Biochemistry
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Item PROTECTIVE EFFECT OF Pterocarpus mildbraedii HARMS EXTRACT ON PROPANIL-INDUCED HEPATOTOXICITY AND ALTERATIONS IN APOPTOTIC-RELATED PROTEINS IN WISTAR RATS(2017-05) OTUECHERE, C.AOne of the probable causes of liver diseases is exposure to environmental chemicals. Agrochemicals containing propanil are known to induce hepatic toxicity. Pterocarpus mildbraedii leaf is used in traditional medicine to treat various disorders without scientific justification. This study was designed to investigate the protective role of extract of Pterocarpus mildbraedii against propanil-induced hepatotoxicity in rats. Pterocarpus mildbraedii leaves, purchased from Oyingbo market, Lagos State, were authenticated at the University of Lagos Herbarium (LUT/5913). Powdered leaf was extracted in soxhlet, using dichloromethane:methanol (1:1), to yield crude extract of Pterocarpus mildbraedii (PME). Sixty-four male Wistar rats (130-160 g), comprising of eight groups (n=8) were used for these experiments. Rats were treated orally with normal saline (control), PME (100 mg/kg), PME (200 mg/kg), PME (400 mg/kg), propanil (200 mg/kg), PME (100 mg/kg) + propanil (200 mg/kg), PME (200 mg/kg) + propanil (200 mg/kg) and PME (400 mg/kg) + propanil (200 mg/kg) for seven consecutive days. Hepatic tissues and serum were assayed for markers of hepatic damage, oxidative stress, inflammation, and apoptosis. Aspartate aminotransferase (AST), lactate dehydrogenase (LDH), bilirubin (BIL), superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), malondialdehyde (MDA), myeloperoxidase (MPO) and nitric oxide (NO) were assayed by spectrophotometry. Inducible Nitric Oxide Synthase (iNOS), Cyclooxygenase-2 (COX-2), Nuclear factor kappa B (NF-κB), Caspase 3, Caspase 9, Bax, Bcl-2 expressions were measured using immunohistochemistry. Tumour suppressor p53, Bcl-2 antagonist of cell death (Bad), NF-κB, inhibitor of total nuclear factor-kappa B α (IκB α), stress activated protein kinase/ C Jun NH2-terminal kinase (SAPK/JNK), p38 mitogen-activated protein kinase (p38) and signal transducer and activator of transcription 3 (STAT 3) were assessed by ELISA. Histopathology of liver was determined by microscopy and apoptosis by TUNEL assay. Data were analysed using ANOVA at α0.05. The yield of PME was 41.9%. Administration of propanil significantly increased AST (132.10±6.32 U/L), LDH (85.70±6.60 U/L), BIL (1.15±0.16 mg/dL), SOD (0.97±0.05 U/mg protein), MDA (1.03±0.08 µgMDA/mg protein), MPO (4.98±0.12 µmol/min/mg protein) and NO (0.38 µmol/mg protein) relative to control (115.90±8.65, 32.84±9.39, 1.15±0.16, 0.38± 0.01, 0.40±0.11, 2.47±0.10 and 0.19± 0.05, respectively). Pre-treatment of propanil-exposed rats with PME (200 mg/kg) significantly decreased LDH (83%), BIL (50%), SOD (50.5%), MDA (33.1%), MPO (63.3%) and NO (59.5%). Further, propanil administration decreased the levels of GSH (2.98±0.24 µg/mg protein) and CAT (52.7±0.24 µmol H202 consumed/min/g tissue) when compared with the controls (2.04±0.09 and 51.00±0.51). However, intervention with PME restored these serum biochemical indices and antioxidant parameters back to normal values. Expressions of iNOS, COX-2, NFκB, Caspase 3, Caspase 9 and Bax were higher in the propanil group relative to control. Levels of signaling mediators p38 (81.28±7.70), STAT 3 (88.80±4.40) and NF-κB (72.76± 5.30) were lower, while SAPK (125.39±9.30), IκB α (115.83±5.60) and Bad (112.48±4.70) were higher in propanil-treated rats relative to control value set at 100. TUNEL-positive nuclei and severe periportal fibrosis were observed in tissues following propanil exposure. However, pre-treatment with PME significantly attenuated the observed propanil-induced inflammation and apoptosis. Pterocarpus mildbraedii extract protected against propanil-induced hepatotoxicity via mechanisms that involved its antioxidant, anti-inflammatory and anti-apoptotic properties.Item THE MODULATION OF RAT LIVER MICROSOMAL CALCIUM ION-PUMPING ATPase BY DICOPHANE