Antineoplastic properties of Maharishi-4 against DMBA-induced mammary tumors in rats

Maharishi-4 (M-4), an ayurvedic food supplement, was tested for anticarcinogenic and anticancer properties against 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in rats. The 6% M-4-supplemented diet protected DMBA-induced carcinogenesis by reducing both tumor incidence and multiplicity during initiation and promotion phases. The control animals who developed tumors when supplemented with M-4 diet for four weeks showed tumor regression in 60% of cases. There was no significant difference in the food intake or weight gain in rats who were on M-4-supplemented diet compared to control group. Possible mechanisms of action of M-4 are discussed.     

In vitro inhibition of cytochrome P-450 reductases from pig liver microsomes by garlic extracts.

The activity of microsomal NADPH-cytochrome-P-450-reductase and NADH-cytochrome-b5-reductase are inhibited after the addition of an aqueous extract of a pharmaceutical preparation of garlic (Allium sativum, L.) to buffer-suspended microsomes. Incubation of garlic extract with isolated pig liver microsomes also decreases the activity of cytochrome P-450-dependent ethoxycoumarin deethylation. As measured by malondialdehyde release, the effects on the enzyme system are evidently not due to lipid peroxidation. No loss of cytochrome P-450 pigment is observed. Moreover, it could be shown that addition of garlic extract displays no protective effect on microsomal lipids when oxidation occurs spontaneously or is enforced by short-wave UV-irradiation. The above findings were reproduced after applying a HPLC-purified preparation of alliin to the incubation mixtures, suggesting that alliin is the active principle for the inhibitory effects observed in vitro.     

Effects of mansonones on lipid peroxidation, P450 monooxygenase activity, and superoxide anion generation by rat liver microsomes

Several structurally related ortho-naphthoquinones isolated from Mansonia altissima Chev (mansonones C, E and F) (a) inhibited NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (b) prevented NADPH-dependent cytochrome P450 destruction; (c) inhibited NADPH-supported aniline 4-hydroxylase activity; (d) inhibited Fe(III)ADP reduction by NADPH-supplemented microsomes; (e) stimulated superoxide anion generation by NADPH-supplemented microsomes; and (f) stimulated ascorbate oxidation. ESR investigation of ascorbate-reduced mansonone F demonstrated semiquinone formation. Mansonone C had a greater effect than mansonones E and F on NADPH-dependent lipid peroxidation, O2- production and ascorbate oxidation, whereas mansonone E was more effective than mansonones C and F on aniline 4-hydroxylase activity. Mansonones E and F did not inhibit hydroperoxide-dependent lipid peroxidation, cytochrome P450 destruction or microsomal aniline 4-hydroxylase activity. Mansonone C inhibited to a limited degree tert-butyl hydroperoxide-dependent lipid peroxidation, this inhibition being increased by NADPH. Mansonone A, a tetrahydro orthonapthoquinone derivative, was in all respects relatively less effective than mansonones C, E and F. It is postulated that mansonones C, E and F inhibited microsomal lipid peroxidation and cytochrome P450 catalyzed reactions by diverting reducing equivalents from NADPH to dioxygen, but mansonone C (including its reduced form) may also exert direct antioxidant activity.     

Antioxidant effects of albumin-bound sulfur in lipid peroxidation of rat liver microsomes.

The antioxidant potential of albumin-bound sulfur (SBA) was investigated in rat liver microsomes using lipid peroxidation systems in vitro. Sulfur bound to protein is a reduced metabolite which is produced from cystine by gamma-cystathionase. Lipid peroxidation was induced either chemically by ferrous ions and ascorbate or enzymatically by carbon tetrachloride or tert-butyl hydroperoxide as indicated by the increase in thiobarbituric acid reactive substances (TBA-RS) and oxygen consumption. Although the antioxidant effect of SBA was weak on the non-enzymatic lipid peroxidation system, the addition of SBA significantly inhibited TBS-RS formation and oxygen consumption compared with non-treated bovine serum alubumin (BSA) in a microsomal lipid peroxidation system induced enzymatically. The sulfur bound to albumin disappeared during incubation with liver microsomes. However, slight differences in the disappearance were observed depending on whether or not lipid peroxidation was induced in the enzymatic systems. In the CCl4-induced lipid peroxidation system, the cytochrome P-450 level was significantly decreased by the addition of SBA. Therefore, in cytochrome P-450 dependent lipid peroxidation system, the potential effects of sulfur bound to albumin are due to an inhibition of cytochrome P-450 rather than by the oxidation itself caused by radical trapping.     

