Application of photoaffinity labeling with [(3)H] all trans- and 9-cis-retinoic acids for characterization of cellular retinoic acid--binding proteins I and II.

Cellular retinoic acid-binding proteins (CRABPs) are carrier proteins thought to play a crucial role in the transport and metabolism of all-trans-retinoic acid (atRA) and its derivatives within the cell. This report describes a novel photoaffinity-based binding assay involving competition between potential ligands of CRABP and [(3)H]atRA or [(3)H]-9-cis-RA for binding to the atRA-binding sites of CRABP I and II. Photoaffinity labeling of purified CRABPs with [(3)H]atRA was light- and concentration-dependent, saturable, and protected by several retinoids in a concentration-dependent manner, indicating that binding occurred in the CRABP atRA-binding site. Structure-function relationship studies demonstrated that oxidative changes to the atRA beta-ionone ring did not affect ligand potency. However, derivatives lacking a terminal carboxyl group and some cis isomers did not bind to CRABPs. These studies also identified two novel ligands for CRABPs: 5,6-epoxy-RA and retinoyl-beta-D-glucuronide (RAG). The labeling of both CRABPs with 9-cis-RA occurred with much lower affinity. Experimental evidence excluded nonspecific binding of RAG to CRABPs and UDP-glucuronosyltransferases, the enzymes responsible for RAG synthesis. These results established that RAG is an effective ligand of CRABPs. Therefore, photoaffinity labeling with [(3)H]atRA can be used to identify new ligands for CRABP and retinoid nuclear receptors and also provide information concerning the identity of amino acid(s) localized in the atRA-binding site of these proteins.

Direct interaction of all-trans-retinoic acid with protein kinase C (PKC). Implications for PKC signaling and cancer therapy.

Protein kinase C (PKC) regulates fundamental cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. All-trans-retinoic acid (atRA) modulates PKC activity, but the mechanism of this regulation is unknown. Amino acid alignments and crystal structure analysis of retinoic acid (RA)-binding proteins revealed a putative atRA-binding motif in PKC, suggesting existence of an atRA binding site on the PKC molecule. This was supported by photolabeling studies showing concentration- and UV-dependent photoincorporation of [(3)H]atRA into PKCalpha, which was effectively protected by 4-OH-atRA, 9-cis-RA, and atRA glucuronide, but not by retinol. Photoaffinity labeling demonstrated strong competition between atRA and phosphatidylserine (PS) for binding to PKCalpha, a slight competition with phorbol-12-myristate-13-acetate, and none with diacylglycerol, fatty acids, or Ca(2+). At pharmacological concentrations (10 micrometer), atRA decreased PKCalpha activity through the competition with PS but not phorbol-12-myristate-13-acetate, diacylglycerol, or Ca(2+). These results let us hypothesize that in vivo, pharmacological concentrations of atRA may hamper binding of PS to PKCalpha and prevent PKCalpha activation. Thus, this study provides the first evidence for direct binding of atRA to PKC isozymes and suggests the existence of a general mechanism for regulation of PKC activity during exposure to retinoids, as in retinoid-based cancer therapy.

4-hydroxyretinoic acid, a novel substrate for human liver microsomal UDP-glucuronosyltransferase(s) and recombinant UGT2B7.

It is suggested that formation of more polar metabolites of all-trans-retinoic acid (atRA) via oxidative pathways limits its biological activity. In this report, we investigated the biotransformation of oxidized products of atRA via glucuronidation. For this purpose, we synthesized 4-hydroxy-RA (4-OH-RA) in radioactive and nonradioactive form, 4-hydroxy-retinyl acetate (4-OH-RAc), and 5,6-epoxy-RA, all of which are major products of atRA oxidation. Glucuronidation of these retinoids by human liver microsomes and human recombinant UDP-glucuronosyltransferases (UGTs) was characterized and compared with the glucuronidation of atRA. The human liver microsomes glucuronidated 4-OH-RA and 4-OH-RAc with 6- and 3-fold higher activity than atRA, respectively. Analysis of the glucuronidation products showed that the hydroxyl-linked glucuronides of 4-OH-RA and 4-OH-RAc were the major products, as opposed to the formation of the carboxyl-linked glucuronide with atRA, 4-oxo-RA, and 5,6-epoxy-RA. We have also determined that human recombinant UGT2B7 can glucuronidate atRA, 4-OH-RA, and 4-OH-RAc with activities similar to those found in human liver microsomes. We therefore postulate that this human isoenzyme, which is expressed in human liver, kidney, and intestine, plays a key role in the biological fate of atRA. We also propose that atRA induces its own oxidative metabolism via a cytochrome P450 (CYP26) and is further biotransformed into glucuronides via UGT-mediated pathways.

