BISPHENOL A DOSE- AND TIME-DEPENDENTLY INDUCES OXIDATIVE STRESS IN RAT LIVER MITOCHONDRIA EX VIVO

Objective: The probable toxic effects of bisphenol A (BPA) on different physiological functions have been reported in animal models. The role of BPA in mitochondrial oxidative stress has not been reported till date. The present study is aimed to elucidate dose- and time-dependent oxidative stress generation by BPA, respectively, in rat liver mitochondria in ex vivo model. Methods: The incubation mixture of BPA-treated groups containing mitochondria, 50 mM potassium phosphate buffer (pH 7.4), and different concentrations of BPA (20–160 μM/ml) (dissolved in 12% DMSO) in a final volume of 1.0 ml was incubated at 37°C in incubator for different time durations (30 min–2 h). Whereas, the incubation mixture of control group contained DMSO (12%), mitochondria and 50 mM potassium phosphatebuffer (pH 7.4).’ will be replaced by ‘Whereas, the incubation mixture of control group contained the same constituents except BPA. Result: We have observed significant decrease in mitochondrial intactness incubated with BPA in dose- and time-dependent manner under bright field and confocal microscopic study compared to control. Further, we have observed a decrease in mitochondrial reduced glutathione (GSH) content and increase in lipid peroxidation and protein carbonylation levels in dose- and time-dependent manner in BPA-exposed mitochondria. We have found a significant increase in the activity of Mn-superoxide dismutase and decrease in the activities of GSH peroxidase, GSH reductase, pyruvate dehydrogenase, and other three enzymes of Kreb’s cycle dose and time dependently in BPA-exposed mitochondria. The results indicate that exposure to BPA leads to decrease in intactness of mitochondria and increase in oxidative stress in mitochondria isolated from rat liver in a dose- and time-dependent manner. Conclusion: It can be concluded that the incubation of mitochondria isolated from rat liver with BPA, caused oxidative stress-mediated damages in mitochondria in both dose- and time-dependent manners.

Sulfidogenic and metal reducing activities of Desulfuromonas genus bacteria under the influence of copper chloride

The selection of strains isolated from technogenically altered ecotopes and resistant to contamination, capable of metabolizing a wide range of pollutants is a task highly relevant for creation of new methods for environmental purification. Sulphur-reducing bacteria of the Desulfuromonas genus carry out dissimilatory reduction not only of S0 but also oxidized forms of metals. Intensity of anaerobic respiration of microorganisms in polluted environments is determined by level of their adaptation to stress factors, in particular, copper (II) compounds. The aim of this work was to investigate the influence of copper (II) chloride on H2S production by Desulfuromonas sp. strains isolated by us from Yavorivske Lake, to determine the efficiency of Cu2+ precipitation by hydrogen sulfide, to analyse the possibility of usage by bacteria of CuCl2 as an electron acceptor of anaerobic respiration and to study the influence of Cu2+ on usage by these microorganisms of ferric (III) citrate, potassium dichromate or manganese (IV) oxide as electron acceptors. Bacteria were grown under anaerobic conditions in Kravtsov-Sorokin medium. To study the influence of Cu2+ on production by bacteria of H2S, their cells were incubated with CuCl2 (0.5–4.0 mM), washed and cultivated in a medium with S0. To determine the level of Cu2+ binding by H2S, produced by bacteria, cells were grown in a medium with CuCl2 (0.5–4.0 mM) and S0. To investigate the ability of bacteria to use copper (II) ions as electron acceptors, they were cultivated in a medium with CuCl2 (1.74–10.41 mM). To study the influence of Cu2+ on usage by bacteria of metal compounds as electron acceptors, their cells were incubated with CuCl2 (0.5–4.0 mM), washed and cultivated in media with C6H5O7Fe, K2Cr2O7 or MnO2 (1.74–10.41 mM). Biomass was determined by the turbidimetric method. In the cultural liquid the content of H2S was determined quantitatively by the spectrophotometric method, qualitatively – presence of Cu2+. Content of CuS in the growth medium was determined by weight method. Desulfuromonas sp. bacteria was revealed to be resistant to 2.0–2.5 mM copper (II) ions. Under the influence of 3.0–4.0 mM CuCl2 in the incubation mixture, sulfidogenic activity of bacteria decreased more than twice. The efficiency of Cu2+ binding in form of CuS by H2S produced by bacteria reached 97.3–100.0% at presence in the medium with S0 of up to 1.5 mM CuCl2. Bacteria used CuCl2 (1.74–10.41 mM) as an electron acceptor in the process of anaerobic respiration. The addition of 2.5–3.0 mM CuCl2 to the incubation mixture caused inhibition of metal reducing activity of cells, growth of all strains in media with 1.74–10.41 mM ferric (III) citrate, potassium dichromate or manganese (IV) oxide as electron acceptors decreased by 2.6 times. Almost complete precipitation up to 1.5 mM copper (II) ions in form of CuS by H2S produced by bacteria and ability to reduce up to 10.41 mM CuCl2, C6H5O7Fe, K2Cr2O7 or MnO2 in the process of anaerobic respiration indicates a high adaptation of the bacteria strains investigated by us to stress factors, in particular, the influence of CuCl2. We have proved the possibility of using Desulfuromonas sp. in biotechnologies for purification of environments with complex contamination from copper (II) compounds.