AND LOW PROTEIN INTAKE(1992-05) ADENUGA, G. A.The effects of the liver tumour promoter, dicophane, with those of low protein intake (LPI) on the functional expression of rat liver microsomal Ca(2+) -ATPase were compared. The effects of dicophane and LPI on the activity of the microsomal enzyme after carcinogenic initiation by pretreatment with aflatoxin B (AFB), a genotoxic liver carcinogen, were also compared. The Status of membrane - bound Ca(2+) -ATPase of erythrocytes of humans having primary liver cancer (PLC) and kwashiorkor was assessed. The specific activity of membrane - bound microsomal Ca(2+) -AT Pase of the livers of untreated rats was 4.543 ± 0.857 µmole P/mg protein/hr. at pH 8.0 and was insensitive to calmodulin. The specific activity of the enzyme was significantly decreased (P < 0.01) following subcutaneous administration of a single dose of 75mg dicophane/kg body wt.; the affinity of the enzyme for Ca(2+) was however unaffected. Similarly, liver microsomal Ca (2+)-ATPase activity was significantly diminished following the ingestion of low protein diet by rats for 12 weeks. The mean Ca(2+) -ATPase activity of AFB -treated animals (in the absence of dicophane) was not significantly different (P > 0.05) from that of AFB-treated rats which subsequently received dicophane. In contrast, liver microsomal Ca(2+)-ATPase activity of animals fed low protein diet prior to and after AFB ingestion was higher (P < 0.05) than that of animals which were on low protein diet only. Basal activity of erythrocyte Ca(2+) -ATPase in paediatric controls and those having kwashiorkor (protein-energy-malnutrition) were similar (P > 0.05); similar observations were made between normal adults and those suffering from PLC. Erythrocyte Ca(2+) -ATPase of either PLC or kwashiorkor patients was however, some- what, less sensitive (15-40 %) to the stimulatory effect of calmodulin, an endogenous activator of the Ca(2+) -pump. These results suggest that liver microsomal Ca(2+) -ATPase could be a useful biochemical marker to determine the onset or occurrence of tumour promotion in liver cells. Finally, chronic dietary protein malnutrition mimics the effect of chemical liver tumour promoters and could possibly enhance the development of human PLC particularly n those areas of the tropics where malnutrition is prevalent. Future confirmatory experiments are however re-quired to fully justify this postulate.Item POST-JUNCTIONAL ALPHA ADRENOCEPTORS IN THE ANOCOCCYGEUS MUSCLE AND VAS DEFERENS: A COMPARATIVE STUDY IN NORMOTENSIVE AND SPONTANEOUSLY HYPERTENSIVE RATS(1984-06) ADENEKAN, O. O.The characteristics of the post-junctional α-adrenoceptors in the isolated anococcygeus muscle and vas deferens were compared in spontaneously hypertensive rats (SHR) and normotensive rats (NCR). Responses to α —adrenoceptor agonists were obtained in the absence and presence of cocaine and of antagonists. Noradrenaline (NA) and phenylephrine (PE) produced concentration related contrations of the preparations which were antagonised by phentolamine, prazosin and yohimbine in both rat strains, indicating α -adrenoceptor mediation. The effects of cocaine revealed the relative efficiency of the uptake mechanism in each preparation. In the anococcygeus NA was equipotent in the NCR and SHR in the absence of cocaine whereas it was less potent in the SHR in the presence of cocaine, PE was less potent in the SHR in the absence and presence of cocaine. Antagonism was assessed by pA2 and K(diss) determinations. Potencies were compared only when antagonism was competitive in both strains. In the anococcygeus low concentrations of prazosin (L-Praz) non—competitively antagonised NA but antagonised PE equally and competitively in both strains. Higher concentrations (H-Praz) competitively antagonised NA in both strains. Phentolamine was competitive against NA in NCR and against PE in both strains. However, it was non-competitive against NA in SHR, Low concentrations of yohimbine (L—YOH) competitively antagonised NA and PE in both the