/nmol of P-450, the preference for formation of the R-enantiomer increased as the R/S ratio changed to 0.92 +/- 0.1 for PB and 1.25 +/- 0.1 for beta NF.(ABSTRACT TRUNCATED AT 250 WORDS)

The perinatal development of epoxide-metabolizing enzyme activities in liver and extrahepatic organs of guinea pig and rabbit.

We have measured the activities of epoxide hydrase in microsomes and glutathione S-epoxidetransferase and glutathione S-aryltransferase in cytosol fractions of liver, lungs, kidneys, and small intestine from fetal and neonatal guinea pigs and rabbits. The rates at which adult values of these enzyme activities are reached in extrahepatic tissues differ from the rates of maturation of the hepatic enzyme activities for both species. In addition, the two pathways of epoxide metabolism studied here developed with age at different rates in any one organ. However, both cytosol glutathione S-transferases showed very similar developmental profiles in any one organ. It was especially interesting that the activities of both glutathione S-transferases were within the adult range in pulmonary cytosol fraction of guinea pig and rabbit before birth. Intestinal microsomes did not have adult values for epoxide hydrase activity until several weeks after birth. A feature common to both epoxide-metabolizing activities in hepatic and extrahepatic organs was a drop in mean specific activity, sometimes not statistically significant, around the time of birth. This decrease appeared to be due to dilution of the active enzyme with other protein, inasmuch as the total organ activity, in general, showed no such decline. We found that the pattern of development of hepatic microsomal epoxide hydrase activity was similar to developmental patterns published by others for hepatic microsomal mixed-function oxidases, and also that development of hepatic cytosol glutathione S-transferase was similar to hepatic development of glutathione S-transferase towards other substrates described in the literature.

Heterogeneity of hepatic aryl hydrocarbon hydroxylase activity in feral winter flounder: relevance to carcinogenicity testing.

Hepatic microsomes from winter flounder (Pseudopleuronectes americanus), treated with polycyclic aromatic hydrocarbon (PAH) inducers, had elevated activities of benzo[alpha]pyrene hydroxylase (AHH) and 7-ethoxyresorufin deethylase (7-ERD). When electrophoresed, they showed a novel or enriched polypeptide species with a monomeric molecular weight of approximately 57,000. These results are consistent with inductive responses already well-characterized in several mammalian and fish species. However, when we studied the urinary clearance of 4-chlorobiphenyl in untreated flounder, wide variations (up to twentyfold) among fish were noted. Subsequent in vitro analysis of AHH and 7-ERD activities in liver demonstrated wide variations in these monooxygenase activities in flounder caught near Mount Desert Island, Maine. In some instances, AHH activities in these feral flounder were as high as those in PAH-induced fish. Based on the response of AHH activity to 7,8-benzoflavone (ANF) added in vitro, flounder could be divided into 2 groups; one had high hepatic AHH activity which was inhibited by ANF, the other had low AHH activity which was enhanced by ANF. Examination of a large number of winter flounder (greater than 400 total) over 4 experimental seasons demonstrated this variability of hepatic AHH activity to be a recurrent characteristic of the flounder population in waters around Mount Desert Island. The hepatic AHH activities did not correlate well with any physical parameter of the fish (e.g., liver, gonad or body weight, length, or sex) or the cytochrome P-450 content of the hepatic microsomes. Our attempt to evaluate the AHH activity (high vs. low) of individual fish in vivo by urinary clearance of antipyrine was unsuccessful, due to the excretion of large amounts of unchanged antipyrine through the gills. Similar studies were performed with another marine teleost, Fundulus heteroclitus. Of the approximately 200 Fundulus examined, almost all had AHH activity inhibited by ANF; in some experiments, hepatic 7-ERD activities were further increased after treatment with the potent PAH-type inducer 3,4,5,3',4',5'-hexachlorobiphenyl but only about twofold. Collectively, these data are consistent with PAH-type induction of the hepatic cytochrome P-450-dependent monooxygenase system in some feral marine teleosts (in winter flounder and Fundulus) from Maine by environmental contaminants or food constituents. It is not known whether these chemicals are of natural or anthropogenic origin. The variation in the response appears to be related to individual exposure level (dose) or sensitivity, or both.(ABSTRACT TRUNCATED AT 400 WORDS)

Prostaglandin H synthase and xenobiotic oxidation.