Biotransformation of 13(Z)-retinoic acid in mouse skin and human keratinocyte cultures.

The metabolism of 13-(CIS) in mouse skin in vivo which was treated with TPA (or vehicle) typically showed that the retinoid was oxidized to 4-hydroxy, 5,6-epoxy-13-CIS, 5,8-oxy-13-CIS and undergoes geometric isomerization to RA. Applied 13-(CIS) in human keratinocyte cultures showed that the retinoid was oxidized to 5,6-epoxy-13-CIS, 5,8-oxy-13-CIS, and isomers. Pretreatment with the antioxidant butylated hydroxyanisole(BHA) resulted in a large decrease in formation of the oxirane and increased formation of the alcohol in mouse skin. Proposed mechanisms suggested the involvement of peroxyl radicals and prostaglandin H synthase for the biotransformation of retinoids.

Peroxidase-catalyzed oxidation of 2,4,6-trichlorophenol.

2,4,6-Trichlorophenol (TCP) is an environmental contaminant that is toxic, mutagenic, and carcinogenic. We have investigated peroxidase-catalyzed oxidation of TCP as an alternative pathway of TCP bioactivation using horseradish peroxidase (HRP) as a model peroxidase. TCP was shown to function as a reducing substarte for HRP as evidenced by TCP-dependent, HRP-catalyzed reduction of 5-phenyl-4-penten-1-yl hydroperoxide (PPHP) to its corresponding alcohol. In addition, TCP was shown to undergo hydroperoxide (H2O2, ethyl hydroperoxide, or PPHP)-dependent metabolism as evidenced by electronic absorption spectroscopic analysis of reaction mixtures. A single major product was detected by reverse phase HPLC and was identified as 2,6-dichloro-1,4-benzoquinone (2,6-dichloro-2, 5-cyclohexadiene-1,4-dione, CAS no. 697-91-6) on the basis of electronic absorption spectroscopy, mass spectrometry, and cochromatography with synthetic standard. In addition, HRP-catalyzed oxidation of TCP yielded EPR-detectable phenoxyl radical intermediates whose EPR spectrum consisted of a 1:2:1 triplet characterized by proton hyperfine coupling constants aH(3,5) = 2.35 gauss. Mechanisms for the hydroperoxide-dependent, HRP-catalyzed oxidation of TCP are proposed that are consistent with these results.

The acetaminophen regioisomer 3'-hydroxyacetanilide inhibits and covalently binds to cytochrome P450 2E1.

3'-Hydroxyacetanilide has been previously studied as a nontoxic regioisomer of the analgesic acetaminophen (4'-hydroxyacetanilide). The radiolabeled derivative has been shown to covalently bind to liver proteins at levels similar to that observed with hepatotoxic doses of radiolabeled acetaminophen with no evidence of hepatic damage. Using an anti-arylacetamide antiserum the primary protein adduct detected following administration of 3'-hydroxyacetanilide (300 and 600 mg/kg) to mice was a 50 kDa microsomal protein that co-migrated with cytochrome P450 2E1. Cytochrome P450 2E1 enzyme activity (p-nitrophenol hydroxylase) was decreased by 79% in the mice treated with 3'-hydroxyacetanilide (600 mg/kg). Incubation of 3'-hydroxyacetanilide with hepatic microsomes resulted in a time dependent 47% decrease in cytochrome P450 2E1 activity. Pre-incubation of acetaminophen with microsomes did not result in covalent binding to the cytochrome P450 nor was there a decrease in p-nitrophenol hydroxylase activity. These data suggest that 3'-hydroxyacetanilide covalently binds to cytochrome P450 2E1 with preferential loss of activity.