NCR and SHR but the K(diss) values were significantly different. Higher concentrations (H—YOH) was competitive against NA in the NCR and PE in both strains. In the vas L-Praz competitively antagonised both NA and PE in the NCR but gave non—competitive antagonism of both strains in the SHR, Phentolamine antagonised NA competitively in the NCR but non-competitively in the SHR. It was equipotent and competitive against PE in both strains. L—YOH non-competitively antagonised NA and PE in the NCR but in the SHR it was competitive. H-YOH antagonism was non—competitive against both NA and PE in both strains. It is suggested that there might be both the α1— and α2 post—junctional adrenoceptor in the NCR anococcygeus muscle, Prazosin and yohimbine seem to be able to differentiate between the two receptor subtypes at low concentrations, It is suggested further that the α2- subpopulation might not possess identical characteristics in the NCR and SHR anococcygeus. Also, there might be an alteration in NA uptake properties in the SHR, In the NCR vas deferens there seems to be a predominance of post-junctional α1-adrenoceptors. In the SHR vas, there might be an increase in the post-junctional α-adrenoceptor population and/or sensitivity. Furthermore, it seems that the post—junctional α2-adrenoceptor characteristics are somewhat different in the SHR, Uptake1 is suggested to be less efficient in the SHR vas.Item SOME CONSEQUENCES OF THE BINDING OF AFLATOXIN B1 WITH PLASMA MEMBRANE ON THE REGULATION OF INTRACELLULAR Ca2+ HOMEOSTASIS(1992-03) ADEBAYO, A. O.The possible influence of aflatoxin B1 a potent hepatocellular carcinogen on the regulation of intracellular Ca2+ homeostasis has been studied using the red cell as a model. Preliminary work on the interaction of the toxin with the red cell membrane using spectrofluometric analysis indicated that the toxin binds spontaneously and irreversibly to the red cell membrane. The binding is highest at pH 4 and least at pH 10. Results obtained from studies using equilibrum dialysis technique show that about 4 nmoles of the toxin bind to one microgram membrane protein. Although the exact membrane component to which aflatoxin B1 binds is not known, experiments carried out to determine the influence of aflatoxin B1 on the activity of the calcium pumping protein revealed that the toxin inhibited the calmodulin-stimulated erythrocyte membrane Ca2+ -ATPase activity by about 50 percent, while it has little or no effect on its basal activity. Kinetic analysis of the inhibition shows that, the toxin reduces the Vmax and Km of the calmodulin-stimulated enzyme by 50 percent in a non-competitive manner, On the other hand, the carcinogen had no significant influence on the kinetic parameters of the enzyme in the non-activated state. Similar results were obtained for the triton X-100 solubilized and calmodulin affinity chromatographed enzyme. In this instance aflatoxin B1 inhibited the calmodulin-stimulated purified enzyme by 50 percent with or without preincubation on ice for half an hour. Again, the toxin had little or no effect on the basal activity of the enzyme in the absence of calmodulin. Analysis of the results obtained using varying concentrations of ATP shows that the Km and Vmax of the non-activated enzyme were not altered by the toxin while both the Vmax and Km values were reduced by about 50 percent in the presence of calmodulin. In addition aflatoxin B1 inhibited Diphosphotidyl glycerol (cardiolipin) by about 28% while it has no effect on the basal activity of the enzyme. Although, the inhibition of the membrane bound or purified Ca2+ ATPase by the toxin is concentration dependent, varying concentrations of phosphatidyl serine and phosphatidyl choline do not affect the inhibition of the purified enzyme by afla toxin B1. Results obtained with triton X-100 solubilized enzyme shows that triton X-100 alone could not activate the enzyme. Thus at triton X-100: protein ratio of 2, the enzyme was stimulated by calmodulin. This activity was sensitive to inhibition