Apoptosis in the Initial Leaf of Etiolated Wheat Seedlings: Influence of the Antioxidant Ionol (BHT) and Peroxides

Apoptosis was observed in the initial leaf of 5-8-day-old etiolated wheat seedlings. A condensation of cytoplasm in apoptotic cells, formation of myelin-like structures, specific fragmentation of cytoplasm, appearance in vacuoles of specific vesicles containing subcellular organelles, condensation and margination of chromatin in the nucleus, and internucleosomal fragmentation of nuclear DNA are ultrastructural features of apoptosis in the initial wheat leaf. Single-membrane vesicles detected in vacuoles of the leaf cells resemble in appearance the vacuolar vesicles in the coleoptile apoptotic cells described earlier (Bakeeva, L. E., et al. (1999) FEBS Lett., 457, 122-125); they contain preferentially plastids but not mitochondria as was observed in coleoptile. The vacuolar vesicles are specific for the apoptotic plant cells. Thus, apoptosis in various tissues is an obligatory element of plant (wheat) growth and development even in the early stages of ontogenesis. Contrary to strong geroprotecting action in coleoptile, the known antioxidant BHT (ionol, 2.27·10^–4 M) does not prevent in the leaf cells the apoptotic internucleosomal DNA fragmentation and appearance of specific vacuolar vesicles containing subcellular organelles. Therefore, the antioxidant action on apoptosis in plants is tissue specific. Peroxides (H₂O₂, cumene hydroperoxide) stimulated apoptosis (internucleosomal DNA fragmentation) in coleoptile and induced it in an initial leaf when apoptosis in a control seedling leaf was not yet detected. Thus, apoptosis that is programmed in plant ontogenesis and controlled by reactive oxygen species (ROS) can be modulated by anti- and prooxidants.     

A new perspective on the role of CuZn superoxide dismutase (SOD1)

The CuZn superoxide dismutase (SOD1), a member of a group of isoenzymes involved in the scavenger of superoxide anions, is a dimeric carbohydrate free protein, mainly localized in the cytosol. The reactive oxygen species (ROS) are involved in many pathophysiological events correlated with mutagenesis, cancer, degenerative processes and aging. In the first part of this mini-review the well known role of SOD1 and ROS are briefly summarized. Following, a potential novel biological action that SOD1 could exert is described, based on the recent researches demonstrating the secretion of this enzyme in many cellular lines. Moreover, the role of impaired mutant SOD1 secretion, associated with cytoplasmic toxic inclusion, which occurs in familial amyotrophic lateral sclerosis (ALS), is summarized. In addition, a depolarization-dependent release of SOD1 in pituitary GH3 cells and in rat synaptosomes through a calcium and SNARE-dependent mechanism is reported.     

Interaction of Yeast Phosphoglyceromutase with Nucleotides

Moderate cell growth occurred after a long lag phase of about 100 hr when oxygen-sensitive hydrogen bacterium N34 was cultivated chemoautotrophically under 40% O₂. A decrease in cell growth or viable count was not observed during the lag phase. These cells grown under 40 % O₂ were oxygen-resistant because when used as inocula for fresh 40 % O₂-culture, the growth lag period was less than 10 hr. Nine oxygen-sensitive colonies developed from a single oxygen-sensitive cell respectively. When these colonies were inoculated into 40% O₂-culture, they showed an almost equal lag period and growth rate. These results suggest that cell growth in 40% O₂-culture inoculated with oxygen-sensitive strain N34 occurred not by selection of oxygen-resistant variants which might preexist but by adaptation of very oxygen-sensitive cells to high oxygen tension. Oxygen-resistance thus developed was maintained after successive subcultures under 10% O₂ for more than one year.     

Effect of exercise on bone and articular cartilage in heterozygous manganese superoxide dismutase (SOD2) deficient mice

Abstract

Reactive oxygen species (ROS) are involved in both bone and cartilage physiology and play an important role in the pathogenesis of osteoporosis and osteoarthritis. The present study investigated the effect of running exercise on bone and cartilage in heterozygous manganese superoxide dismutase (SOD2)-deficient mice. It was hypothesized that exercise might induce an increased production of ROS in these tissues. Heterozygous SOD2-deficient mice should exhibit an impaired capability to compensate, resulting in an increased oxidative stress in cartilage and bone. Thirteen female wild type and 20 SOD2^+/? mice (aged 16 weeks) were randomly assigned to a non-active wild type (SOD2^+/+Con, n = 7), a trained wild type (SOD2^+/+Run, n = 6), a non-active SOD2^+/? (SOD2^+/?Con, n = 9) and a trained SOD2^+/? (SOD2^+/?Run, n = 11) group. Training groups underwent running exercise on a treadmill for 8 weeks. In SOD2^+/? mice elevated levels of 15-F_2t-isoprostane and nitrotyrosine were detected in bone and articular cartilage compared to wild type littermates. In osteocytes the elevated levels of these molecules were found to be reduced after exercise while in chondrocytes they were increased by aerobic running exercise. The observed changes in oxidative and nitrosative stress did neither affect morphological, structural nor mechanical properties of both tissues. These results demonstrate that exercise might protect bone against oxidative stress in heterozygous SOD2-deficient mice.     