Influence of prenylated and non-prenylated flavonoids on liver microsomal lipid peroxidation and oxidative injury in rat hepatocytes.

Prenylated chalcones from hops and beer were compared with non-prenylated flavonoids [chalconaringenin (CN), naringenin (NG), genistein (GS) and quercetin (QC)] for their ability to inhibit lipid peroxidation in rat liver microsomes. Chalcones with prenyl- or geranyl-groups (5 and 25 microM) were more effective inhibitors of microsomal lipid peroxidation than CN, NG or GS induced by Fe(2+)/ascorbate. Prenylated chalcones were effective inhibitors of microsomal lipid peroxidation induced by Fe(3+)-ADP/NADPH and by tert-butyl hydroperoxide (TBH) but to a lesser extent compared to the Fe(2+)/ascorbate system. An increase of prenyl substituents decreased antioxidant activity in the lipid peroxidation systems. Certain flavonoids behaved as prooxidants in the iron-dependent lipid peroxidation systems. For example, at 5 microM, NG enhanced iron/ascorbate-induced lipid peroxidation whereas CN, diprenylxanthohumol and tetrahydroxanthohumol enhanced Fe(3+)-ADP/NADPH-induced lipid peroxidation. None of the flavonoids (25 microM), except QC, inhibited NADPH cytochrome P450-reductase activity of rat liver microsomes, suggesting that the mechanism of inhibition of lipid peroxidation induced by Fe(3+)-ADP/NADPH is not due to inhibition of the reductase enzyme. Chalcones exhibiting antioxidant activity against TBH-induced lipid peroxidation such as xanthohumol and 5'-prenylxanthohumol, and NG, with no antioxidant property at 5 microM concentration protected cultured rat hepatocytes from TBH toxicity. Other antioxidants (desmethylxanthohumol and CN) in the TBH system were not cytoprotective. These results demonstrate the importance of prenyl groups in the antioxidant activity of hop chalcones in the various in vitro systems of lipid peroxidation. Furthermore, the antioxidant activity of the flavonoids has little or no bearing on their ability to protect rat hepatocytes from the toxic effects of TBH.     

Novel 6-hydroxychroman-2-carbonitrile inhibitors of membrane peroxidative injury

Novel 6-hydroxychroman-2-carbonitrile compounds have been synthesized, and their antiperoxidant activity against superoxide-dependent, iron-promoted mycocardial phospholipid peroxidation has been evaluated quantitatively. With few exceptions, these compounds afforded significant, concentration-dependent antiperoxidant protection to myocardial-membrane phospholipid at sub- to low-micromolar concentrations. Structure-activity correlation demonstrated that R1-, R2-, and R3-methyl groups in the aromatic ring enhanced antiperoxidant activity, whereas hydrophobic groups at either R4 or R5 of the pyran ring compromised antiperoxidant efficacy. The most efficacious antiperoxidant synthesized contained a catechol moiety at R4 and was some 10-fold more potent than alpha-tocopherol. None of the 6-hydroxychroman-2-carbonitrile antiperoxidants scavenged superoxide or inhibited the enzymatic superoxide generator, xanthine oxidase, at effective antiperoxidant concentrations. The ability of these compounds to interrupt the propagatory phase of an on-going peroxidation reaction indicated that they acted as antiperoxidants by trapping chain-carrying lipid peroxyl radicals. Since a number of the 6-hydroxychroman-2-carbonitriles were most potent antiperoxidants than a variety of known chain-breaking compounds, this new class of phenolic antioxidants may represent a novel approach to the design of therapeutics against diseases in which lipid peroxidation is a causative factor or in which lipid peroxidases serve as mediators.     