by the toxin. In this instance, the calmodulin-stimulated activity was inhibited by about 50%, while at lower ratios of the triton X-100 to protein there was no significant inhibition of enzyme. Results of experiments carried out on the 124KDa fragment, which was produced as a result of exposure to calpain a Ca2+ - dependent cysteine protease, indicated that the toxin has no effect whatsoever on the activity of the fragmented enzyme, Similarly experiments on limited proteolysis of the Ca2+ ATPase by trypsin to give the 90KDa fragment which still retains its calmodulin binding domain and the 76KDa fragment which has lost its calmodulin binding domain revealed that the aflatoxin inhibited the 90KDa fragment by about 50% while the 76KDa fragment is not affected at all. Altogether, -these findings show that aflatoxin B1 inhibits the plasma membrane Ca2+ - pumping ATPase by interacting with the enzyme at the calmodulin binding domain. The nature of the exact amino acid residue to which the toxin binds is however not known. The implication of these observations is that Ca2+ extrusion may be hampered in situations where the cell is poisoned by the aflatoxinItem EFFECTS OF LAMIVUDINE ON BIOCHEMICAL INDICES IN RATS.(2014-05) OLANIYAN, LAMIDI WAHEED BABATUNDELamivudine (L(-)21,31–dideoxy–3-thiacytidine) is an antiretroviral drug which exhibits tissue toxicity leading to peripheral neuropathy and parkinsonism. The exact mechanism of cytotoxicity and effects on target tissues are not well understood. This study was designed to elucidate the effects of lamivudine on biochemical indices in the liver, kidney and brain of rats. Female Wistar rats (180-200g) were randomly assigned into 5 groups of 6 rats each treated orally for 45 days with normal saline (Control), 4 mg/kg, 20 mg/kg, 100 mg/kg and 500 mg/kg lamivudine respectively. Rats were sacrificed after 12 hours fast and blood (6 mL) collected. Serum obtained was used for biochemical analysis. Serum Alanine and Aspartate Aminotransferases (ALT and AST), Quinine Oxidase (QO), γ–GlutamylTransferase(GGT), urinary trehalase activities as well as urinary creatinine and protein were determined by spectrophotometric techniques while urinary magnesium (Mg2+) level was determined by atomic absorption spectrophotometry. In the liver, kidney and brain, the activities of Superoxide Dismutase (SOD), Glutathione-S-Transferase (GST) and levels of malondialdehyde (MDA, index of lipid peroxidation) were determined. Histology of the liver and kidney was assessed using hematoxylin and eosin stain. Data were analysed using ANOVA at p=0.05. Lamivudine (500 mg/kg) produced significant increases in the activities of serum ALT, AST, GGT and QO (33.2±3.9, 56.4±7.2, 16.3±1.8 IU/L and 10.1±1.7 Baier’s Unit (BU)) relative to controls (21.3±1.5, 42.6±1.9, 11.1±0.7 IU/L and 4.9±1.0 BU) respectively. The drug at 20, 100 and 500 mg/kg increased hepatic GGT activities by 3.1, 4.0 and 5.2 folds while hepatic GST increased by 1.7, 1.8 and 2.0 folds relative to controls respectively. Renal GST activities significantly increased in lamivudine (100 and 500 mg/kg) treated rats (3.15±0.12, 3.57±0.23x 10-2 U/mg protein) relative to control (1.74±0.23x 10-2 U/mg protein). The level of hepatic MDA in control was 0.06±0.01 nmol/mg protein while lamivudine at 100 and 500 mg/kg significantly increased MDA levels (0.17±0.03, 0.22±0.04 respectively). Also, lamivudine at 100 and 500 mg/kg significantly increased renal MDA levels by 125% and 189% respectively when compared with the control. The activity of hepatic SOD in control was 9.9±0.7 U/mg protein. Treatment with lamivudine (20, 100 and 500 mg/kg) significantly increased SOD activities (14.7±1.5, 18.4±1.2, 16.5±1.3 U/mg protein). Similar trend was observed for renal SOD UNIVERSITY OF IBADAN LIBRARY activities of rats treated with lamivudine. Administration of lamivudine at 100 mg/kg significantly increased urinary trehalase activity (324.1±15.3 vs 157.8±18.6 U/mg per ml), urinary Mg2+ concentration (8.6±0.4 vs 4.8±0.5x10-3/mg/ml) and