Synthesis and evaluation of N-substituted indole-3-carboxamide derivatives as inhibitors of lipid peroxidation and superoxide anion formation

The in vitro antioxidant effects of novel N-substituted indole-3-carboxamides (I3CDs) 1-10 on rat liver microsomal NADPH-dependent lipid peroxidation (LP) levels and their free radicals scavenging properties were determined by the inhibition of superoxide anion formation (SOD). Among the synthesized compounds, 4, 5, 8 and 9 significantly inhibited SOD with an inhibition range at 84–100% at 10^{? 3} M concentration. The presence of halo substituents both ortho- and para- positions of these compounds resulted 100% inhibition of SOD. Comparison the activity results of halogenated and non-halogenated derivatives suggested that the halogenated compounds are more active than the non-halogenated compounds. On the other hand, the introduction of a para fluoro benzyl in the 1-position of indole (compounds 7, 8) has more impact on the SOD inhibition when the benzamide ring was mono halogenated. However, none of other compounds had a significant inhibitory effects on the level of lipid peroxidation.     

Identification of superoxide dismutase isoenzymes in tobacco pollen

We investigated the possible existence of superoxide dismutase (SOD; EC 1.15.1.1) isoenzymes in the pollen of Nicotiana tabacum (Petit Havana SR-1 cultivar). To detect SOD activity, crude extracts from tobacco pollen were subjected to native polyacrylamide gel electrophoresis followed by staining with nitroblue tetra-zolium (NBT). The presence of six SOD isoenzymes was detected in tobacco pollen. Treatment with SOD inhibitors indicated the presence of one manganese SOD (Mn SOD), five copper-zinc SOD (Cu/Zn SOD) isoenzymes, and the absence of iron SOD (Fe SOD).     

Trace element concentrations and superoxide dismutase and catalase activities in benign and malignant larynx tumors

The plasma and erythrocyte levels of zinc, copper, and magnesium and the activities of red-cell copper-zinc superoxide dismutase (Cu/Zn-SOD) and catalase (CAT) were determined in patients with benign and malignant tumors of the larynx. Blood samples from patients and healthy controls were drawn using heparinized tubes. The erythrocyte Cu/Zn-SOD and CAT activities were determined spectrophotometrically and the zinc, copper, and magnesium concentrations were determined in erythrocyte and plasma by atomic absorption spectrometry. Variance analysis was employed in the statistical evaluation of the findings. There was a significant increase in red-cell Cu/Zn-SOD activity in the subjects with malignant and benign tumors compared to controls (p<0.001). The CAT activity increased only in the benign tumor group (p<0.01). The plasma zinc concentrations were significantly lower in the malignant tumor group (p<0.05) and significantly higher in the benign tumor group (p<0.01). The erythrocyte copper concentrations were significantly lower in both benign and malignant tumor groups (p<0.001). The plasma copper and magnesium and the erythrocyte magnesium concentrations did not show significant differences relative to controls (p>0.05). The increases in the activities of SOD and CAT activities and the changes in trace elements concentrations can indicate the presence of increased reactive oxygen species that might play a part in the pathogenesis larynx tumors. Presented at the IX Asian-Pacific Congress of Clinical Biochemistry, March 9–14, 2002, New Delhi, India.     

Triarylmethyl-based biradical as a superoxide probe

Abstract

Superoxide radical represents one of the most biologically relevant reactive oxygen species involved in numerous physiological and pathophysiological processes. Superoxide measurement through the decay of an electron paramagnetic resonance (EPR) signal of a triarylmethyl (TAM) radical possesses the advantage of a high selectivity and relatively high rate constant of TAM reaction with the superoxide. Hereby we report a straightforward synthesis and characterization of a TAM–TAM biradical showing a high reactivity with superoxide (second-order rate constant, (6.7?±?0.2)?×?10³ M^?1 s^?1) enabling the measurement of superoxide radical by following the increase of a sharp EPR signal associated with the formation of a TAM-quinone-methide monoradical product.     