Effect of seminal plasma zinc–binding proteins on motility and membrane integrity of canine spermatozoa stored at 5°C

Sperm surface binding sites for non-zinc-binding proteins (nZnBPs) and zinc-binding proteins (ZnBPs) were studied by the fluorescence technique with biotin-labelled proteins. The nZnBPs binding pattern was unspecific, no characteristic sites on plasmalemma were found. ZnBPs were attached mainly to the acrosomal region of sperm head and to the sperm flagellum. ZnBPs added to the incubation mixture of the canine spermatozoa allowed the preservation of higher values for total motility, progressive motility, curvilinear line velocity, straight line velocity, and beat cross frequency (P < 0.05), both at time 0 and after 1 h incubation at 5ºC. The addition of nZnBPs to the incubation mixture caused only weak positive effects when compared with control sample (PBS). A higher percentage of canine-ejaculated spermatozoa with intact membranes were observed when ejaculate was incubated with ZnBPs in comparison to control sample stored with PBS (P < 0.05) or nZnBPs (P < 0.05). Spermatozoa diluted with ZnBPs and nZnBPs exhibited a higher percentage of cells with active mitochondria when compared with control, both at time 0 and after 1 h; however, no statistical differences were observed. Our results emphasise the role of seminal plasma protein in securing the correct quantity and availability of zinc ions as a component regulating the motility of canine spermatozoa. The protective effect of ZnBPs against the cooling effect may be due to their ability of preventing sperm membrane damage.

The role of malate in the synthesis of glutamate in Pisum arvense roots

The in vivo and in vitro activities of NADH-dependent glutamate synthase in excised <em>Pisum arvense</em> roots increased several-fold under the influence of malate while pyruvate oxaloacctate. citrate and succinate inhibited this entyme. The plastids isolated from <em>Pisum arvense</em> root,. ahen incubated with glutamine and α-ketoglutarate, released glutamate into the medium Malate clearly stimulated this process. Albizziin (25 mM) completely reduced the presence of glutamate in the incubation mixture. These results indicate that reduced pyridine nucleotides arising in <em>P. arvense</em> root plastids during oxidation of malic acid may constitute the indispensable source of electrons for glutamic acid synthesis.

Sources of reducing equivalents for nitrite reductase in Pisum arvense roots

Glucose-6-phosphate and NADP+ as well as malic acid and NADP⁺ present in the incubation mixture enhanced nitrite reductase (EC 1.6.6.4) activity in <em>Pisum arvense</em> roots. This was manifested by a depression of the nitrite level in the tissues and an increased reduction of nitrites by plastids isolated from <em>P. arvense</em> roots. A marked stimulation of plastid malate dehydrogenase was also observed under the influence of nitrates present in the medium. These results suggest that pyridin nucleotides utilised by NiR during nitrite reduction may be formed not only in processes of glucose-6-phosphate oxidation in the pentosephosphate cycle, but also of malic acid by NADP⁺-dependent malate dehydrogenase.