urinary protein (2.9±0.29 mg/ mlvs 1.4±0.14mg/ml). However, there were no significant differences in the values of brain SOD, MDA, and urinary creatinine in lamivudine-treated animals relative to controls. Histological sections of rats treated with lamivudine (100 and 500 mg/kg) showed visible lesions in the liver (hydropic degeneration) and kidney (cortical congestions). Repeated administration of lamivudine altered biochemical indices accompanied by visible histologic effects in the liver and kidney of the rats. The biochemical alterations appear to be mediated by oxidative stress. Keywords: Biochemical Indices, Cytotoxicity, Lamivudine, Oxidative stress. Word count: 484Item ANTIMALARIAL AND ANTITUBERCULAR ACTIVITIES OF CRUDE METHANOL EXTRACT AND FRACTIONS OF THE BULB OF CRINUM JAGUS ( Linn.)(2015-02) KOLAWOLE, ADEBOLA OLAYEMICrinum jagus is a medicinal plant used traditionally to treat tuberculosis, malaria and other bacterial infections. However, there are limited documented scientific studies to substantiate the use of this plant. Due to increase in resistance to malaria and tuberculosis drugs, the need for the development of other drugs is pertinent. This study was designed to determine the pharmacological activities of extract and fractions of Crinum jagus. Methanol extract of C. jagus obtained by soxhlet extraction was subjected to phytochemical analysis and fractionated using column chromatography. Antitubercular and antimicrobial activities of the extract and its fractions were evaluated against isolates of Mycobacterium tuberculosis and selected microorganisms using the disc and agar diffusion methods. Antimalarial activity was assessed in vivo using Rane’s test in Plasmodium berghei infected mice (n = 80 in 10 groups) treated orally with tween 80 (control), 10, 25, 50 and 75 mg/kg of extract and its fractions at 10 mg/kg respectively, while chloroquine (10 mg/kg) and arteether (3 mg/kg) groups served as positive controls. Anti-inflammatory potential was assessed in rats using carrageenan-induced paw inflammatory model. In vitro antioxidant potentials were determined spectrophotometrically using 1,1-diphenyl-2-picryl hydrazyl (DPPH), hydroxyl radical scavenging activities, Total Flavonoids Contents (TFC) and Phenolic Contents (TPC) Antioxidant indices- Superoxide dismutase (SOD) and Catalase (CAT) activities and levels of Malondialdehyde (MDA) and reduced Glutathione (GSH) were determined by spectrophotometry. Aspartate (AST) and Alanine (ALT) amino transferases and Alkaline Phosphatase (ALP) were estimated spectrophotometrically. Data were analysed by Student’s t test at p = 0.05. Phytochemical analysis revealed the presence of alkaloids, flavonoids, phenols and steroids in the crude extract. The extract and its fractions (F1, F2 and F3) showed a concentration- dependent inhibition of Mycobacterium tuberculosis, with F1 having the lowest IC50of : 0.22mg/mL relative to rifampicin (IC50 : 0.19mg/mL) and isoniazid (0.23mg/mL). The extract at 10, 25, 50, 75 mg/kg and F1, F2 and F3 at 10 mg/kg suppressed parasitaemia in Plasmodium berghei infected mice by 70.0, 76.0, 79.0, 87.0% and 89.0, 76.0, 78.0% respectively relative to chloroquine (100%) and arteether (89.0%). The extract at 10, 25, 50, 75 mg/kg and F1, F2 and F3 at 10 mg/kg inhibited oedema in rat paws by 26.0, 30.0, 32.0, 66.0% and 80.0, 25.0, 52.0% UNIVERSITY OF IBADAN LIBRARY iii respectively when compared with indomethacin (95.0%). The extract and its fractions significantly scavenged DPPH and hydroxyl radical in vitro. The TPC and TFC of extract, F1, F2 and F3 at 500 μg/ml were 0.310, 0.460, 0.240, 0.380 μg/mg and 0.523, 0.864, 0.396, 0.643 μg/g respectively. The extract and its fractions significantly reduced MDA level while GSH, SOD and CAT levels were increased. Activities of AST, ALT and ALP were significantly increased at 50 and 75 mg/kg body weight of extract . Crinum jagus exhibited antitubercular, antimalarial and anti-inflammatory activities via scavenging of radicals and antioxidative mechanism. This indicates a promising potential of the plant for drug development. Keywords: Crinum jagus, antituberculosis, antimalarial, antioxidant. Word Count: 471 .Item CYTOTOXICITY OF HEXAVALENT CHROMATE COMPOUNDS IN CH310T1/2 CELLS AND CYTOMODULATION BY SODIUM ARSENITE AND METHANOL EXTRACT OF Rauvolfia vomitora (Afzel) IN MICE.