Effect of ethylene producer ethrel and antioxidant ionol (BHT) on the proteolytic apparatus in coleoptiles of wheat seedlings during apoptosis

It was established that total proteolytic activity in etiolated wheat seedlings changes in ontogenesis in cycles: peaks of proteolytic activity correspond to the 3rd, 5th, and 8th days of seedling growth, respectively. The maximum of proteolytic activity preceded the maximum of nuclease activity, which may be due to activation of nucleases by proteolytic enzymes. According to inhibitory analysis the cysteine and serine proteases play the main role in apoptosis in wheat coleoptiles. Growing of seedlings in the presence of ethrel stimulated apoptosis in the coleoptile, and it increased (almost 6-fold) the proteolytic activity in its cells. On the other hand, the antioxidant ionol (BHT) suppressed the induction of proteases, particularly at the second stage of coleoptile development, and it slowed down the increase in the nuclease activity after 6th day of the seedling life. It is suggested that phytohormones and antioxidants participate in regulation of apoptosis in the ageing coleoptile, directly or indirectly effecting the proteolytic apparatus in the coleoptile cells.     

Subcutaneous administration of bovine superoxide dismutase protects lungs from radiation-induced lung injury

Abstract

Background. The objective of the present study was to determine whether single administration of the antioxidant enzyme bovine superoxide dismutase (bSOD) after radiation therapy (RT) mitigates development of pulmonary toxicity in rats. Methods. Female F344 rats (n = 60) were divided among six experimental groups: (1) RT, single dose of 21 Gy to the right hemithorax; (2) RT + 5 mg/kg bSOD; (3) RT + 15 mg/kg bSOD; (4) No RT; (5) sham RT + 5 mg/kg bSOD; and (6) sham RT + 15 mg/kg bSOD. A single subcutaneous injection of bSOD (5 or 15 mg/kg) was administered 24 h post-radiation. The effects of bSOD on radiation-induced lung injury were assessed by measurement of body weight, breathing frequency, and histopathological changes. Immunohistochemistry was used to evaluate oxidative stress (8-OHdG⁺, NOX4⁺, nitrotyrosine⁺, and 4HNE⁺ cells), macrophage activation (ED1⁺), and expression of profibrotic transforming growth factor-β or TGF-β in irradiated tissue. Results. Radiation led to an increase in all the evaluated parameters. Treatment with 15 mg/kg bSOD significantly decreased levels of all the evaluated parameters including tissue damage and breathing frequency starting 6 weeks post-radiation. Animals treated with 5 mg/kg bSOD trended toward a suppression of radiation-induced lung damage but did not reach statistical significance. Conclusions. The single application of bSOD (15 mg/kg) ameliorates radiation-induced lung injury through suppression of reactive oxygen species/reactive nitrogen species or ROS/RNS-dependent tissue damage.     

Radical-scavenging activity of butylated hydroxytoluene (BHT) and its metabolites

To clarify the radical-scavenging activity of butylated hydroxytoluene (BHT), a food additive, stoichiometric factors (n) and inhibition rate constants (k_inh) were determined for 2,6-di-tert-butyl-4-methylphenol (BHT) and its metabolites 2,6-di-tert-butyl-p-benzoquinone (BHT-Q), 3,5-di-tert-butyl-4-hydroxybenzaldehyde (BHA-CHO) and 3,5-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadiene-1-one (BHT-OOH). Values of n and k_inh were determined from differential scanning calorimetry (DSC) monitoring of the polymerization of methyl methacrylate (MMA) initiated by 2,2′-azobis(isobutyronitrile) (AIBN) or benzoyl peroxide (BPO) at 70 °C in the presence or absence of antioxidants (BHT-related compounds). The n values declined in the order BHT (1–2) > BHT-CHO, BHT-OOH (0.1–0.3) > BHT-Q (0). The n value for BHT with AIBN was approximately 1.0, suggesting dimerization of BHT. The k_inh values declined in the order BHT-Q ((3.5–4.6)×10⁴ M⁻¹ s⁻¹) > BHT-OOH (0.7–1.9×10⁴ M⁻¹ s⁻¹) > BHT-CHO ((0.4–1.7)×10⁴ M⁻¹ s⁻¹) > BHT ((0.1–0.2)×10⁴ M⁻¹ s⁻¹). The k_inh for metabolites was greater than that for the parent BHT. Growing MMA radicals initiated by BPO were suppressed much more efficiently by BHT or BHT-Q compared with those initiated by AIBN. BHT was effective as a chain-breaking antioxidant.     

