Marta Dubin

Inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions by β-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.

Redox Cycling of β-Lapachone and Structural Analogues in Microsomal andCytosol Liver Preparations☆

Publisher Summary Quinones are widely distributed in nature and make up an important group of substrates for flavoenzymes. Lipophilic o-naphthoquinones possess antibacterial, antifungal, trypanocidal, and cytostatic effects. Among those quinones, β-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho [1,2b]pyran- 5,6-dione) isolated from the lapacho tree ( Tabebuia avellanedae ) has proved to be an effective cytostatic agent in different human tumor cells, such as murine leukemia, melanoma, hepatoma, human leukemia, colon carcinoma, lymphoma, and glioma, as well as epidermoid laryngeal, ovarian, breast, lung, and prostate cancer . On these grounds, β-lapachone is suggested for clinical use; its effects have often been described as apoptosis or necrosis, depending on target cells, time, and drug dose. In the presence of molecular oxygen, the semiquinone radical can transfer an electron and generate the O 2 - . Unlike most other cellular reductases, two-electron reduction of quinine can also be catalyzed by cytosolic and mitochondrial DT-diaphorase (DTD), quinone oxidoreductase, E.C. 1.6.99.2 (NQO1), directly to the hydroquinone. Observations with β-lapachone and related o-naphthoquinones indicate that the corresponding hydroquinones must be included in the second group in agreement with (1) the semiquinone spectrum, demonstrated by ESR spectroscopy; (2) semiquinone (or quinone) production, demonstrated by optical spectroscopy; and (3) the effect of dicoumarol on the quinone redox cycling and oxygen consumption by the NADPH/o-naphthoquinone/ DTD system. These reactions associated with DTD activity seem to rule out the contention proposing DTD as an antioxidant enzyme protecting against quinone toxicity.

Novel o-naphthoquinones induce apoptosis of EL-4 T lymphoma cells through the increase of reactive oxygen species.

Novel β-lapachone analogs 2-phenyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (NQ1), 2-p-tolyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (NQ3) and 2-methyl-2-phenyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (NQ7), which have trypanocidal activity, were assayed for cytotoxic effects on murine EL-4 T lymphoma cells. The NQs inhibited the proliferation of EL-4 cells at concentrations above 1μM. Nuclear staining of the EL-4 cells revealed chromatin condensation and a nuclear morphology compatible with the induction of apoptosis. Flow cytometry assays with annexin V-FITC and propidium iodide confirmed the cell death by apoptosis. Using electron paramagnetic resonance (EPR), a semiquinone radical was detected in EL-4 cells treated with NQs. In addition, a decrease in the GSH level in parallel with reactive oxygen species (ROS) production was observed. Preincubation with n-acetyl-l-cysteine (NAC) was able to reverse the inhibitory effects of the NQs on cell proliferation, indicating that ROS generation is involved in NQ-induced apoptosis. In addition, the NQs induced a decrease in the mitochondrial membrane potential and increased the proteolytic activation of caspases 9 and 3 and the cleavage of Poly (ADP-Ribose) Polymerase (PARP). In conclusion, these results indicate that redox cycling is induced by the NQs in the EL-4 cell line, with the generation of ROS and other free radicals that could inhibit cellular proliferation as a result of the induction of the intrinsic apoptosis pathway.

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.

Inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions by nitrofuran compounds.

(5-Nitro-2-furfurylidene)amino compounds bearing triazol-4-yl, benzimidazol-1-yl, pyrazol-1-yl, triazin-4-yl or related groups (a) stimulated superoxide anion radical generated by rat liver microsomes in the presence of NADPH and oxygen; (b) inhibited the NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (c) prevented the NADPH-dependent destruction of cytochrome P-450; (d) inhibited the NADPH-dependent microsomal aniline 4-hydroxylase activity; (e) failed to inhibit either the cumenyl hydroperoxide-dependent lipid peroxidation or the aniline-4-hydroxylase activity, except for the benzimidazol-1-yl and the substituted triazol-4-yl derivatives, which produced minor inhibitions. Reducing equivalents enhanced the benzimidazol-1-yl derivative inhibition of the cumenyl hydroperoxide-induced lipid peroxidation. The ESR spectrum of the benzimidazol-1-yl derivative, reduced anaerobically by NADPH-supplemented microsomes, showed characteristic spin couplings. Compounds bearing unsaturated nitrogen heterocycles were always more active than those bearing other groups, such as nifurtimox or nitrofurazone. The energy level of the lowest unoccupied molecular orbital was in fair agreement with the capability of nitrofurans for redox-cycling and related actions. It is concluded that nitrofuran inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions was mostly due to diversion of reducing equivalents from NADPH to dioxygen. Trapping of free radicals involved in propagating lipid peroxidation might contribute to the overall effect of the benzimidazol-1-yl and substituted triazol-4-yl derivatives.

Generation of Radical Anions of Nifurtimox and Related Nitrofuran Compounds By Ascorbate

Nifurtimox analogues bearing triazol-4-yl, benzimidazol-1-yl, triazin-4-yl or related groups as counterpart of the (5-nitro-2-furfurylidene) amino group were reduced to their nitro anion radicals by ascorbate in anaerobic solutions at high pH. The ESR spectra of the radical anions showed hyperfine spin couplings restricted to the nitrofuran moiety. With these compounds, the spin density at the nitro group was greater than with nifurtimox, nitrofurazone and nitrofurantoin. At neutral pH, solutions containing ascorbate and nitrofuran derivatives consumed oxygen, the compounds bearing unsaturated nitrogen heterocycles being the most effective. Superoxide dismutase and catalase decreased the rate of oxygen consumption, thus demonstrating the production of superoxide and hydrogen peroxide, respectively. NMR spectra of the triazol-4-yl and triazin-4-yl nitrofuran derivatives showed a deshielding effect for the azomethine proton, which was undetectable with nifurtimox and nitrofurazone.