Andrographolide and kalmegh (Andrographis paniculata) extract: in vivo and in vitro effect on hepatic lipid peroxidation

Single or repeated (for 15 consecutive days) oral administration of kalmegh leaf extract (500 mg/kg) or its bitter principle, andrographolide (5 mg/kg), to adult male albino rats (b.wt. 125-150 g) produced no significant change in NADPH induced hepatic microsomal lipid peroxidation. Carbontetrachloride (5 ml/kg) induced hepatic microsomal lipid peroxidation was decreased when the rats were pretreated (for 4 hr), but only with a single dose and not with long term administration of kalmegh or andrographolide. In vitro carbontetrachloride (1 microliter) induced hepatic microsomal lipid peroxidation was completely normalized by kalmegh leaf extract (0.5 and 5.0 micrograms/mg protein) or andrographolide (0.5 and 5.0 micrograms/mg protein). At the higher concentration of carbontetrachloride (2 microliter), hepatic microsomal lipid peroxidation remained significantly increased (25 %) in the presence of andrographolide (0.5 microgram/mg protein), but not in the presence of kalmegh extract (0.5 microgram/protein). These results suggest that kalmegh leaf extract has more protective action on carbontetrachloride-induced hepatic toxicity than its bitter principle, andrographolide.     

Intervention in free radical mediated hepatotoxicity and lipid peroxidation by indole-3-carbinol

The cytoprotective effect of the natural dietary constituent indole-3-carbinol (I-3-C) on carbon tetrachloride (CCl4) mediated hepatotoxicity in mice was examined. I-3-C pretreatment by gavage 1 hr prior to intraperitoneal injection of CCl4 produced a 63% decrease in CCl4-mediated centrolobular necrosis and a related 60% decrease in plasma alanine aminotransferase activity (a marker of liver necrosis). Since the toxicological effects of CCl4 are mediated by radical species generated during reductive metabolism by cytochrome P-450, we examined the potential ability of I-3-C to scavenge reactive radicals. Three systems were used to evaluate the ability of I-3-C to intervene in free radical mediated lipid peroxidation. These systems consisted of the following: (1) phospholipid dissolved in chlorobenzene, with peroxidation initiated by the thermal and photo decomposition of azobisisobutyronitrile (AIBN); (2) sonicated phospholipid vesicles in phosphate buffer (pH 7.4), with peroxidation initiated by ferrous/ascorbate; and (3) mouse liver microsomes containing an NADPH-regenerating system, with peroxidation initiated with CCl4. Lipid peroxidation was measured in these three systems as thiobarbiturate-reacting material. In the AIBN and ferrous/ascorbate systems, I-3-C inhibited lipid peroxidation, with greater inhibition under conditions of low rates of free radical generation. I-3-C was not as effective an antioxidant as butylated hydroxytoluene (BHT) or tocopherol, but it inhibited peroxidation in a dose-response manner. I-3-C was most effective as a radical scavenger in the microsomal CCl4-initiated system by inhibiting lipid peroxidation in a dose-dependent fashion, with 50% inhibition at 35-40 microM I-3-C. This concentration is about one-third of the concentration of I-3-C achieved in liver after treatment of mice by gavage with 50 mg I-3-C/kg body weight. These data suggest that I-3-C may be a natural antioxidant in the human diet and, as such, may intervene in toxicological or carcinogenic processes that are mediated by radical mechanisms.     

