Ayodele O. Kolawole

Insecticides and bio-insecticides modulate the glutathione-related antioxidant defense system of cowpea storage bruchid (Callosobruchus maculatus).

The possible cellular involvements of cowpea storage bruchid (Callosobruchus maculatus (Fab.) [Coleoptera: Chrysomelidae]) glutathione and its related enzymes system in the cellular defense against insecticides (Cypermethrin and λ-cyhalothrin) and bio-insecticides (ethanolic extract of Tithonia diversifolia, Cyperus rotundus, Hyptis suavolens leaves, and Jatropha curcas seed) were investigated. The results showed that the effect of insecticides and bio-insecticides on the C. maculatus is a function of oxidative and nitrosative stresses generated in vivo. A significant (p < 0.05) increase in carbonyl protein (CP) and lipid peroxidation (LPO) contents in bio-insecticides and insecticides exposed groups compared to the control indicates the extent of vital organs damage. These stresses caused similar and significant increase of glutathione peroxidase and glutathione synthetase in response to insecticides and bio-insecticide exposure in a dose-dependent manner. There was no post-translational modification of glutathione transferases expression induced. The alterations of the insect glutathione-dependent antioxidant enzyme activities reflect the presence of a functional defense mechanism against the oxidative and nitrosative stress and are related firmly to the glutathione demands and metabolism but appear inadequate by the significant reduction in glutathione reductase (GR) activity to prevent the damages. Exogenous application of reduced glutathione (GSH), to complement the in vivo demand, could not protect against the onslaught.

Some physicochemical properties of two major soluble hepatic glutathione transferases of tilapia (Tilapia zilli)

Two distinct glutathione transferases from the liver of adult Tilapia zilli were identified and purified to apparent homogeneity by ion-exchange chromatography on DEAE-cellulose and by gel filtration on Sephadex G-150. These major GST forms labeled tzGST1 and tzGST2 accounted for approximately 42% of the activity detectable with 1-chloro-2,4-dinitrobenzene (CDNB) as a typical electrophilic substrate. Apparent subunit and molecular mass values, substrate specificities and sensitivity to inhibitors as well as kinetic studies were used to differentiate the GST forms. SDS/PAGE indicated subunit molecular masses of 22.0 kDa (tzGST1) and 26.1 kDa (tzGST2) while native molecular weight by gel-filtration on sephadex G-100 indicated native molecular masses of 46.8 kDa and 48.0 kDa for tzGST1 and tzGST2 respectively. They appeared to be homodimers. Inhibition studies showed that tzGST1 was more sensitive to ethacrynic acid (EA), hematin, tributyltinacetate (TBTA), triethyltinbromide (TETB), and triphenyltinchloride (TPTC) than tzGST2 with TPTC being the most potent inhibitor. T. zilli GSTs could conjugate CDNB, DCNB, ρ-NBC, and EA with GSH but displayed no observable conjugating activity with NBDCl. The K(m) and V(max) for tzGST1 and tzGST2 with CDNB were 0.56 ± 0.05 mM; 0.24 ± 0.03 μmol/min/ml and 0.91 ± 0.07 mM; 0.14 ± 0.05 μmol/min/ml respectively while K(m) and V(max) with GSH were 0.46 ± 0.02 mM; 0.19 ± 0.20 μmol/min/ml and 1.32 ± 0.15 mM; 0.21 ± 0.07 μmol/min/ml respectively. Denaturation and renaturation studies with guanidine hydrochloride (Gdn-HCl) revealed that concentration of 4.0 M Gdn-HCl completely denatured tzGST1 and the possible isoenzyme was able to renature to 92% of the original activity. The renaturation process was dependent on temperature. The outcome of this study indicated that tzGSTs are possible GST isoenzymes actively present and involve in the detoxification process in the liver of tilapia when the subject is exposed to chemical toxins. The wide range of chemical toxins encountered in the polluted environment may have directed the selection of multiple tilapia GST isoforms with broad substrate specificity via gene duplication. Consequently, tzGST1 has a better chemical toxin bio-transforming capacity than tzGST2 due to its higher affinity for its substrates--a form of adaption to the polluted environment.