Retinoic acid-dependent stimulation of 2,2'-azobis(2-amidinopropane)-initiated autoxidation of linoleic acid in sodium dodecyl sulfate micelles: a novel prooxidant effect of retinoic acid.

(E)-Retinoic acid (RA) was shown to stimulate the rate of 2,2'-azobis(2-amidinopropane) (AAPH)-initiated autoxidation of linoleic acid (18:2) in sodium dodecyl sulfate (SDS) micelles. RA-dependent stimulation of 18:2 autoxidation was characterized by enhanced rates of dioxygen uptake which were linear with retinoid concentration. In contrast, 5,6-epoxy-RA, a major oxidation product of RA, failed to affect the rate of dioxygen consumption at all concentrations tested. RA was also shown to stimulate peroxyl radical-dependent oxidation of styrene to the corresponding oxirane when styrene was included in the micellar system as a molecular probe. Furthermore, unequivocal evidence of RA-dependent stimulation of 18:2 autoxidation was obtained by relative quantitation of 13-hydroxy-(9Z, 11E)-octadecadienoic acid (13-HODE) plus 9-hydroxy-(10E,12Z)-octadecadienoic acid (9-HODE) production. In addition, enhanced carbon-centered radical formation was demonstrated in the presence of RA by EPR spectroscopy using alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (4-POBN) as a spin trap. Analysis and quantitation of RA oxidation products indicated that RA was oxidized to one primary product, 5,6-epoxy-RA, which was identified on the basis of cochromatography with synthetic standard (in a reverse-phase HPLC system), electronic absorption spectroscopy, and positive chemical ionization mass spectrometry of the corresponding methyl ester. Other minor oxidation products were also detected but not characterized. In contrast, reaction mixtures devoid of 18:2 failed to demonstrate significant retinoid oxidation. Mechanisms are proposed to account for the prooxidant effects of RA in this system.

Synthesis and 32P-postlabeling/high-performance liquid chromatography separation of diastereomeric 1,N2-(1,3-propano)-2'-deoxyguanosine 3'-phosphate adducts formed from 4-hydroxy-2-nonenal.

4-Hydroxy-2-nonenal (HNE), a major electrophilic byproduct of lipid peroxidation, is mutagenic and cytotoxic. The two pairs of HNE-derived diastereomeric 1,N2-propanodeoxyguanosine 3'-monophosphate adducts were synthesized from reaction of HNE with 2'-deoxyguanosine 3'-monophosphate. After HPLC separation, these adducts were characterized by UV-visible absorption and negative ion electrospray ionization MS/MS analysis. To further characterize the structures, these adducts were dephosphorylated to the corresponding HNE-modified deoxyguanosine adducts and their HPLC retention times and UV spectra were compared with those of the synthetic standards prepared from reaction of HNE with 2'-deoxyguanosine. Separation of these adducts by 32P-postlabeling/HPLC was developed. Reaction of HNE with calf thymus DNA resulted in only one pair of diastereomeric adducts, with one adduct predominantly formed with a modification level of 1.2 +/- 0.5 adducts/10(7) nucleotides.

Regiospecificity of peroxyl radical addition to (E)-retinoic acid.

The regiochemistry of peroxyl radical addition to (E)-retinoic acid (RA) was investigated. Peroxyl radicals, generated by reaction of 13-hydroperoxy-(9Z,11E)-octadecadienoic acid with hydroxo(porphyrinato)iron(III) in Tween 20 micelles, were reacted with RA. The major, and virtually exclusive, RA oxidation product was 5,6-epoxy-RA which was identified on the basis of cochromatography with the synthetic synthetic oxirane (in a reverse phase HPLC system), electronic absorption spectroscopy, high-field 1H-NMR, and EI mass spectrometry. These results suggest that peroxyl radicals react with RA by regioselective addition to either C5 or C6 yielding an endocyclic tertiary allylic or tertiary carbon-centered radical adduct, respectively. Subsequent beta-elimination of an alkoxyl radical yields the oxirane. Computational studies were carried out in order to gain mechanistic insights into the observed regiospecificity of the peroxyl radical-dependent epoxidation reaction; molecular mechanics and semiempirical quantum mechanical calculations were carried out using Tripos force field parameters and AM1, respectively. The results suggest that the regiospecific epoxidation may be influenced by the 5,6-olefinic function behaving as a partially-isolated double bond as well as inherent allylic A1,2 strain in the substituted cyclohexene ring as a consequence of substitutions at C1 and C6. In addition, calculated heats of formation indicated preferential peroxyl radical addition to C5 versus C6; this may reflect differences in the geometries of sp2-orbitals containing the radical densities rather than resonance contributions by the highly conjugated polyene system.