Dissociation of superoxide production by mitochondrial complex I from NAD(P)H redox state

The relationship between the rate of superoxide production by complex I and NAD(P)H redox state was investigated in rat skeletal muscle mitochondria. A high rate of superoxide production was observed during succinate oxidation; the rate during pyruvate oxidation was over fourfold lower. However, the NAD(P)H pool was significantly less reduced during succinate oxidation than during pyruvate oxidation. We conclude that there is no unique relationship between superoxide production by complex I and the reduction state of the NAD(P)H pool. Our data suggest that less than 10% of the superoxide originates from the flavin site during reverse electron transport from succinate.     

Quantitative generation of singlet (1Δg) oxygen from acidified aqueous peroxynitrite produced by the reaction of nitric oxide and superoxide anion

Simple acidification of aqueous alkaline peroxynitrite quantitatively generates singlet (¹Δ_g) molecular oxygen, detected and quantitated spectroscopically (1270 nm). This observation provides a chemical basis for physiological cytotoxicity of ONOO^? generated in the diffusion - controlled reaction of cellular NO^? and O. The experiments consist of (i) chemical generation of ONOO^? from NO^? gas and KO₂ powder in alkaline aqueous solution; (ii) absorption spectral identification of ONOO^? in the near-UV with maximum at 302 nm; (iii) spectroscopic identification of ¹O₂ by its emission band at 1200–1340 nm with maximum at 1275 nm; and (iv) quantitation of ¹O₂ generated in ONOO^?/H⁺ reaction by comparison of the chemiluminescence intensity at 1270 nm with that from H₂O₂/OCl^? reaction that generates ¹O₂ with unit efficiency at alkaline pH. ¹O₂ was generated with unit efficiency with respect to ONOO^? concentration by the ONOO^?/H⁺ reaction.     

Time-related alterations of superoxide radical levels in diverse organs of bile duct-ligated rats

The time-related alterations of superoxide radical measured in vivo by employing an ultrasensitive fluorescent assay in the liver, intestine, kidney and brain of rats with experimentally induced obstructive jaundice was investigated. Eighteen rats were randomly divided into Group A, rats subjected to sham operation, and Group B, rats subjected to bile duct ligation (BDL). Three rats from each group were subsequently killed at different time points post-operatively (1, 5 and 10 days). As compared to sham-operated, BDL rats showed a gradual increase with time of superoxide radical in the intestine, liver, kidney and brain: for animals sacrificed on the 1^st, 5^th and 10^th day the increase was 45%, 50% and 96% in the liver, 76%, 81% and 118% in the intestine, 64%, 71% and 110% in the kidney and 76%, 95% and 142% in the brain, respectively. This study provides direct evidence of an early appearance of oxidative stress in diverse organs, implying a uniform systemic response to biliary obstruction and emphasizing the need of early bile flow restoration.     

Inhibition of cell growth by overexpression of manganese superoxide dismutase (MnSOD) in human pancreatic carcinoma

Manganese superoxide dismutase (MnSOD) levels have been found to be low in human pancreatic cancer [Pancreas26, (2003), 23] and human pancreatic cancer cell lines [Cancer Res.63, (2003), 1297] when compared to normal human pancreas. We hypothesized that stable overexpression of pancreatic cancer cells with MnSOD cDNA would alter the malignant phenotype. MIA PaCa-2 cells were stably transfected with a pcDNA3 plasmid containing sense human MnSOD cDNA or containing no MnSOD insert by using the lipofectAMINE method. G418-resistant colonies were isolated, grown and maintained. Overexpression of MnSOD was confirmed in two selected clones with a 2-4-fold increase in MnSOD immunoreactive protein. Compared with the parental and neo control cells, the MnSOD-overexpressing clones had decreased growth rates, growth in soft agar and plating efficiency in vitro, while in vivo, the MnSOD-overexpressing clones had slower growth in nude mice. These results suggest that MnSOD may be a tumor suppressor gene in human pancreatic cancer.     

Antioxidative enzymes and the Russian wheat aphid (Diuraphis noxia) resistance response in wheat (Triticum aestivum)

A crucial function of antioxidative enzymes is to remove excess reactive oxygen species (ROS), which can be toxic to plant cells. The effect of Russian wheat aphid (RWA), Diuraphis noxia (Mordvilko), infestation on the activities of antioxidative enzymes was investigated in the resistant (cv. Tugela DN) and the near-isogenic susceptible (cv. Tugela) wheat (Triticum aestivum L.). RWA infestation significantly induced the activity of superoxide dismutase, glutathione reductase and ascorbate peroxidase to higher levels in the resistant than in susceptible plants. These findings suggest the involvement of antioxidative enzymes in the RWA-wheat resistance response, which was accompanied by an early oxidative burst. The results are consistent with the role of ROS in the resistance response and the control of their levels to minimise toxic effects.