In vivo production of nitric oxide after administration of cyclohexanone oxime.

Cyclohexanone oxime (CHOX), an intermediate used in the synthesis of polycaprolactam (Nylon-6), has been reported to be hematotoxic in Fischer rats. The in vivo metabolism of CHOX was found to release nitric oxide, which was detected in venous blood by electron paramagnetic resonance spectroscopy as the nitrosylhemoglobin complex. In vitro incubation of CHOX with venous blood resulted in the formation of the characteristic nitrosylhemoglobin complex, suggesting that the blood was a possible site for metabolism. Excessive nitric oxide production may, in part, contribute to the observed toxicity of CHOX.

The fate of the oxidizing tyrosyl radical in the presence of glutathione and ascorbate. Implications for the radical sink hypothesis.

Cellular systems contain as much as millimolar concentrations of both ascorbate and GSH, although the GSH concentration is often 10-fold that of ascorbate. It has been proposed that GSH and superoxide dismutase (SOD) act in a concerted effort to eliminate biologically generated radicals. The tyrosyl radical (Tyr.) generated by horseradish peroxidase in the presence of hydrogen peroxide can react with GSH to form the glutathione thiyl radical (GS.). GS. can react with the glutathione anion (GS-) to form the disulfide radical anion (GSSG-). This highly reactive disulfide radical anion will reduce molecular oxygen, forming superoxide and glutathione disulfide (GSSG). In a concerted effort, SOD will catalyze the dismutation of superoxide, resulting in the elimination of the radical. The physiological relevance of this GSH/SOD concerted effort is questionable. In a tyrosyl radical-generating system containing ascorbate (100 microM) and GSH (8 mM), the ascorbate nearly eliminated oxygen consumption and diminished GS. formation. In the presence of ascorbate, the tyrosyl radical will oxidize ascorbate to form the ascorbate radical. When measuring the ascorbate radical directly using fast-flow electron spin resonance, only minor changes in the ascorbate radical electron spin resonance signal intensity occurred in the presence of GSH. These results indicate that in the presence of physiological concentrations of ascorbate and GSH, GSH is not involved in the detoxification pathway of oxidizing free radicals formed by peroxidases.

Photochemical reactions and phototoxicity of sterols: novel self-perpetuating mechanisms for lipid photooxidation.

Sterols are important lipid components that may contribute to phototoxicity. We have found that phototoxic response in earthworms is related to sterols extractable with lipophilic solvents. The photochemically active compounds in worm lipids are 5,7,9(11),22-ergostatetraen-3 beta-ol (9-DHE) and 5,7,9(11)-cholestartien-3 beta-ol (9-DDHC), respectively. Human skin lipids are known to contain 9-DHE. We have also found 9-DDHC in human skin, which is reported here for the first time. In the presence of an excess of the corresponding 5,7-dienes (ergosterol of 7-dehydrocholesterol), these photoactive sterols constitute a self-regenerating source of singlet molecular oxygen (1O2) during irradiation in vivo or in vitro with UVA (315-400 nm). The quantum yield for photosensitization of 1O2 by 9-DHE was estimated to be 0.09. The 1O2 is scavenged by the dienes and the rate constant for 1O2 quenching by ergosterol was found to be 1.2 x 10(7) M-1 s-1 in methyl t-butyl ether (MTBE). This scavenging ultimately leads to the production of 5,8-endoperoxide and hydrogen peroxide. Photochemically induced superoxide radical was also produced on irradiation of sterol 5,7,9-trienes and trapped with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The production of singlet oxygen, peroxides and radicals by the sterols may be significant in the cell damaging and tumor promoting action of UVA light on skin.

In vitro free radical metabolism of phenolphthalein by peroxidases.