Covalent binding and inhibition of cytochrome P4502E1 by trichloroethylene.

1. To investigate the effects of trichloroethylene on cytochrome P4502E1 (CYP2E1), an isozyme responsible for its metabolic activation, mice were treated with trichloroethylene and Western blot staining with both anti-dichloroacetyl and anti-CYP2E1 antisera detected a comigrating 50 kDa protein band. There was a dose-dependent increase in the intensity of the 50 kDa protein adduct stained immunochemically with anti-dichloroacetyl. 2. CYP2E1 enzyme activity was decreased from control levels in a dose-dependent manner in mice treated with 250-500 mg/kg TRI. 3. Microsomal incubations with trichloroethylene resulted in covalent binding to several proteins including a 50 kDa adduct, which is in contrast with the selective binding to the 50 kDa protein observed in vivo. 4. CYP2E1 enzyme activity levels were significantly decreased following microsomal incubation with NADPH and trichloroethylene, and additionally there was a time- and NADPH-dependent decrease in enzyme activity indicating that trichloroethylene is a mechanism-based inhibitor of CYP2E1.

Prostaglandin H synthase-catalyzed oxidation of all-trans- and 13-cis-retinoic acid to carbon-centered and peroxyl radical intermediates.

Due to the importance of all-trans-retinoic acid (RA) in the treatment of various dermatological conditions and the wide distribution of prostaglandin H synthase (PGHS) in tissues, we have further examined the mechanisms involved in the hydroperoxide-dependent cooxidation of RA and its isomer, 13-cis-retinoic acid ((13Z)-RA), by PGHS. Hydroperoxide-dependent, PGHS-catalyzed oxidation of RA and (13Z)-RA was shown to form free radical adducts, using electron spin resonance (ESR) spin trapping techniques and 5-phenyl-4-penten-1-yl hydroperoxide (PPHP) or 13-hydroperoxy-9-cis-11-trans-octadecadienoic acid (13-OOH-18:2) as hydroperoxide substrates. Utilization of the spin trap alpha-phenyl-N-tert-butylnitrone (PBN) resulted in the detection of (13Z)-RA-PBN and RA-PBN adducts whose spectra were characterized by hyperfine coupling constants of aH = 4.16/aN = 15.69 and aH = 3.01/aN =15.92, respectively. Identical experiments under anaerobic conditions were carried out using the spin trap 2-methyl-2-nitrosopropane (NtB) which yielded nitroxide adducts whose spectra were characterized by a triplet of doublets with values of aH = 3.49/aN = 15.84 for the (13Z)-RA adduct and aH = 3.49/aN = 15.88 for the RA adduct. These results are indicative of secondary carbon-centered radical formation. We also used (+)-benzo[a]pyrene 7(S),8(S)-dihydrodiol ((+)-BP-7,8-diol) as a peroxyl radical probe. The results demonstrated the formation of (+)-BP-7,8-diol-derived tetrols, with the trans-anti tetrol representing the major oxidation product in systems undergoing PPHP-dependent, PGHS-catalyzed oxidation of (13Z)-RA or RA. These results are consistent with the formation of peroxyl radicals in these systems. In all experiments, the (13Z)-RA isomer appeared to be a better substrate for the enzyme compared to the all-trans isomer. Collectively these results provide further evidence to support the previously proposed mechanism for retinoid oxidation by PGHS involving the intermediacy of C4 carbon-centered radicals which subsequently react with dioxygen, yielding retinoid-derived peroxyl radicals.

Mechanism of acetaminophen-induced hepatotoxicity: covalent binding versus oxidative stress.