Initiation of in vitro lipid peroxidation by N-hydroxynorcocaine and norcocaine nitroxide

Norcocaine nitroxide and N-hydroxynorcocaine were found to stimulate hepatic microsomal lipid peroxidation in vitro, as measured by spin-trapping techniques using the spin trap alpha-[4-pyridyl-1-oxide]-N-tert-butylnitrone. It was determined that either norcocaine nitroxide or N-hydroxynorcocaine markedly enhanced the rate of spin trapping of lipid peroxyl radicals when added to hepatic microsomal preparations. Glutathione, in the presence of dialyzed cytosol, inhibited the formation of lipid peroxyl spin-trapped adducts. This finding suggests that cytosolic glutathione-dependent enzymes perhaps including glutathione peroxidase play an important role in the prevention of norcocaine nitroxide-or N-hydroxynorcocaine-mediated lipid peroxidation.     

Protection of cardiac membrane phospholipid against oxidative injury by calcium antagonists

Calcium antagonists representative of the four major chemical classes were assessed for their abilities to prevent peroxidation of rat heart membrane lipids through xanthine oxidase-dependent, superoxide-driven, iron-promoted oxygen radical chemistry. The dihydropyridines nifedipine and nitrendipine did not affect peroxidation, even at a concentration (500 microM) approaching their solubility limit. The benzothiazepine diltiazem did protect the cardiac lipids against oxidative injury, but at high micromolar concentrations: 50% inhibition of peroxidation (antiperoxidant IC50) required 510 microM diltiazem. The phenylalkylamines verapamil and gallopamil (D-600) were likewise weak antiperoxidants (approximately 35% inhibition of peroxidation at 500 microM). In contrast, two other alkylamines, bepridil and prenylamine, were very effective membrane lipid protectants with respective antiperoxidant IC50 values of 55 and 75 microM. The diphenylpiperazines flunarizine (IC50 = 190 microM) and cinnarizine (IC50 = 180 microM) displayed moderate antiperoxidant activity. No Ca2+ antagonist inhibited xanthine oxidase under conditions whereby 10 microM allopurinol inhibited enzyme activity by 50%. The effects of the Ca2+ antagonist-antiperoxidants on the kinetics of cardiac membrane lipid peroxidation indicate that they inhibit peroxidation by intercepting oxy- and/or lipid free radical intermediates. These data raise the possibility that antiperoxidant action may contribute to the spectrum of pharmacologic and therapeutic activities of certain Ca2+ antagonists.     

A labile sulfur in trisulfide affects cytochrome P-450 dependent lipid peroxidation in rat liver microsomes

The effects of trisulfide derivatives were studied on cytochrome P-450-dependent lipid peroxidation using rat liver microsomal systems. Cytochrome P-450-dependent lipid peroxidation was induced by carbon tetrachloride or tert-butylhydroperoxide and was evident by an increase in thiobarbituric acid-reactive substances (TBA-RS) and oxygen consumption. In these cytochrome P-450-dependent lipid peroxidation systems, pretreatment of microsome with trisulfide derivatives (cystine trisulfide and thiocyclam) significantly inhibited TBA-RS formation and oxygen consumption compared with disulfide or thiol analogs (cystine, nereistoxin, or cysteine). The labile sulfur contained in trisulfide disappeared during incubation with liver microsomes. In the CCl₄-induced lipid peroxidation system, the cytochrome P-450 level and NAD(P)H-cytochrome P-450 reductase activity were significantly decreased by the addition of trisulfide derivatives. Therefore, in cytochrome P-450-dependent lipid peroxidation system, the potential effects of trisulfide appear to be mediated via enzyme inhibition. These results suggest that pretreatment of the trisulfide derivatives may affect the toxic function of exogenous xenobiotics or drugs, which are reduced by the liver enzyme cytochrome P-450 to radical species.     