(2015-02) AKINWUMI, KAZEEM AKINYINKAExposure to certain hexavalent chromate compounds (HCC) causes lung and colon cancers. Their mechanisms of cytotoxicity are unclear, but believed to be affected by ascorbate and particle size. However, their role is not clearly defined. Co-exposure with sodium arsenite (SA) is common, but its effect on HCC toxicity is unknown. Current therapy has side effects, necessitating the search for antidote from unexplored natural products such as Rauvolfia vomitora (RV). This study therefore investigates the effect of particle size and ascorbate on cytotoxity of selected HCC [lead chromate (PbCrO4), barium chromate (BaCrO4), strontium chromate (SrCrO4) and potassium dichromate (K2Cr2O7)] in C3H10T1/2 cells and cytomodulatory effects of SA and RV in mice. The effect of ascorbate, dehydroascorbate and particle size on HCC cytotoxicity in C3H10T1/2 cells was determined by measuring survival fraction and yield of foci by microscopy. Actin and cellular ultrastructure disruption and induction of cell death were assessed by electron and fluorescent microscopy. The molecular mechanisms of cytotoxicity and transformation were evaluated in eighty-four cell death genes using real time (RT2) gene array, while cell cycle analysis was done by flow cytometry. Leaves of RV were air dried, powdered and extracted with methanol. Forty male mice (20-25g) were divided into 8 groups of 5 Swiss albino mice each and treated with water (control), RV (275 mg/Kg), SA (2.5 mg/kg), K2Cr2O7 (12 mg/Kg), SA + K2Cr2O7, RV + SA, RV + K2Cr2O7, RV + SA + K2Cr2O7. Rauvolfia vomitora was given orally for seven days, while K2Cr2O7 and SA were administered on day seven. Serum aspartate and alanine aminotransferases (AST and ALT), catalase, glutathione-S-transferase (GST), glutathione and malondialdehyde (MDA) levels were determined by spectrophotometry. Micronucleated polychromatic erythrocytes (mPCEs) were evaluated by microscopy. Data were analysed using ANOVA and Student‟s t- test at p= 0.05. Survival fraction of control cells was 1.0, treatment with PbCrO4 and ≤ 12.5 μM ascorbate or ≤ 2 μM dehydroascorbate decreased it to 0.4. The 15-20 μM ascorbate and 3-4 μM dehydroascorbate reversed it to 0.7. Exposure of cells to small (≤ 3 μm) and large particles (≤ 8 μm) of PbCrO4, BaCrO4 and SrCrO4 resulted in a dose-dependent decrease in survival. The total foci were higher for PbCrO4 (3.8) with large particles and BaCrO4 (6.6) with small particles. Phagocytosis of particles was time-dependent. The HCC treatment led to G2/M and S phase arrest, anucleation, actin disruption and mixed cell death. Thirty-four cell death genes including Bax and Casp3 were up-regulated by 4 folds and six including Bcl-2 and Traf2 were down- regulated in treated cells. Twenty-one anti-apoptotic and autophagy genes including Atg5 and Bcl-2 were up-regulated in PbCrO4 transformed cells. The K2Cr2O7 and/ or SA significantly increased mPCEs, AST, ALT, catalase and MDA levels while glutathione and GST were reduced. The RV restored the markers towards normal values. Cytotoxicty of chromate compounds is particle size and ascorbate dependent. The cytotoxicity might be due to actin disruption, micronuclei induction and cell cycle arrest. Methanol extract of Rauvolfia vomitora modulated the toxicity in mice. Keywords: Hexavalent chromate compounds, Sodium arsenite, Rauvolfia vomitora, Cytotoxicity Word counts: 494Item Influence of Chloramphenicol and Amoxicillin on Rat Liver Microsomal Enzymes and Lipid Peroxidation(2014-09) Adesanoye, O. A.; Ifezue, A. O. C.; Farombi, E. O.Generation of reactive oxygen species beyond the antioxidant capacity of biological system has been reported to give rise to oxidative