The hepatotoxicity of acetaminophen is believed to be mediated by the reactive metabolite N-acetyl-p-benzoquinone imine; however, the mechanism by which this metabolite produces the toxicity is unknown. The metabolite, which is both an electrophile and an oxidizing agent, may covalently bind to critical proteins, or it may initiate oxidative damage. We have previously developed a Western blot assay for detection of acetaminophen covalently bound to protein and have reported the relationship between covalent binding and the development of hepatotoxicity. Recently, we developed a Western blot assay for protein aldehyde formation, which may occur via the reactive oxygen species, the hydroxyl radical. In this paper, we have compared covalent binding to protein aldehyde formation. Toxic doses of acetaminophen (400 mg/kg) were administered to mice, and the mice were subsequently killed at 0, 1, 2, 4, and 6 h. Since the oxidizing agent FeSO4 has been reported to potentiate lipid peroxidation when administered with acetaminophen, other mice received FeSO4 (100 mg/kg) plus acetaminophen. Compared to saline-treated control mice, acetaminophen treatment significantly increased serum alanine aminotransferase levels, an index of hepatotoxicity, at 4 and 6 h, but not at 1 or 2 h. Acetaminophen plus FeSO4 treatment of mice significantly increased serum alanine aminotransferase levels at 2, 4, and 6 h compared to controls. Levels of alanine aminotransferase in serum of acetaminophen plus ferrous sulfate-treated mice were higher at 4 and 6 h than those of acetaminophen-treated mice, but not significantly different. FeSO4 alone did not increase alanine aminotransferase levels. Western blot assays revealed that acetaminophen did not cause an increase in protein aldehydes over control at any time, nor did acetaminophen plus FeSO4; however, FeSO4 alone increased the intensity of staining of the immunoblot for protein aldehydes over control at all times after 0 time. Acetaminophen-protein adducts were detected in acetaminophen- and acetaminophen plus FeSO4-treated mice. In vitro experiments indicated that FeSO4 plus tert-butyl hydroperoxide in the presence of bovine serum albumin increased protein aldehyde formation. Inclusion of acetaminophen in the incubation mixture inhibited protein oxidation of bovine serum albumin in a concentration dependent manner. The data indicate that acetaminophen quenches protein oxidation, presumably by reacting with the hydroxyl radical. These data are consistent with the theory that acetaminophen covalent binding is the primary mechanism of toxicity and argue against a role for protein oxidation in acetaminophen hepatotoxicity.

Nonenzymatic reduction of tetrachloro-1, 4-benzoquinone by reduced nicotinamide adenine dinucleotide phosphate in an aqueous system.

NADPH was shown to reduce tetrachloro-1,4-benzoquinone (TCBQ) to tetrachloro-1,4-benzene diol which was identified on the basis of cochromatography with synthetic standard in a reverse phase HPLC system, electronic absorption spectroscopy, and mass spectrometry. Conversely, NADPH was shown to undergo TCBQ-dependent oxidation as evidenced by uv spectroscopic analysis. ESR spectroscopy demonstrated that NADPH-dependent reduction of TCBQ proceeds through the intermediacy of a semiquinone radical intermediate. In addition, molecular mechanics and molecular orbital calculations were carried out on TCBQ to obtain partial atomic charges and the results suggest that the first electron transfer from the reduced nicotinamide moiety to TCBQ occurs at a carbonyl carbon. Kinetic analysis indicated that the rate of NADPH oxidation is first order with respect to [TCBQ], second order with respect to [NADPH], and an Arrhenius plot yielded an activation energy of 26 kJ x mol (-1) for the rate limiting step. Mechanisms are proposed which are consistent with these results.

Free radical oxidation of (E)-retinoic acid by prostaglandin H synthase.