The inhibition of lipid peroxidation by cinnarizine. Possible implications to its therapeutic and side-effects

Cinnarizine has antivasoconstrictor properties and improves red-cell deformability. Its major side-effects are the induction of extrapyramidal reactions. It is a calcium antagonist, but it was suggested that its effects may depend on other mechanisms, namely on antiperoxidant properties. We have studied these properties in different biological systems, intact red-cells included. The occurrence of lipid peroxidation was determined by the formation of 2-thiobarbituric acid reactive products. Cinnarizine was found to inhibit spontaneous lipid peroxidation in rat liver homogenates, copper-induced lipid peroxidation in human plasma and copper-induced and hydrogen peroxide-induced lipid peroxidation in human red-cells. In red-cells, the inhibition of lipid peroxidation is accompanied by the inhibition of hemolysis. Copper-induced red-cell lipid peroxidation is 85% inhibited by as little as 5 microM cinnarizine. The antioxidant activity of cinnarizine may contribute to explain some of the effects of this drug.     

Antioxidant and pro-oxidant properties of active rosemary constituents: carnosol and carnosic acid.

1. Carnosol and carnosic acid have been suggested to account for over 90% of the antioxidant properties of rosemary extract. 2. Purified carnosol and carnosic acid are powerful inhibitors of lipid peroxidation in microsomal and liposomal systems, more effective than propyl gallate. 3. Carnosol and carnosic acid are good scavengers of peroxyl radicals (CCl3O2.) generated by pulse radiolysis, with calculated rate constants of 1-3 x 10(6) M-1 s-1 and 2.7 x 10(7) M-1 s-1 respectively. 4. Carnosic acid reacted with HOCl in such a way as to protect the protein alpha 1-antiproteinase against inactivation. 5. Both carnosol and carnosic acid stimulated DNA damage in the bleomycin assay but they scavenged hydroxyl radicals in the deoxyribose assay. The calculated rate constants for reaction with .OH in the deoxyribose system for carnosol and carnosic acid were 8.7 x 10(10) M-1 s-1 and 5.9 x 10(10) M-1 s-1 respectively. 6. Carnosic acid appears to scavenge H2O2, but it could also act as a substrate for the peroxidase system. 7. Carnosic acid and carnosol reduce cytochrome c but with a rate constant significantly lower than that of O2(-.).     

N-acetylserotonin suppresses hepatic microsomal membrane rigidity associated with lipid peroxidation.

N-acetylserotonin, the immediate precursor of melatonin in the tryptophan metabolic pathway in the pineal gland, has been reported to be an antioxidant. The aim of this work was to test the effect of N-acetylserotonin in stabilizing biological membranes against oxidative stress. Hepatic microsomal membranes from male adult rats were incubated with N-acetylserotonin (0.001-3 mM) before inducing lipid peroxidation using FeCl(3), ADP and NADPH. Control experiments were done by incubating microsomal membranes with N-acetylserotonin in the absence of lipid peroxidation-inducing drugs. Membrane fluidity was assessed by fluorescence spectroscopy and malonaldehyde plus 4-hydroxyalkenals concentrations were measured to estimate the degree of lipid peroxidation. Free radicals induced by the combination of FeCl(3)+ADP+NADPH produced a significant decrease in the microsomal membrane fluidity, which was associated with an increase in the malonaldehyde plus 4-hydroxyalkenals levels. These changes were suppressed in a concentration-dependent manner when N-acetylserotonin was added in the incubation buffer. In the absence of lipid peroxidation, N-acetylserotonin (0.001-3 mM) did not change membrane fluidity nor malonaldehyde plus 4-hydroxyalkenals levels. These results suggest that the protective role of N-acetylserotonin in preserving optimal levels of fluidity of the biological membranes may be related to its ability to reduce lipid peroxidation.     

Stimulative effects of chelating agents, 2,2'-bipyridine and 1,10-phenanthroline, on lipid peroxidation in rat liver microsomes.