stress which through a series of events deregulates cellular functions, leading to oxidative damage and various pathological conditions. This study examined the effect of chloramphenicol and amoxicillin on liver microsomal enzymes Ca2+-ATPase and Glucose-6-Phosphatase (G-6-P) and lipid peroxidation in rats. Male Wistar strain rats weighing 120 – 195 g were divided into four groups. Group one, the control group, received physiological saline, group two received Amoxicillin at 10.71 mg/kg, group three received Chloramphenicol at 28.57 mg/kg, while group four was administered combination of chloramphenicol and amoxicillin. Drugs were administered for ten days and the animals sacrificed on the eleventh day. Detection of oxidation in liver microsomal fraction was carried out by assessment of lipid peroxidation and conjugated diene. Ca2+-ATPase and G-6-P activities and total protein content were also measured. Data were analysed by ANOVA and Student’s T-Test. Significant (p<0.05) decreases in G-6-P activity by 55.30%, 38.37%, 55.30% and Ca2+-ATPase activity by 38.99%, 30.16%, 26.88% were recorded with chloramphenicol, amoxicillin and chloramphenicol/amoxicillin treatments respectively when compared with the control group while total microsomal protein content was depleted by 70.50% 79.27%, 75.87% respectively. TBARS and Diene Conjugation were significantly (p<0.05) elevated in the treated groups. Findings from this study suggest that Chloramphenicol and Amoxicillin induced oxidative stress in rats and perturbed Ca2+ homeostasis presumably due to generation of free radicals.Item PROTECTIVE EFFECT OF AQUEOUS EXTRACTS OF AFRAMOMUM LONGISCAPUM SEED AGAINST SODIUM ARSENITE - INDUCED HEPATOTOXICITY AND CLASTOGENICITY IN WISTAR RATS(2014-03) ANIFOWOSE, O. J.The use of medicinal plants has been found to be effective in the treatment of diseases such as cancer. Aframomum longiscapum seed has been used in the treatment of cancer in ethno medicine but has not been subjected to appropriate scientific evaluation. This study was therefore designed to evaluate the protective effect of Aqueous Extract of Aframomum longiscapum (AEAL) seed against sodium arsenite- and ethanol-induced hepatotoxicity and clastogenicity in male Wistar rats. Phytochemical composition of dried milled seeds of Aframomum longiscapum was evaluated using standard procedures. Twenty five male albino Wistar rats (150-250 g) were randomly divided into five groups of five rats each: Group I (control) - distilled water, Group II- sodium arsenite (2.5mg/kg), Group III - sodium arsenite (2.5mg/kg) in 3% ethanol (v/v), Group IV- AEAL (122.5 mg/kg), Group V- sodium arsenite (2.5mg/kg) in 3% ethanol + AEAL (122.5mg/kg body weight). After five weeks of treatment, hepatotoxicity was assessed using serum activities of Alkaline Phosphatase (ALP), Alanine amino Transferase (ALT), Gamma Glutamyl Transferase (GGT) and Aspartate amino Transferase (AST) by spectrophotometry. Clastogenicity was determined using bone marrow cytology to identify Micronucleated Polychromatic Erythrocytes (MnPCEs) by microscopy. Sperm counts, motility, viability and morphological abnormalities were estimated using microscopic techniques. Histology was carried out using Haematoxylin and Eosin. Data were analysed using Student’s t test at p = 0.05. Phytochemical screening revealed the presence of alkaloids, saponins, flavonoids and cardenolides. There were increased levels of liver enzymes on exposure to sodium arsenite compared with the control (ALP = 236.8 ± 15.2 vs 432.2 ± 41.4, ALT = 23.1 ± 5.9 vs 29.9 ± 2.8, GGT = 2.6 ± 1.9 vs 4.6 ± 1.6 and AST = 47.5 ± 9.2 vs 66.0 ± 15.6 U/L). The elevated levels of these enzymes were significantly reduced by about 2 folds in AEAL–treated rats compared with the sodium arsenite group (29.9 ± 2.8 vs 19.8 ± 3.1). The degree of reduction of MnPCEs was 2 folds in the treated-animals (15.4 ± 5.2 vs 26.5 ±2.1). The AEAL reversed the severe hepatic degeneration and necrosis induced by sodium arsenite and caused a significant decrease in