Janusz Z. Byczkowski

Hydrogen peroxide: A potent activator of dioxygenase activity of soybean lipoxygenase

Hydrogen peroxide, an ubiquitous biologically occurring peroxide, was found to stimulate the dioxygenase activity of soybean lipoxygenase at the physiologically attainable concentration. The increase in enzyme specific activity was directly proportional to hydrogen peroxide concentration up to 0.5 nM. A decrease in the stimulation of dioxygenase activity was observed at higher concentrations. At low enzyme concentration up to 28-fold stimulation was noted when the formation of lipid hydroperoxide was monitored spectrophotometrically. The stimulation was further confirmed by increased oxygen uptake. It is proposed that the mechanism for in vivo activation involves hydrogen peroxide.

Lipoxygenase-catalyzed epoxidation of benzo(a)pyrene-7,8-dihydrodiol

Metabolism of resolved radioactive stereoisomer, [14C](+)-benzo-(a)pyrene-trans-7,8-dihydrodiol by highly purified soybean lipoxygenase plus linoleic acid was investigated. Trans-anti-7,8,9,10-tetrahydrotetrol, the product of hydrolytic breakdown of ultimate mutagenic benzo(a)pyrene-anti-7,8-dihydrodiol,9,10-epoxide, was detected as a major metabolite. The epoxidation, depended on the enzyme concentration and was inhibited by nordihydroguaiaretic acid. This study provides evidence on the ability of lipoxygenase to catalyze the epoxidation of benzo(a)pyrene-7,8-dihydrodiol.

Vanadium-mediated lipid peroxidation in microsomes from human term placenta

Vanadium is considered an essential element present in living organisms in trace amounts but it is toxic when introduced in excessive doses to animals and humans. Vanadium compounds are extensively used in modern industry and occupational exposure to high doses of vanadium is quite common. In pregnant mice, vanadium accumulates preferentially in the placenta and to lower extent in fetal skeleton and mammary gland during exposure to radioactive vanadium. Accumulation of vanadium in fetoplacental unit may present threat to the fetus by interacting with enzymes and ion-transporting systems in membranes. It is also possible that accumulation of vanadium with its concomitant reduction to vanadyl may lead to lipid peroxidation, followed by damage to biological membranes, lysosomal enzymes release and destruction of placental tissue. To explore some of these possibilities the authors decided to examine whether vanadate can undergo redox cycling in microsomes from human term placenta (HTP) that can lead to lipid peroxidation.

Bioactivation of benzo(a)pyrene-7,8-dihydrodiol catalyzed by lipoxygenase purified from human term placenta and conceptal tissues

Bioactivation of 14C-benzo(a)pyrene-7,8-dihydrodiol catalyzed by lipoxygenase purified from human term placenta of nonsmoking women and intrauterine conceptal tissues (at 4 weeks of gestation) was investigated. Incubation of 14C-benzo(a)pyrene-7,8-dihydrodiol with 3 mM linoleic acid in the presence of lipoxygenase purified from either human term placenta or intrauterine conceptal tissues resulted in co-oxidation generating several soluble and protein-bound metabolites of benzo(a)pyrene-7,8-dihydrodiol. The co-oxidation was inhibited significantly by the specific lipoxygenase inhibitor, nordihydroguaiaretic acid. Substitution for the active enzyme in the reaction mixture with heat denatured enzyme resulted in almost complete abolition of benzo(a)pyrene-7,8-dihydrodiol co-oxidation. These results suggest that lipoxygenase in the placentas and intrauterine conceptal tissues is capable of metabolizing benzo(a)pyrene-7,8-dihydrodiol to several reactive metabolites and may represent one of the major xenobiotic metabolizing pathways of bioactivating chemicals in the intrauterine compartment.

Toxic effects of long-term intratracheal administration of vanadium pentoxide in rats

The toxic effects of vanadium pentoxide were investigated following chronic exposure. Rats were exposed intratracheally once a month (0.56 mg V2O5/kg) for 12 months. Body weight gain of exposed animals slowed down following the 10th treatment when compared to the corresponding controls. Lung weights were significantly greater than controls; however, other organ weights were not changed. Blood glucose of treated animals was slightly decreased whereas blood total cholesterol was reduced markedly. Thein vitro experiments were performed to explain the mechanism of chronic toxic effects. The results of these experiments confirmed that vanadium(V) undergoes one-electron redox cycling in rat lung biomembranes and that reoxidation of vanadium(IV) initiates lipid peroxidation under aerobic conditions. The lung is the primary target organ during the intratracheal exposure to V2O5. It is postulated that free-radical redox cycling of vanadium may be responsible for the observed pulmonary toxicity.

