http://ir.library.ui.edu.ng/handle/123456789/497 Biochemistry 2026-04-08T18:46:12Z http://ir.library.ui.edu.ng/handle/123456789/9395 Title: PROTECTIVE EFFECT OF Pterocarpus mildbraedii HARMS EXTRACT ON PROPANIL-INDUCED HEPATOTOXICITY AND ALTERATIONS IN APOPTOTIC-RELATED PROTEINS IN WISTAR RATS Authors: OTUECHERE, C.A Abstract: One 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. Description: A Thesis in The Department Of Biochemistry submitted To The Faculty Of Basic Medical Sciences In Partial Fulfilment Of The Requirements For The Degree Of Doctor Of Philosophy Of The University Of Ibadan 2017-05-01T00:00:00Z http://ir.library.ui.edu.ng/handle/123456789/5111 Title: THE MODULATION OF RAT LIVER MICROSOMAL CALCIUM ION-PUMPING ATPase BY DICOPHANE AND LOW PROTEIN INTAKE Authors: ADENUGA, G. A. Abstract: 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. Description: A THESIS IN THE DEPARTMENT OF BIOCHEMISTRY SUBMITTED TO THE FACULTY OF BASIC MEDICAL SCIENCES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY OF THE UNIVERSITY OF IBADAN 1992-05-01T00:00:00Z http://ir.library.ui.edu.ng/handle/123456789/4846 Title: POST-JUNCTIONAL ALPHA ADRENOCEPTORS IN THE ANOCOCCYGEUS MUSCLE AND VAS DEFERENS: A COMPARATIVE STUDY IN NORMOTENSIVE AND SPONTANEOUSLY HYPERTENSIVE RATS Authors: ADENEKAN, O. O. Abstract: 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. Description: A THESIS IN THE DEPARTMENT OF PHARMACOLOGY AND THERAPEUTICS SUBMITTED TO THE FACULTY OF BASIC MEDICAL SCIENCES OF THE COLLEGE OF MEDICINE IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY OF THE UNIVERSITY OF IBADAN 1984-06-01T00:00:00Z http://ir.library.ui.edu.ng/handle/123456789/4389 Title: SOME CONSEQUENCES OF THE BINDING OF AFLATOXIN B1 WITH PLASMA MEMBRANE ON THE REGULATION OF INTRACELLULAR Ca2+ HOMEOSTASIS Authors: ADEBAYO, A. O. Abstract: 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 aflatoxin Description: A THESIS IN THE DEPARTMENT OF BIOCHEMISTRY SUBMITTED TO THE FACULTY OF BASIC MEDICAL SCIENCES IN PARTIAL FULFILMENT OF REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY OF THE UNIVERSITY OF IBADAN 1992-03-01T00:00:00Z