sperm counts (126.1 ± 8.5 vs 85.5 ± 14.9) and motility (90.0 ± 10.0 vs 52.5 ± 15.0). However, there were significant increases in sperm viability (92.5 ± 3.5 vs 93.3 ± 4.0) and morphological abnormalities (10.1 ± 1.9 vs 13.4 ± 2.4). Aframomum longiscapum seed extract has protective effect against sodium-arsenite induced hepatotoxicity and clastogenicity in intoxicated rats. The extract however had deleterious side effects on male fertility in the treated rats. This plant extract should be administered with caution to human subjects given the provisional nature of these data.Item CYTOTOXICITY OF HEXAVALENT CHROMATE COMPOUNDS IN CH310T1/2 CELLS AND CYTOMODULATION BY SODIUM ARSENITE AND METHANOL EXTRACT OF Rauvolfia vomitora (Afzel) IN MICE.(2015-02) AKINWUMI, K. A.Exposure to certain hexavalent chromate compounds (HCC) causes lung and colon cancers. Their mechanisms of cytotoxicity are unclear, but believed to be affected by ascorbate and particle size. However, their role is not clearly defined. Co-exposure with sodium arsenite (SA) is common, but its effect on HCC toxicity is unknown. Current therapy has side effects, necessitating the search for antidote from unexplored natural products such as Rauvolfia vomitora (RV). This study therefore investigates the effect of particle size and ascorbate on cytotoxity of selected HCC [lead chromate (PbCrO4), barium chromate (BaCrO4), strontium chromate (SrCrO4) and potassium dichromate (K2Cr2O7)] in C3H10T1/2 cells and cytomodulatory effects of SA and RV in mice. The effect of ascorbate, dehydroascorbate and particle size on HCC cytotoxicity in C3H10T1/2 cells was determined by measuring survival fraction and yield of foci by microscopy. Actin and cellular ultrastructure disruption and induction of cell death were assessed by electron and fluorescent microscopy. The molecular mechanisms of cytotoxicity and transformation were evaluated in eighty-four cell death genes using real time (RT2) gene array, while cell cycle analysis was done by flow cytometry. Leaves of RV were air dried, powdered and extracted with methanol. Forty male mice (20-25g) were divided into 8 groups of 5 Swiss albino mice each and treated with water (control), RV (275 mg/Kg), SA (2.5 mg/kg), K2Cr2O7 (12 mg/Kg), SA + K2Cr2O7, RV + SA, RV + K2Cr2O7, RV + SA + K2Cr2O7. Rauvolfia vomitora was given orally for seven days, while K2Cr2O7 and SA were administered on day seven. Serum aspartate and alanine aminotransferases (AST and ALT), catalase, glutathione-S-transferase (GST), glutathione and malondialdehyde (MDA) levels were determined by spectrophotometry. Micronucleated polychromatic erythrocytes (mPCEs) were evaluated by microscopy. Data were analysed using ANOVA and Student’s t- test at p= 0.05. Survival fraction of control cells was 1.0, treatment with PbCrO4 and ≤ 12.5 µM ascorbate or ≤ 2 µM dehydroascorbate decreased it to 0.4. The 15-20 µM ascorbate and 3-4 µM dehydroascorbate reversed it to 0.7. Exposure of cells to small (≤ 3 µm) and large particles (≤ 8 µm) of PbCrO4, BaCrO4 and SrCrO4 resulted in a dose-dependent decrease in survival. The total foci were higher for PbCrO4 (3.8) with large particles and BaCrO4 (6.6) with small particles. Phagocytosis of particles was time-dependent. The HCC treatment led to G2/M and S phase arrest, anucleation, actin disruption and mixed cell death. Thirty-four cell death genes including Bax and Casp3 were up-regulated by 4 folds and six including Bcl-2 and Traf2 were down- regulated in treated cells. Twenty-one anti-apoptotic and autophagy genes including Atg5 and Bcl-2 were up-regulated in PbCrO4 transformed cells. The K2Cr2O7 and/ or SA significantly increased mPCEs, AST, ALT, catalase and MDA levels while glutathione and GST were reduced. The RV restored the markers towards normal values. Cytotoxicty of chromate compounds is particle size and ascorbate dependent. The cytotoxicity might be due to actin disruption, micronuclei induction and cell cycle arrest. Methanol extract of Rauvolfia vomitora modulated the toxicity in mice.