Interaction of Aldehyde dehydrogenase with acetaminophen as examined by spectroscopies and molecular docking

The interaction of acetaminophen, a non-substrate anionic ligand, with Aldehyde Dehydrogenase was studied by fluorescence, UV–Vis absorption, and circular dichroism spectroscopies under simulated physiological conditions. The fluorescence spectra and data generated showed that acetaminophen binding to ALDH is purely dynamic quenching mechanism. The acetaminophen-ALDH is kinetically rapid reversible interaction with a binding constant, Ka, of 4.91×103 L mol−1. There was an existence of second binding site of ALDH for acetaminophen at saturating acetaminophen concentration. The binding sites were non-cooperative. The thermodynamic parameters obtained suggest that Van der Waal force and hydrogen bonding played a major role in the binding of acetaminophen to ALDH. The interaction caused perturbation of the ALDH structures with an obvious reduction in the α-helix. The binding distance of 4.43 nm was obtained between Acetaminophen and ALDH. Using Ficoll 400 as macro-viscosogen and glycerol as micro-viscosogen, Stoke-Einstein empirical plot demonstrated that acetaminophen-ALDH binding was diffusion controlled. Molecular docking showed the participation of some amino acids in the complex formation with −5.3 kcal binding energy. With these, ALDH might not an excipient detoxifier of acetaminophen but could be involved in its pegylation/encapsulation.

Activity of the Antioxidant Defense System in a Typical Bioinsecticide- and Synthetic Insecticide-treated Cowpea Storage Beetle Callosobrochus maculatus F. (Coleoptera: Chrysomelidae)

The non-enzymatic and enzymatic antioxidant defense systems play a major role in detoxification of pro-oxidant endobiotics and xenobiotics. The possible involvement of beetle non-enzymatic [α-tocopherol, glutathione (GSH), and ascorbic acid] and enzymatic [catalase (CAT), superoxide dismutase (SOD), peroxidase (POX), and polyphenol oxidase (PPO)] antioxidant defense system on the insecticidal activity of synthetic insecticides (cypermethrin, 2,2-dicholorovinyl dimethyl phosphate, and λ-cyhalothrin) and ethanolic plant extracts of Tithonia diversifolia, Cyperus rotundus, Hyptis suaveolens leaves, and Jatropha Curcas seeds was investigated. 2,2-Dicholorovinyl dimethyl phosphate (DDVP; 200 ppm, LC50 = 13.24 ppm) and T. diversifolia (20,000 ppm) resulted in 100% beetle mortality at 96-hour post-treatment. The post-treatments significantly increased the beetle α-tocopherol and GSH contents. Activities of CAT, SOD, POX, and PPO were modulated by the synthetic insecticides and bioinsecticides to diminish the adverse effect of the chemical stresses. Quantitative and qualitative allelochemical compositions of bioinsecticides and chemical structure of synthetic insecticides possibly account and for modulation of their respective enzyme activities. Altogether, oxidative stress was enormous enough to cause maladaptation in insects. This study established that oxidative imbalance created could be the molecular basis of the efficacy of both insecticides and bio-insecticides. Two, there was development of functional but inadequate antioxidant defense mechanism in the beetle.

Physicochemical properties of malted finger millet (Eleusine coracana) and pearl millet (Pennisetum glaucum)