Inhibitory effects of vanadium pentoxide on respiration of rat liver mitochondria

The bioenergetic functions of liver mitochondria were studied following the acute and chronic exposure of rats to vanadium pentoxidevia respiratory tract. The mitochondrial respiration with glutamate or succinate as substrate was inhibited significantly when compared to the control animals. No inhibition was found with ascorbate. The same effects were observedin vitro. Vanadium (V) was responsible for these inhibitory effects. It is postulated that significant amounts of vanadate are accumulated in the intermembrane space of liver mitochondria of the exposed rats. The process of “detoxification” by reduction of vanadate in the tissue may be insufficient to prevent the deleterious action of this compound on liver mitochondria.

NADPH-dependent drug redox cycling and lipid peroxidation in microsomes from human term placenta.

1. NADPH-dependent iron and drug redox cycling, as well as lipid peroxidation process were investigated in microsomes isolated from human term placenta. 2. Paraquat and menadione were found to undergo redox cycling, catalyzed by NADPH:cytochrome P-450 reductase in placental microsomes. 3. The drug redox cycling was able to initiate microsomal lipid peroxidation in the presence of micromolar concentrations of iron and ethylenediaminetetraacetate (EDTA). 4. Superoxide was essential for the microsomal lipid peroxidation in the presence of iron and EDTA. 5. Drastic peroxidative conditions involving superoxide and prolonged incubation in the presence of iron were found to destroy flavin nucleotides, inhibit NADPH:cytochrome P-450 reductase and inhibit propagation step of lipid peroxidation. 6. Reactive oxo-complex formed between iron and superoxide is proposed as an ultimate species for the initiation of lipid peroxidation in microsomes from human term placenta as well as for the destruction of flavin nucleotides and inhibition of NADPH:cytochrome P-450 reductase as well as for impairment of promotion of lipid peroxidation under drastic peroxidative conditions.

Vanadium redox cycling, lipid peroxidation and co-oxygenation of benzo(a)pyrene-7,8-dihydrodiol

Mechanism of lipid peroxidation triggered by vanadium in human term placental microsomes was reinvestigated in vitro. Production of lipid peroxyl radicals was estimated from co-oxygenation of benzo(a)pyrene and benzo(a)pyrene-7,8-dihydrodiol. Vanadyl(IV), but not vanadate(V) caused a dose-dependent co-oxygenation. Vanadate(V) required the presence of reduced nicotinamide adenine dinucleotide phosphate to trigger co-oxygenation of benzo(a)pyrene-7,8-dihydrodiol. To determine the role of pre-formed lipid hydroperoxides, the results obtained with partially peroxidized linoleic acid were compared with those of fresh linoleate. Superoxide dismutase inhibited the co-oxygenation of reaction when fresh linoleic acid was used. To further characterize the role of superoxide anion-radical in the vanadium redox cycling, the increase of optical density of vanadate(V) dissolved in Tris buffer was measured at 328 nm during the addition of KO2. The rate of this reaction producing peroxy-vanadyl complex was decreased by superoxide dismutase, especially, in the presence of catalase. It is suggested that vanadium catalyzes two separate processes, both leading to enhanced lipid peroxidation: (i) initiation, dependent on superoxide and triggered by peroxy-vanadyl; (ii) propagation, dependent on pre-formed lipid hydroperoxide not sensitive to superoxide dismutase. It is postulated that the vanadium-triggered initiation of lipid peroxidation may be crucial for toxicity in organs with limited endogenous lipid peroxidation.

Some aspects of activation and inhibition of rat brain lipoxygenase.

1. Regulatory properties of lipoxygenase activity in rat brain cytosol were studied using linoleic acid (LA) as a substrate. 2. A change in the absorbance at 234 nm was biphasic when a mixture of LA and pre-formed hydroperoxide (LA-OOH) was incubated with freshly isolated native brain cytosol. Initially, a rapid depletion of LA-OOH was observed with a concomitant formation of LA-oxo compounds. This phase was followed by LA dioxygenation. 3. Both hydroperoxidase and dioxygenase activities of lipoxygenase were inhibited by micromolar concentrations of classic lipoxygenase inhibitors (phenidone, 5,8,11-eicosatriynoic acid and nordihydroguaiaretic acid). 4. The dioxygenase activity in dialysed cytsool was stimulated by nanomolar concentrations of H2O2 and micromolar concentrations of LA-OOH and it was inhibited by serotonin, dopamine and norepinephrine (IC50 25-43 microM).

Comparative study of respiratory chain inhibition by DDT and DDE in mammalian and plant mitochondria

The problem of DDT 1 as an environmental component that affects living organisms (for review see BYCZK0WSKI 1974 and 1976) wi11 be acotual for a long rime at least some 30 years. A few recent studies by BYCZKOWSKI (1976a) have showed some data suggestin~ that toxicity of DDT in mammals may be related to disruption of ener&~y conservation process and inhibition of electron transs in mitochondria. If has been also shown that DDT and DDE affected the eleetron-transfeF chain in chloroplasts (for review see BYCZKOWSKI 1974). However, es of these compounds on plant mitoohondria has been not established since now. The persistance of DDT in soil is well known ands ther~/ore it was of interest to investigate the efs oF this pesticide on mitochondria from germinating cereals seedlings.