We have attempted in this article to summarize and review cooxidation reactions that occur during the metabolism of AA and potential roles that these reactions can play in the activation and detoxification of chemicals. This review summarizes approximately 15 years of intensive investigation by a number of laboratories, and as such not all studies are cited, and in some cases data are not discussed with the emphasis that the original investigators may have intended. The major focus of many of these studies has been toward carcinogenesis. In the future, emphasis may shift to the formation of metabolites that will lead to other toxic effects. The cooxidation reactions that occur during AA metabolism are dependent upon the peroxidase activity of PHS. For some chemicals that are not cosubstrates, the epoxidation reactions that occur are dependent upon the subsequent formation of peroxyl radicals. A large and diverse number of chemicals are metabolized by an equally large and diverse number of chemical reactions. The unifying theme is the free radical nature of these oxidations. The subsequent reactions that these chemicals undergo is dictated by the nature of the free radical and the environment in which it is generated. Ample evidence now exists for the contribution of these free radical-mediated reactions not only in the formation of toxic metabolites, but also in some cases in the detoxification of chemicals. The overriding factor for this type of metabolism to occur is the relative concentrations in the specific tissue of PHS and peroxyl radicals with respect to other activating systems, particularly the monooxygenase system. In vivo investigations support the importance of the peroxidase and peroxyl radical systems in both activation and detoxification of chemicals in extrahepatic tissues.

Xenobiotic metabolism in Clara cells and alveolar type II cells isolated from lungs of rats treated with beta-naphthoflavone.

An investigation of several pathways for xenobiotic metabolism in rat lung cells was carried out using enriched fractions of alveolar type II cells (80% purity) and Clara cells (50% purity) which had been prepared from either untreated (control) rats or animals which had been treated with beta-naphthoflavone. Monooxygenase activities (7-ethoxycoumarin deethylase; aryl hydrocarbon [benzo(a)pyrene] hydroxylase) and activities of conjugating enzymes (glutathione transferase; glucuronosyl transferase) were found to be much higher in fractions enriched in Clara cells than in either the crude cell digest or in fractions enriched in type II cells. This was also found to be true for epoxide hydrolase activity. beta-Naphthoflavone treatment of animals was found to increase the monooxygenase and glucuronosyl transferase activities in all cell fractions, but no effect was seen on either glutathione transferase or epoxide hydrolase activity, each of which was extremely low in type II cells.

The metabolism and excretion of 14C-styrene oxide-glutathione adducts administered to the winter flounder, Pseudopleuronectes americanus, a marine teleost. Identification of the corresponding S-cysteine derivatives as major urinary metabolites.

The metabolism and excretion of intramuscularly administered 14C-glutathione conjugates of styrene oxide (Ia and IIa) were studied in the winter flounder at three dose levels. The various radiolabeled thioether metabolites excreted were separated by reverse-phase high pressure liquid chromatography, and identified and quantitated by cochromatography with synthetic standards. The urine was the major excretion route for radioactivity derived from the glutathione conjugates (up to 90%) at each dose level (1.0, 3.9, and 24.4 mg/fish) studied. The corresponding cysteine derivatives (Ic, IIc) were the major urinary metabolites although the N-acetylcysteine derivatives (Id, IId), or mercapturic acids, were also present in significant amounts at each dose and excretion interval examined. Unchanged glutathione conjugates of styrene oxide were the major radioactive constituents of 24-hr bile samples from the treated flounder, although significant amounts of the cysteinylglycine (Ib, IIb), cysteine, and N-acetylcysteine derivatives were also present in bile. Bile was a minor excretory route relative to urine. The oxidation of 14C-styrene to styrene 7,8-oxide by the cytochrome P-450-dependent monooxygenase system of hepatic microsomes of winter flounder was also demonstrated; likewise, styrene was converted to the diastereomeric glutathione conjugates of styrene 7,8-oxide by 9,000g supernatant fractions of flounder liver supplemented with glutathione. This study demonstrated that flounder liver can convert styrene to glutathione conjugates to styrene oxide and that mercapturic acid biosynthesis occurs after parenteral administration of a xenobiotic-glutathione adduct to this marine species, although the major urinary metabolites were the cysteine conjugates rather than the anticipated mercapturic acid derivatives.