Phenolphthalein, a widely used laxative, is the active ingredient in more than a dozen commercial nonprescription formulations. Fast-flow EPR studies of the reaction of phenolphthalein with horseradish peroxidase (HRP) and hydrogen peroxide permit the direct detection of two free radicals. One has EPR parameters characteristic of phenoxyl radicals. The other has a broad unresolved spectrum, possibly arising from free radical polymeric products of the initial phenoxyl radical. EPR spin-trapping studies of incubations of phenolphthalein with lactoperoxidase, reduced glutathione (GSH), and hydrogen peroxide with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) demonstrate stimulated production of DMPO/.SG compared with an identical incubation lacking phenolphthalein. In the absence of DMPO, measurements with a Clark-type oxygen electrode show that molecular oxygen is consumed by a sequence of reactions initiated by the glutathione thiyl radical. Enhanced production of DMPO superoxide radical adduct is also found in a system of phenolphthalein, NADH, and lactoperoxidase. In this system the phenolphthalein phenoxyl radical abstracts hydrogen from NADH to generate NAD., which is not spin trapped by DMPO, but reacts with molecular oxygen to produce the superoxide radical detected by EPR. In the absence of DMPO, the oxygen consumption is measured using the Clark-type electrode. Production of ascorbate radical anion is also enhanced in a system of phenolphthalein, ascorbic acid, hydrogen peroxide, and lactoperoxidase. Ascorbate inhibits oxygen consumption when phenolphthalein is metabolized in the presence of either glutathione or NADH by reducing radical intermediates to their parent molecules and forming the relatively stable ascorbate anion radical. The detection of enhanced free radical production in these three systems, a consequence of futile metabolism (or redox cycling), suggests that phenolphthalein may be a significant source of oxidative stress in physiological systems. Parallel EPR and oxygen consumption studies with phenolphthalein glucuronide give analogous results, but with lesser enhancement of free radical production.

Self-peroxidation of metmyoglobin results in formation of an oxygen-reactive tryptophan-centered radical.

In the reaction between hydrogen peroxide and metmyoglobin, the heme iron is oxidized to its ferryl-oxo form and the globin to protein radicals, at least one of which reacts with dioxygen to form a peroxyl radical. To identify the residue(s) that forms the oxygen-reactive radical, we utilized electron spin resonance (ESR) spectroscopy and the spin traps 2-methyl-2-nitrosopropane and 3,5-dibromo-4-nitrosobenzenesulfonic acid (DB-NBS). Metmyoglobin radical adducts had spectra typical of immobilized nitroxides that provided little structural information, but subsequent nonspecific protease treatment resulted in the detection of isotropic three-line spectra, indicative of a radical adduct centered on a tertiary carbon with no bonds to nitrogen or hydrogen. Similar isotropic three-line ESR spectra were obtained by spin trapping the oxidation product of tryptophan reacting with catalytic metmyoglobin and hydrogen peroxide. High resolution ESR spectra of DBNBS/.trp and of the protease-treated DBNBS/.metMb were simulated using superhyperfine coupling to a nitrogen and three non-equivalent hydrogens, consistent with a radical adduct formed at C-3 of the indole ring. Oxidation of tryptophan by catalytic metMb and hydrogen peroxide resulted in spin trap-inhibitable oxygen consumption, consistent with formation of a peroxyl radical. The above results support self-peroxidation of a tryptophan residue in the reaction between metMb and hydrogen peroxide.

Doubly allylic hydroperoxide formed in the reaction between sterol 5,7-dienes and singlet oxygen.

Ergosterol and 7-dehydrocholesterol, common 5,7-conjugated diene sterols, react with photochemically produced singlet oxygen very efficiently to yield, in parallel pathways, the corresponding 5,8-endoperoxides and the 7 beta-hydroperoxy-5,8(9),22-trienol or -5,8(9)-dienol, respectively. The hydroperoxides decompose in an acid-catalyzed reaction to generate hydrogen peroxide and the 5,7,9(11),22-tetraenol or 5,7,9(11) trienol, respectively, with 1:1 stochiometry. The molar ratio of endoperoxide to hydroperoxide was constant (16:5) with two different reaction solvents, two different photosensitizers, and at all time points between 5 min and 3 h from the start of irradiation. Ergosterol did not react with either hydrogen peroxide or superoxide ion under our reaction conditions. Inhibition studies with nitrogen, 2,5-dimethylfuran, beta-carotene, and tert-butanol confirmed the involvement of singlet oxygen in these reactions. The unstable hydroperoxide would be expected to have undesirable biological consequences if formed in vivo.

Endogenous lipids of the earthworm Lumbricus terrestris.

Earthworms (Lumbricus terrestris) were given [1-14C]-labeled palmitic acid by gavage on days 0 and 3, and sacrificed on day 7. The distribution of label among lipid classes indicated that glycerides, sterol esters, cerebrosides, sulfatides, phosphatidylethanolamine, phosphatidylserine and (or) phosphatidylinositol, phosphatidylcholine, and sphingomyelin turn over in, or are synthesized by, the earthworm. Free fatty acids still had the highest specific radioactivity of any lipid class at the end of the experiment. Incorporation of label into sterol and hydrocarbon fractions was insignificant and there was no detectable label incorporated into gangliosides. Phosphatidylethanolamine apparently turned over quite slowly compared with other lipid classes, while the cerebroside fraction became highly labeled. Elongation of palmitic acid to stearate and oxidation to CO2 occurred extensively, but there was no evidence for desaturation.

The metabolism of di(2-ethylhexyl)phthalate in the earthworm Lumbricus terrestris.