Well known lipid peroxidation inhibitors, 1,10-phenanthroline and 2,2'-bipyridine, stimulated microsomal NADPH- and ascorbic acid-dependent lipid peroxidation when low concentrations of these chelating agents were added to incubation mixture. The stimulatory effects of the chelating agents on lipid peroxidation were enhanced when ferrous ion was added together with the chelating agents to the mixture at a molar ratio of 1:1. Ethylenediaminetetraacetic acid (EDTA) had no stimulatory effect on lipid peroxidation. Ferrous ion-EDTA complex increased lipid peroxidation by only 20-30%, which was lower than that obtained by addition of the same concentration of ferrous ion alone. On the other hand, manganese and calcium ions, which are also inhibitors of lipid peroxidation, had no ability to stimulate lipid peroxidation even in the presence of extra ferrous ions. Changes in the lipid peroxidation by chelating agents affected the apparent activity of ethylmorphine N-demethylation.     

NADPH- and linoleic acid hydroperoxide-induced lipid peroxidation and destruction of cytochrome P-450 in hepatic microsomes

Temporal aspects of the effects of inhibitors on hepatic cytochrome P-450 destruction and lipid peroxidation induced by NADPH and linoleic acid hydroperoxide (LAHP) were compared. In the absence of added Fe2+, NADPH-induced lipid peroxidation in hepatic microsomes exhibited a slow phase followed by a fast phase. The addition of Fe2+ eliminated the slow phase, thus demonstrating that iron is a rate-limiting component in the reaction. EDTA, which complexes iron, and p-chloromercurobenzoate (pCMB), which inhibits NADPH-cytochrome P-450 reductase, inhibited both phases of the reaction. Catalase as well as scavengers of hydroxyl radical, inhibited NADPH-induced lipid peroxidation almost completely. GSH also inhibited the NADPH-dependent reaction but only when added at the beginning of the reaction. In contrast with NADPH-dependent lipid peroxidation, the autocatalytic reaction induced by LAHP was not biphasic, NADPH-dependent or iron-dependent, nor was it inhibited by hydroxyl radical scavengers, catalase or GSH. A synergistic effect on lipid peroxidation was observed when both NADPH and LAHP were added to microsomes. It is concluded that both the fast and slow phases of NADPH-dependent microsomal lipid peroxidation are catalyzed enzymatically and are dependent upon Fe2+, whereas LAHP-dependent lipid peroxidation is autocatalytic. Since the fast phase of enzymatic lipid peroxidation occurred during the fast phase of destruction of cytochrome P-450, it is postulated that iron made available from cytochrome P-450 is sufficient to promote optimal lipid peroxidation. Since catalase and hydroxyl radical scavengers inhibited NADPH-dependent but not LAHP-dependent lipid peroxidation, it is concluded that the hydroxyl radical derived from H2O2 is the initiating active-oxygen species in the enzymatic reaction but not in the autocatalytic reaction.     

DPPH and oxygen free radicals as pro-oxidant of biomolecules.

Numerous investigations exist about the alterations that oxygen free radicals can provoke on biomolecules; these modifications can be prevented and/or reversed by different antioxidants agents. On the other hand, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), a stable nitrogen synthetic radical, is used to evaluate the antioxidant capacity of medicinal herbal products; however, the structural changes that this radical provoke on the herbal active principles are not clear yet. In this work, we compared the redox reactivity of oxygen free radicals and DPPH radical on phospholipids and protein thiol groups present in rat liver microsomes. Cu2+/ascorbate was used as generator system of oxygen free radical and as antioxidant, an extract of Buddleja globosa's leaves. Cu2+/ascorbate provoked microsomal lipid peroxidation, microsomal thiols oxidation and oxygen consumption; all of these phenomena were inhibited by B. globosa extract. On the other hand, DPPH was bleached in different extension by the herbal extract and phosphatidyl choline; beside, DPPH decreased microsomal thiols content, but this phenomenon were not prevented by the herbal extract. Furthermore, DPPH did not induce oxygen consumption and neither modified the oxygen consumption induced by Cu2+/ascorbate. Distinct redox mechanisms may explain the differences between the reactivity of DPPH and oxygen free radicals on biomolecules, which is discussed.     