Cooxidative metabolism of all-trans (E)-retinoic acid (RA) by prostaglandin H synthase was investigated employing ram seminal vesicle microsomes (RSVM) or purified, RSVM-derived enzyme. RA was shown to undergo hydroperoxide [H2O2 or 5-phenyl-4-penten-1-yl hydroperoxide (PPHP)]- or arachidonic acid-dependent cooxidation by microsomal prostaglandin H (PGH) synthase as evidenced by UV spectroscopic analysis of reaction mixtures. Cooxidation of RA by microsomal or purified PGH synthase, using PPHP as substrate, was characterized by uptake of dioxygen which was first order with respect to enzyme concentration. Dioxygen uptake was inhibited by the peroxidase reducing substrate 2-methoxyphenol. In addition, O2 uptake was inhibited by the spin trap nitrosobenzene. ESR spin trapping studies, using alpha-phenyl-N-tert-butylnitrone (PBN) as the spin trap, demonstrated the formation of RA-PBN adducts, characterized by hyperfine coupling constants of alpha H = 3.2 G and alpha N = 15.8 G. Reverse phase HPLC analysis of reaction mixtures demonstrated the formation of 4-hydroxy-RA, 5,6-epoxy-RA, 4-oxo-RA, (13Z)-retinoic acid, and other geometric isomers which were identified on the basis of cochromatography with synthetic standards, UV spectroscopy, and/or mass spectrometry. Mechanisms are proposed for the hydroperoxide-dependent, PGH synthase-catalyzed oxidation of RA that are consistent with these results.

Peroxidase-catalyzed oxidation of pentachlorophenol.

Pentachlorophenol (PCP) was shown to function as a reducing substrate for horseradish peroxidase (HRP) and to stimulate the HRP-catalyzed reduction of 5-phenyl-4-penten-1-yl hydroperoxide (PPHP) to 5-phenyl-4-penten-1-ol. HRP catalyzed the hydroperoxide-dependent oxidation of PCP, using H2O2, PPHP, or ethyl hydroperoxide as substrates, as evidenced by UV spectroscopic and reverse phase HPLC analysis of reaction mixtures. The major oxidation product was tetrachloro-1,4-benzoquinone which was identified on the basis of electronic absorption spectroscopy, mass spectrometry, and cochromatography with authentic standard. HRP-catalyzed oxidation of PCP yielded relatively stable, ESR-detectable pentachlorophenoxyl radical intermediates whose ESR spectra consisted of a symmetrical single line without hyperfine structure. Substitution of natural abundance isotopically-labeled PCP with 13C-labeled PCP resulted in broadening of the ESR signal line width from 6.1 G to 13.5 G. ESR spin trapping studies, with alpha-(1-oxy-4-pyridyl)-N tert-butylnitrone (4-POBN) as the spin trap demonstrated identical spectra using natural abundance isotopically-labeled PCP versus 13C-labeled PCP, suggesting oxyl addition, rather than carbon-centered radical addition to 4-POBN. The computer simulation of the observed spectra is consistent with two distinct 4-POBN adducts, with relative abundances of approximately 3:1, and hyperfine coupling constants of alpha N = (14.61 G)/alpha H = 1.83 G and alpha N = (14.76 G)/alpha H = 5.21 G, respectively. Mechanisms for the hydroperoxide-dependent, HRP-catalyzed oxidation of PCP are presented that are consistent with these results.

Topology of the chloroperoxidase active site: regiospecificity of heme modification by phenylhydrazine and sodium azide.

Chloroperoxidase (CLP) from Caldariomyces fumago is rapidly and irreversibly inactivated by phenylhydrazine and H2O2 but not by H2O2 alone. Inactivation is characterized by a phenylhydrazine-to-CLP partition ratio of approximately 15, formation of trans-azobenzene, and formation of a sigma-bonded phenyl-iron heme complex with a characteristic absorption maximum of 472 nm. Anaerobic extraction of the heme complex from the protein, followed by exposure to dioxygen under acidic conditions, shifts the phenyl group from the iron to the porphyrin nitrogens and yields the four possible N-phenylprotoporphyrin IX regioisomers. Oxidation of the iron-phenyl complex within the intact protein by ferricyanide or high peroxide concentrations results in protein-directed phenyl migration to give exclusively the N-phenylprotoporphyrin IX regioisomers with the phenyl group on pyrrole rings A and C. CLP also catalyzes the H2O2-dependent oxidation of azide to the azidyl radical and is inactivated by azide in the presence of H2O2. Inactivation of CLP by azide and H2O2 results in loss of heme Soret absorbance and formation of delta-meso-azidoheme. These results suggest a topological model for the CLP active site and indicate that the tertiary structure of the enzyme permits substrates to interact with both the delta-meso heme edge and catalytic ferryl (FeIV = O) species, in agreement with the fact that CLP catalyzes both H2O2-dependent peroxidation and monooxygenation reactions.