Haloalkane Dehalogenase LinB Is Responsible for β- and δ-Hexachlorocyclohexane Transformation in Sphingobium indicum B90A

ABSTRACT Incubation of resting cells of Sphingobium indicum B90A, Sphingobium japonicum UT26, and Sphingobium francense Sp+ showed that they were able to transform β- and δ-hexachlorocyclohexane (β- and δ-HCH, respectively), the most recalcitrant hexachlorocyclohexane isomers, to pentachlorocyclohexanols, but only resting cells of strain B90A could further transform the pentachlorocyclohexanol intermediates to the corresponding tetrachlorocyclohexanediols. Moreover, experiments with resting cells of Escherichia coli expressing the LinB proteins of strains B90A, UT26, and Sp+ indicated that LinB was responsible for these transformations. Purified LinB proteins from all three strains also effected the formation of the respective pentachlorocyclohexanols. Although the three LinB enzymes differ only marginally with respect to amino acid sequence, they showed interesting differences with respect to substrate specificity. When LinB from strain B90A was incubated with β- and δ-HCH, the pentachlorocyclohexanol products were further transformed and eventually disappeared from the incubation mixtures. In contrast, the LinB proteins from strains UT26 and Sp+ could not catalyze transformation of the pentachlorocyclohexanols, and these products accumulated in the incubation mixture. A mutant of strain Sp+ lacking linA and linB did not degrade any of the HCH isomers, including β-HCH, and complementation of this mutant by linB from strain B90A restored the ability to degrade β- and δ-HCH.

Accelerated Denaturation of Hemoglobin and the Antimalarial Action of Chloroquine

ABSTRACT To study the antimalarial action of chloroquine, normal mouse erythrocytes were used as surrogates for erythrocytoid bodies. These bodies form in the endosomes of intraerythrocytic malaria parasites as they feed on their host and consist of erythrocyte cytoplasm enclosed in a vestige of the erythrocyte membrane. In suspensions of normal erythrocytes or lysates (equivalent to 5 μl of erythrocytes per ml in each case), hemoglobin underwent denaturation when it was incubated at 38°C in 150 mM sodium acetate (pH 5). It is reasonable to assume that the same phenomenon occurs in acidic endosomes. Addition of 100 μM chloroquine to the incubation mixture caused the rate of hemoglobin denaturation to double to 40 nanomoles per hour per ml of packed erythrocytes. This effect required the presence of erythrocyte stroma and was inhibited by reducing the temperature to 24°C or increasing the pH to 6. We propose that the primary antimalarial action of chloroquine is to bind to ferriprotoporphyrin IX (FP) and remove it from oxidized hemoglobin, thus producing toxic FP-chloroquine complexes and an excess of denatured globin. Furthermore, we suggest that these substances inhibit endosomal maturation and thereby cause hemoglobin accumulation in immature endosomes and masking of the lipids needed for FP dimerization. The term “masking” is used to signify that unsaturated lipids are present in parasitized erythrocytes but are specifically unavailable to promote FP dimerization.

Possibility of the Nonenzymatic Conversion of 6-(1'-Oxo-2'-hydroxypropyl)- Tetrahydropterin to Sepiapterin

Tetrahydrobiopterin (BH4) is a cofactor for aromatic amino acid hydroxylases and nitric oxide synthase. The biosynthetic pathway of BH4 from 6-pyruvoyl-tetrahydropterin (PPH4) includes two reduction steps catalyzed by sepiapterin rcductasc (SPR). PP1I4 is reduced to 6-( 1 '-oxo-2'-hydroxypiOpyl)-tetrahydropterin (l'-OXPH4 ) or 6- ( r-hydroxy-2'-oxopropyl)-tetrahydropterin (2'-OXPH4), which is further converted to BH4. However, patients with SPR deficiency show normal urinary excretion of pterins without hyperphenylalaninemia, suggesting the existence of another BH4 biosynthetic pathway, which is not concerned with SPR in humans. Blau et al. proposed a BH4 biosynthetic salvage pathway containing nonenzymatic conversion of l'-OXPH4 to sepiapterin. In this study, the possibility of the nonenzymatic conversion of l'-OXPH4 to sepiapterin was examined by using silkworm carbonyl reductase (CR 1) and human monomeric carbonyl reductase. Since l'-OXPH4 has been suggested to be nonenzymatically converted into sepiapterin, in an incubation mixture containing PPH4 and silkworm CR I, sepiapterin was determined by its derivative as biopterin. No sepiapterin was detected in the incubation mixture when the mixture, containing 800 pmol of l'-OXPH4, was further incubated at 37°C for 2 h in darkness. This result suggests that the rate of nonenzymatic formation of sepiapterin from l'-OXPH4 is quite low. The findings obtained here indicate that the proposed pathway in which a nonenzymatic conversion of l'-OXPH4 to sepiapterin occurs may be difficult or unlikely to proceed in humans.