PHENOLIC CONTENTS AND ANTIOXIDANT ACTIVITY IN CENTRAL-SOUTHERN URUGUAYAN PROPOLIS EXTRACTS

A study on the phenolic content and antioxidant properties of Uruguayan propolis is presented. Identification and quantification was carried out by Reverse Phase High Performance Liquid Chromatography (RP-HPLC). Antioxidant activity was determined by Antiradical Activity Determination (AAD) measured with DPPH and Microsomal Lipid Peroxidation (MLP) assays. Two phenolic acids (gentistic and p-coumaric) and eight flavonoids (fisetin, myricetin, luteolin, quercetin, kaempferol, pinocembrin, chrysin and tectochrysin) were identified. The existence of five other phenols is postulated. The large difference between range of values of scavenger activity AAD (between 60% and 100%) as compared with lipoperoxidation inhibition (between 15% and 92%), suggests that the complexity of the biologic matrix could modulate the antioxidant effect. From a commercial standpoint, research such as the one presented here endows the product with added value, making it more appealing to the interest of industry and entrepreneurs.

Effect of 5-nitroindole on adenylate energy charge, oxidative phosphorylation, and lipid peroxidation in rat hepatocytes

5-Nitroindole (NI), a mutagenic nitroarene, was assayed for cytotoxic effects on rat hepatocytes. After incubation with 25-100 microM NI, the adenylate energy charge of the hepatocytes decreased significantly as a result of the decrease in ATP and the increase in AMP. ATP depletion correlated well with the effects of NI on mitochondrial electron transfer and energy transduction in hepatocytes. Thus, NI (a) inhibited the antimycin-sensitive hepatocyte respiration; (b) inhibited NADH oxidation by disrupted hepatocyte mitochondria; (c) inhibited L-malate-L-glutamate oxidation by ADP-supplemented mitochondria; (d) in the absence of ADP, stimulated the same substrates and also succinate oxidation by mitochondria; (e) released the latent ATPase activity of mitochondrial F1F0-ATP synthase; (f) shifted the redox level of reduced cytochromes (c + c1) and b towards the oxidized state; (g) inhibited NADH oxidation by disrupted mitochondria in the vicinity of the NADH-dehydrogenase flavoprotein; (h) inhibited Ca2+ uptake by mitochondria using L-malate-L-glutamate as an energy source; (i) inhibited valinomycin-induced, endogenously energized K+ uptake, with little effect on the ATP-induced uptake; and (j) inhibited the MgATP-dependent contraction of Ca(2+)-swollen mitochondria. NI inhibited lipid peroxidation in hepatocytes and also in substrate-supplemented liver microsomes and mitochondria, thus ruling out hydroperoxides as a cause of NI cytotoxicity. Long-term incubation with NI produced loss of hepatocyte viability, as indicated by lactate dehydrogenase leakage.

Nitrofuran inhibition of microsomal lipid peroxidation

Two nitrofuran compounds, nifurtimox and nitrofurantoin, inhibited in a concentration‐dependent manner the NADPH‐, iron‐induced lipid peroxidation in rat liver microsomes, as shown by the decreased rate of MDA accumulation. Other nitro compounds (benznidazole and chloramphenicol) were relatively inactive. Nifurtimox inhibition affected polyenoic fatty acids and cytochrome P‐450 degradation that follows lipid peroxidation. The ascorbate‐ or tert‐butyl hydroperoxide‐dependent lipid peroxidations were much less inhibited than the NADPH‐dependent one. Nifurtimox and nitrofurantoin, but not benznidazole and chloramphenicol, strongly stimulated the microsomal NADPH‐oxidase activity, thus supporting electron diversion, as the main cause of the inhibition of peroxidation initiation.

The metabolism of 9-chloro-β-lapachone and its effects in isolated hepatocytes. The involvement of NAD(P)H:quinone oxidoreductase 1 (NQO1).

A β-lapachone analogue (3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione) (9-chloro β-lapachone), named CGQ, with antitumoral, antiviral and antitrypanocidal activities was assayed for cytotoxic effects on isolated rat hepatocytes. The incubation of hepatocytes with this o-naphthoquinone showed (a) decreased adenylate energy charge, as a result of a decrease in ATP, and an increase in AMP levels; (b) increased NADP(+) content, with a concomitant decrease of NADPH, NADH and NAD(+) content; (c) decreased GSH content, accompanied by an increase in GSSG formation; (d) stimulated oxygen uptake as well as increased superoxide anion production and hydrogen peroxide formation; (e) inhibited lipid peroxidation; (f) hepatocyte viability was not reduced unless the NQO1 inhibitor dicoumarol was present. We hypothesize that the cytotoxicity of CGQ in dicoumarol-treated hepatocytes was the result of inhibition of the NQO1 detoxification pathway, thus allowing more quinone to be metabolized towards the one-electron pathway to form reactive semiquinones and/or reactive oxygen species. The results obtained indicate a protective role of NQO1 in preventing CGQ cytotoxicity in isolated rat hepatocytes.