1. Earthworms can hydrolyze di-(2-ethylhexyl) phthalate (DEHP) to mono-2-ethylhexyl phthalate (MEHP) and phthalic acid (PA). 2. They apparently cannot produce the side-chain-oxidized derivatives of MEHP that constitute the major DEHP metabolites in higher animals. 3. With the assistance of intestinal bacterial Pseudomonas, the worm-derived PA is degraded through protocatechuic and beta-carboxymuconic acids to CO2. 4. There is an indication of a second pathway for degradation of PA leading through benzoic acid.

Isolation and characterization of the initial radical adduct formed from linoleic acid and alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone in the presence of soybean lipoxygenase.

The spin trapping agent alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) was used to trap the initial radical formed from [U-14C]linoleic acid in the reaction with soybean lipoxygenase. By using low levels of enzyme and relatively short incubation times it was possible to avoid the formation of secondary oxidation products and polymers. The adduct was extracted after methyl esterification, and isolated by a combination of open column chromatography on silicic acid and high pressure liquid chromatography on Spherisorb S5 CN with non-aqueous solvents. The 1:1 POBN-linoleate adduct was characterized by UV, IR and ESR spectra of the appropriate HPLC column fraction, by the ratio of the UV absorption to 14C content, and by mass spectrometry of the reduced (hydroxylamine) form. The results indicated that POBN trapped a linoleic acid carbon-centered radical such that POBN was attached to the fatty acid chain at C-13 or C-9 (two isomers), the linoleate double bonds having become conjugated in the process. The exact locations of the bridges in the two isomers were only tentatively determined. There was no evidence for the presence of oxygen-bridged adducts. The trapped linoleoyl radical adduct provides evidence for the production of a free radical as part of the enzymatic mechanism of soybean lipoxygenase.

Chamber and gavage technique for metabolic studies of earthworms.

Earthworms make very suitable laboratory animals for metabolic studies in vivo using radiolabeled test chemicals. We describe the construction and operation of a metabolic chamber to enable the collection of labeled CO2, volatile organics, material excreted into the bedding, and labeled material remaining in the worms. A gavage technique has been developed that permits the administration of water-soluble and lipid-soluble test chemicals in spite of the extremely low level of triglyceride lipase activity in the earthworm gut. This technique is less likely to puncture the worm tissue than previous methods. Radiolabeled DDT and diethylhexyl adipate were used to provide examples of the use of these techniques and the metabolic chamber. Results were qualitatively similar to those that have been noted in vertebrates.

Lipids of the earthworm Lumbricus terrestris.

The lipid composition of the earthworm Lumbricus terrestris has been reexamined under conditions intended to avoid enzymatic and chemical alterations during storage, extraction, and fractionation procedures. The simple lipids included aliphatic hydrocarbons, steryl esters, glycerides, and at least nine different sterols, all thought to be derived from the diet. Free fatty acids, previously considered to be major components of worm lipids, comprised only 0.3% of the total lipid weight. Phospholipids included (in order of relative abundance) phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, as well as sphingomyelin. Glycolipids included cerebrosides and sulfatides containing both glucose and galactose, and gangliosides containing glucosamine and sialic acid. The fatty acid compositions of these lipid classes appeared to be a mixture of what are considered typical plant, bacterial, and animal acids. Several fatty acids found in the worms, including cis-vaccenic and eicosapentaenoic acids, were essentially absent from the dietary components, and it is concluded that these acids were synthesized in the worms. The earthworm derives much of its lipid adventitiously, but exerts at least some control over its tissue lipid composition.

Mono-2-ethylhexyl phthalate, a metabolite of di-(2-ethylhexyl) phthalate, causally linked to testicular atrophy in rats.

Acute testicular atrophy results when appropriate dosages of di-(2-ethylhexyl) phthalate (DEHP) or its hydrolysis product mono-2-ethylhexyl phthalate (MEHP) are given to male rats. Events thought to be involved in this pathological effect also occur in cultures of testicular cells in vitro, but require MEHP rather than DEHP. Primary cultures of hepatocytes, Sertoli cells, and Leydig cells were incubated with 14C-labeled MEHP [8 microM] for up to 24 hr. No significant reduction in viability was produced under these conditions. In contrast to the hepatocytes, which extensively metabolized MEHP to a variety of products in 1 hr, the testicular cell cultures were apparently unable to metabolize MEHP (beyond a slight hydrolysis to phthalic acid by Sertoli cells) in 18-24 hr. MEHP was efficiently taken up by hepatocytes, but much less so by testicular cells. These results, combined with related observations from the literature, support the hypothesis that MEHP itself is the metabolite of DEHP responsible for testicular atrophy in rats.