Effects of ceruloplasmin on superoxide-dependent iron release from ferritin and lipid peroxidation.

Ceruloplasmin (CP) effectively inhibited superoxide and ferritin-dependent peroxidation of phospholipid liposomes, using xanthine oxidase or gamma irradiation of water as sources of superoxide. In addition, CP inhibited superoxide-dependent mobilization of iron from ferritin, suggesting that CP inhibited lipid peroxidation by decreasing the availability of iron from ferritin. CP also exhibited some superoxide scavenging activity as evidenced by its inhibition of superoxide-dependent cytochrome c reduction. However, superoxide scavenging by CP did not quantitatively account for its inhibitory effects on iron release. The effects of CP on iron-catalyzed lipid peroxidation in systems containing exogenously added ferrous iron was also investigated. CP exhibited prooxidant and antioxidant effects; CP stimulated at lower concentrations, reached a maximum, and inhibited at higher concentrations. However, the addition of apoferritin inhibited CP and Fe(II)-catalyzed lipid peroxidation at all concentrations of CP. In addition, CP catalyzed the incorporation of Fe(II) into apoferritin. Collectively these data suggest that CP inhibits superoxide and ferritin-dependent lipid peroxidation via its ability to incorporate reductively-mobilized iron into ferritin.

Effects of (+)-1,2-bis(3,5-dioxopiperazin-1-yl)propane (ADR-529) on iron-catalyzed lipid peroxidation.

ADR-529 [(+)-1,2-bis(3,5-dioxopiperazin-1-yl)propane], a nonpolar, cyclic analogue of EDTA, protects against anthracycline cardiotoxicity in vivo. The protective mechanism presumably involves chelation of iron by a hydrolysis product of ADR-529, thus preventing the formation of reactive iron/oxygen species which can damage membrane lipids. We investigated the effects of ADR-529 and its hydrolysis products (the tetraacid and the diacid diamide) on NADPH- and ADP-Fe(3+)-dependent lipid peroxidation of rat liver microsomes and liposomes in the presence of cytochrome P-450 reductase. Hydrolyzed ADR-529 products caused inhibition of lipid peroxidation when in excess of the iron concentration. However, no inhibition of lipid peroxidation was detected by similar concentrations of nonhydrolyzed ADR-529. Microsomes did not affect the inhibition of lipid peroxidation, suggesting that rat liver microsomes do not hydrolyze ADR-529. Similarly, the diacid diamide hydrolysis product of ADR-529 inhibited ferritin- and adriamycin-iron-dependent liposomal lipid peroxidation in a concentration-dependent manner. No correlation between partially reduced oxygen species (O2.- and .OH; as measured by electron spin resonance) and lipid peroxidation (as assayed by malondialdehyde formation) was observed, suggesting that liposomal lipid peroxidation was strictly an iron-dependent phenomenon. These results suggest that inhibition of lipid peroxidation by iron chelation may be related to the protective effects of ADR-529 on in vivo anthracycline toxicity.

Inhibition of liver microsomal lipid peroxidation by 13-cis-retinoic acid.

The effects of 13-cis-retinoic acid on iron/ascorbate-dependent lipid peroxidation were investigated with rat liver microsomes. 13-cis-retinoic acid effectively inhibited malondialdehyde generation and molecular oxygen consumption associated with lipid peroxidation. Under the conditions employed, inhibition was complete at concentrations as low as 25 microM and the IC50 was 10 microM. Evidence for concomitant retinoid oxidation by microsomal unsaturated fatty acid-derived peroxyl radicals was demonstrated by detection of several retinoid-derived metabolites, including 5,8-oxy-13-cis-retinoic acid, generated during lipid peroxidation. The data indicate that 13-cis-retinoic acid inhibits lipid peroxidation by scavenging lipid peroxyl radicals with its conjugated polyene system. Its antioxidant properties may contribute to the pharmacological activities of this and related retinoids.