Nitroaryl-1,4-dihydropyridines as antioxidants against rat liver microsomes oxidation induced by iron/ascorbate, Nitrofurantoin and naphthalene

1,4-Dihydropyridines (DHPs) used in the treatment of cardiovascular diseases, are calcium channel antagonists and also antioxidant agents. These drugs are metabolized through cytochrome P₄₅₀ oxidative system, majority localized in the hepatic endoplasmic reticulum. Several lipophilic drugs generate oxidative stress to be metabolized by this cellular system. Thus, DHP antioxidant properties may prevent the oxidative stress associated with hepatic biotransformation of drugs.

In this work, we tested the antioxidant capacity of several synthetic nitro-phenyl-DHPs. These compounds (I–IV) inhibited the microsomal lipid peroxidation, UDPGT oxidative activation and microsomal thiols oxidation; all phenomena induced by Fe³⁺/ascorbate, a generator system of oxygen free radicals. As the same manner, these compounds inhibited the oxygen consumption induced by Cu²⁺/ascorbate in the absence of microsomes. Furthermore, compound III (2,6-dimethyl-4-(4-nitrophenyl)-1,4-dihydropyridin-3,5-ethyl-dicarboxylate) and compound V (N-ethyl-2,6-dimethyl-4-(4-nitrophenyl)-1,4-dihydropyridin-3,5-methyl-dicarboxylate) inhibited the microsomal lipid peroxidation induced by Nitrofurantoin and naphthalene in the presence of NADPH. Oxidative stress induced on endoplasmic reticulum may alter the biotransformation of drugs, so, modifying their plasmatic concentrations and therapeutic effects. When drugs which are activated by biotransformation are administered together with antioxidant drugs, such as DHPs, oxidative stress induced in situ may be prevented.     

The role of iron in 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced lipid peroxidation by rat liver microsomes

Treatment of female Sprague-Dawley rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) enhances hepatic lipid peroxidation, and the role of iron in TCDD-induced lipid peroxidation was examined. Ferrous and ferric ions, and the chelators adenosine diphosphate (ADP), ethylenediaminetetraacetic acid (EDTA) and desferrioxamine (DFX) were added to an in vitro microsomal lipid peroxidation system using microsomes from control and TCDD-treated animals. Both ferrous and ferric ions enhanced microsomal lipid peroxidation, with the greatest effect being produced by the combination of ferrous and ferric ions. The addition of ADP and EDTA produced modest increases in microsomal lipid peroxidation, suggesting that these chelators facilitated formation of reactive oxygen species by iron. The addition of DFX markedly inhibited microsomal lipid peroxidation, with greatest inhibition occurring with microsomes from TCD-treated animals. The results indicate that iron is involved in TCDD-induced lipid peroxidation.     

Inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions by beta-lapachone and related naphthoquinones

The lipophilic o-naphthoquinones beta-lapachone, 3,4-dihydro-2-methyl-2-ethyl-2H-naphtho[1,2b]pyran-5,6-dione (CG 8-935), 3,4-dihydro-2-methyl-2-phenyl-2H-naphtho[1,2b]pyran-5,6-dione (CG 9-442), and 3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione (CG 10-248) (a) inhibited NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (b) prevented NADPH-dependent cytochrome P-450 destruction; (c) inhibited microsomal aniline 4-hydroxylase, aminopyrine N-demethylase and 7-ethoxycoumarin deethylase; (d) did not inhibit the ascorbate- and tert-butyl hydroperoxide-dependent lipid peroxidation and the cumenyl hydroperoxide-linked aniline 4-hydroxylase reaction; and (e) stimulated NADPH oxidation, superoxide anion radical generation and Fe(III)ADP reduction by NADPH-supplemented microsomes. In the presence of ascorbate, the same o-naphthoquinones stimulated oxygen uptake and semiquinone formation, as detected by ESR measurements. The p-naphthoquinones alpha-lapachone and menadione were relatively less effective than the o-naphthoquinones. These observations support the hypothesis that, in the micromolar concentration range, o-naphthoquinones inhibit microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions, by diverting reducing equivalents from NADPH to dioxygen.