The scopoletin assay for hydrogen peroxide. A review and a better method.

Scopoletin, 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one, a naturally occurring component in cotton leaf and citrus peel is a fluorescent substrate for peroxidase which has been used by many investigators for the determination of hydrogen peroxide concentration. The technical details of these investigations are application-specific and rather critical, making it difficult to apply the scopoletin assay to alternative systems without extensive modification. Although such factors as interfering substances and optimum conditions have been discussed in many publications, these discussions tend to be application-specific. The present paper attempts to provide a technical review of scopoletin applications, add a few new experimental observations, and discuss general parameters which must be carefully controlled for reliable results.

In vitro studies of the inhibition of protein kinase C from rat brain by di-(2-ethylhexyl)phthalate.

The environmental contaminant di(2-ethylhexyl)phthalate (DEHP) has been shown to inhibit the phosphorylation of histone by purified protein kinase C (PK-C) from rat brain in a concentration-dependent manner. The inhibition does not involve making the substrate unavailable, although DEHP does bind to some extent to histone. DEHP displaces phorbol dibutyrate from PK-C, indicating that DEHP binds to the regulatory domain of the enzyme. Since DEHP does not affect the PK-C dependent phosphorylation of protamine, DEHP probably does not bind at the catalytic site. DEHP non-competitively blocked activation of PK-C by either phosphatidyl serine or calcium ion. Inhibition of histone phosphorylation by DEHP was enhanced if diglyceride was present, and the enhancement was stereoselective for the isomeric form of the diglyceride. The mechanism of the inhibition is thought to involve interference with the interaction between calcium ion and the regulatory domain of PK-C, and would have significance only for those PK-C substrates that require calcium activation of the enzyme. Thus the presence of DEHP in the high nanomolar concentration range alters the effective substrate specificity of PK-C.

Comparison of the effects of carbon tetrachloride and of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the disposition of linoleic acid in rat liver in vitro.

Both 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and carbon tetrachloride (CCl4) have conspicuous effects on lipid metabolism in rat liver. Although it is generally accepted that CCl4 administration leads to hepatic lipid peroxidation in vivo, conflicting reports from different laboratories make it unclear whether or not lipid peroxidation is involved in the mechanism of toxicity of TCDD. The present study involved pretreating F344 rats with CCl4 or TCDD, then at predetermined times thereafter, giving [U-14C]linoleic acid. A variety of compound classes were monitored in extracts of liver taken 30 min after the label was given. A previously unreported effect of CCl4 was a conspicuous increase in turnover of 1,2-diglycerides. That CCl4 did cause lipid peroxidation was evident from the presence of allylic hydroxyacids not seen in vehicle-treated controls, greatly increased radioactivity in protein-bound material, and decreased levels of arachidonate without decreased synthesis from linolate. Where effects of TCDD pretreatment could be seen, they were much less than the corresponding effects of CCl4. No allylic hydroxyacids were detected in livers of TCDD-treated rats. The concentration of arachidonate was not reduced, and elongation of linolate was not stimulated, indicating that TCDD did not cause extensive-but-repaired peroxidation. It is concluded that while TCDD may slightly increase hepatic lipid peroxidation in rats in vivo, the extent of such stimulation appears to be too slight to account for the toxicity of TCDD.

Rapid isolation of microsomes for studies of lipid peroxidation.

Conventional isolation of microsomes by high-speed centrifugation from isotonic sucrose requires exposure to air for several hours, leading to the formation of low levels of lipid peroxidation products. Sucrose interferes in protein and malondialdehyde assays and provides no protection against lipid peroxidation during workup. A new procedure for the purification of microsomes from rat liver substitutes mannitol (a hydroxyl radical scavenger) for sucrose and takes advantage of the properties of morpholinopropane sulfonic acid (MOPS) buffer and triethylenetetramine to provide protection against lipid peroxidation during the rapid (less than one hour) workup and subsequent low-temperature storage. The microsomal fractions prepared by the proposed method are free of detectable mitochondrial contamination and at least as pure overall as those prepared by the conventional method, but they have higher glucose-6-phosphatase and laurate hydroxylase activities and significantly less malondialdehyde than conventional microsomes at the time isolation is complete. Laurate hydroxylase activity is more stable during frozen storage in mannitol medium. The kinetics of lipid peroxidation in vitro are quite different for microsomes prepared by the two methods.

Beta-oxidation of 2-ethyl-5-carboxypentyl phthalate in rodent liver.