Abstract Germinated and raw finger millet (Eleusine coracana) and pearl millet (Pennisetum glaucum) were investigated for their physicochemical (pH, total titratable acidity (TTA), proximate, mineral analysis), phytochemical, and antioxidant properties. The results showed that there were decreases in pH (8.50–7.60) with a corresponding increase in TTA (0.0038–0.18 g/L) during germination of the millets. Proximate composition of the millets revealed slight increases in protein (7.61%–7.81%; 10.57%–11.87%) and crude fiber (5.54%–8.81%; 1.07%–2.55%) with reductions in fat (3.84%–2.73%; 7.69%–2.30%) after germination for finger and pearl millet, respectively. The millets were found to be rich sources of minerals, which include magnesium (1,028.42–1,763.50 ppm), calcium (36.42–4,158.40 ppm), sodium (150.00–510.00 ppm), potassium (470.00–4,500 ppm), zinc (20.00–40.00 ppm), and iron (66.00–121.00 ppm) which either decreased or increased with germination. The results of the phytochemical composition revealed that during germination, alkaloid (36.03–74.53 mg/g) and saponin (4.46–31.91 mg/g) contents were found to increase while there were reductions in tannin (0.88–1.64 mg/g) and phytate (7.00–17.72 mg/g) content of the flour. For finger millet, DPPH ranged from 70.00% to 72.14% and pearl millet (49.95%–64.01%), while for FRAP, pearl millet (53.69–53.76 mg/g) demonstrated better activity compared to finger millet (46.91–53.54 mg/g). Findings from this work may suggest that further studies should be carried out on germinated finger and pearl millets to examine their abilities to serve as functional foods.

Spectroscopic characterisation of interaction of ferulic acid with aldehyde dehydrogenase (ALDH)

Interaction of a pharmacological important phenolic, ferulic acid, with Aldehyde dehydrogenase (ALDH) at the simulative pH condition, was studied using spectroscopic approach. Ferulic acid caused a decrease in the fluorescence intensity formed from ALDH-ferulic acid complex resulting in mixed inhibition of ALDH activity (IC50=30.65μM). The intrinsic quenching was dynamic and induced altered conformation of ALDH and made the protein less compact but might not unfold it. ALDH has two binding sites for ferulic acid at saturating concentrations having association constant of 1.35×103Lmol-1 and a dissociation constant of 9.7×107Lmol-1at 25°C indicating ALDH-ferulic acid complex formation is more favourable than its dissociation. The interaction was not spontaneous and endothermic and suggests the involvement of hydrophobic interactions with a FRET binding distance of 4.49nm. Change in pH near and far from isoelectric points of ferulic acid did not affect the bonding interaction. Using trehalose as viscosogen, the result from Stoke-Einstein hypothesis showed that ferulic acid-ALDH binding and dissociation equilibrium was diffusion controlled. These results clearly suggest the unique binding properties and lipophilicity influence of ferulic acid.

Catalysis of Silver catfish Major Hepatic Glutathione Transferase proceeds via rapid equilibrium sequential random Mechanism

Fish hepatic glutathione transferases are connected with the elimination of intracellular pollutants and detoxification of organic micro-pollutants in their aquatic ecosystem. The two-substrate steady state kinetic mechanism of Silver catfish (Synodontis eupterus) major hepatic glutathione transferases purified to apparent homogeneity was explored. The enzyme was dimeric enzyme with a monomeric size of 25.6 kDa. Initial-velocity studies and Product inhibition patterns by methyl glutathione and chloride with respect to GSH-CDNB; GSH-ρ-nitrophenylacetate; and GSH-Ethacrynic acid all conforms to a rapid equilibrium sequential random Bi Bi kinetic mechanism rather than steady state sequential random Bi Bi kinetic. α was 2.96 ± 0.35 for the model. The pH profile of Vmax/KM (with saturating 1-chloro-2,4-dinitrobenzene and variable GSH concentrations) showed apparent pKa value of 6.88 and 9.86. Inhibition studies as a function of inhibitor concentration show that the enzyme is a homodimer and near neutral GST. The enzyme poorly conjugates 4-hydroxylnonenal and cumene hydroperoxide and may not be involved in oxidative stress protection. The seGST is unique and overwhelmingly shows characteristics similar to those of homodimeric class Pi GSTs, as was indicated by its kinetic mechanism, substrate specificity and inhibition studies. The rate- limiting step, probably the product release, of the reaction is viscosity-dependent and is consequential if macro-viscosogen or micro-viscosogen.