[7-14C]-2-Ethyl-5-carboxypentyl phthalate was isolated and purified from urine of rats given [7-14C]-di-(2-ethylhexyl) phthalate. This metabolite was shown to serve as a precursor for 2-ethyl-3-carboxypropyl phthalate in vivo. 2-Ethyl-5-carboxypentyl phthalate was oxidized to 2-ethyl-3-carboxypropyl phthalate in liver slices from control or, much more rapidly, from clofibrate-pretreated rats. Inhibition by KCN in liver slices from untreated rats, and strong inhibition by acrylate, suggested that formation of 2-ethyl-3-carboxypropyl phthalate involved mitochondrial beta-oxidation. The strong enhancement of the production of this compound by clofibrate (a very weak inducer for mitochondrial dehydrogenases), and strong inhibition by chlorpromazine suggested that peroxisomes may also be able to oxidize 2-ethyl-5-carboxypentyl phthalate. We were able to detect beta-oxidation of 2-ethyl-5-carboxypentyl phthalate to 2-ethyl-3-carboxypropyl phthalate using purified mitochondria, but strong phthalate monoester hydrolase activity observed during incubation of the former compound with purified peroxisomes made it impossible to determine whether 2-ethyl-3-carboxypropyl phthalate could be produced in the latter organelle or not. 2-Ethyl-5-carboxypentyl phthalate was such an inefficient substrate for beta-oxidation compared to palmitic acid that it is unlikely that it contributes significantly to the production of H2O2 in rats chronically exposed to di-(2-ethylhexyl) phthalate. Normal fatty acids are most likely to serve as the dominant substrates for peroxisomal beta-oxidase.

Application of the thiobarbiturate assay to the measurement of lipid peroxidation products in microsomes.

By applying two different thiobarbiturate assay procedures in parallel to aliquots of a microsomal incubation mixture one can simultaneously monitor free malondialdehyde and malondialdehyde plus labile lipid peroxidation products. The levels of malondialdehyde increase continuously during the incubation of microsomes, NADPH and ferrous-ADP complex, while the lipid precursors of MDA stop forming when the system becomes depleted in NADPH. In contrast to systems in which lipids are undergoing autooxidation, NADPH-dependent lipid peroxidation does not appear to generate significant amounts of water-soluble malondialdehyde precursors. As a result, quantitative interpretation of results is straightforward in the microsomal system. In spite of the lack of specificity of the thiobarbiturate coupling reaction, interferences can be easily compensated for by using zero time controls.

Generation of hydrogen peroxide by incidental metal ion-catalyzed autooxidation of glutathione.

Autooxidation of reduced glutathione in 50 mM buffer at pH 7.9 is indetectably slow in the presence of 1 mM DETAPAC, EDTA, TET, or tripyridine, but passing buffer through Chelex resin was insufficient to remove traces of catalytically active metals. Production of hydrogen peroxide during glutathione autooxidation was catalyzed by traces of Fe+2 or Cu+2, and to a much lesser extent by Cu+1 and Ni+2, but not to a detectable extent by Na+1, K+1, Fe+3, Al+3, Cd+2, Zn+2, Ca+2, Mg+2, Mn+2, or Hg+2. Cysteine was a much better precursor for hydrogen peroxide production than were cysteine sulfinic or sulfonic acids. The chelators EGTA, NTA, bipyridine, dimethyl glyoxime, salicylate, and Desferal were ineffective at preventing autooxidation. EDDA and 8-hydroxyquinoline were partially effective. Catalase could completely prevent the accumulation of detectable H2O2, but superoxide dismutase was only slightly inhibitory. Hydroxyl radical and singlet oxygen quenching agents (mannitol and histidine) stimulated. A mechanism for the production of H2O2 during trace metal catalyzed oxidation of glutathione is proposed, involving glutathione-complexed metal and dissolved oxygen. Although a radical intermediate can not be ruled out, no radical initiated chain reaction is necessary.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on lipid peroxidation in microsomal systems in vitro.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) when added to suspensions of rat hepatic microsomes in the presence of NADPH has little influence on the peroxidation of microsomal lipids unless the system also contains complexed ferric ion, in which case TCDD stimulates. This stimulation does not appear to require metabolism of the TCDD. Peroxidation was monitored by production of thiobarbiturate-reactive substances (malondialdehyde and dienals), production of conjugated dienes, and disappearance of polyunsaturated fatty acids. Stimulation of lipid peroxidation by TCDD in a mixed lysosome-microsome preparation resulted in significantly decreased 'leakage' of acid phosphatase into the medium, implying an effect on lysosomal membranes. Consideration both of the present results and data in the literature leads to the conclusion that it is premature to attempt to define the relationship between enzyme induction, lipid peroxidation and TCDD lethality.