Background: Evidence from our laboratories indicates that triterpene constituents of Syzygium cordatum (Hochst.) [Myrtaceae] crude leaf extracts can be used to treat diabetes mellitus. For the plant derived triterpenes to have further potential in diabetes management, they should, however, additionally alleviate or prevent some of the complications of diabetes mellitus such as impaired kidney function and cardiovascular disorders. Accordingly, this study was designed to isolate the triterpene, oleanolic acid (OA) from S. cordatum leaves and evaluate its effects on blood glucose, renal function and blood pressure in streptozotocin (STZ)-induced diabetic rats. OA was studied because it is the major constituent of many African plant species used in traditional medicine. Materials and Methods S. cordatum crude leaf ethyl acetate solubles (EAS) were obtained after defatting the leaves with hexane followed by dichloromethane before maceration with ethyl acetate. Preliminary experiments indicated that EAS contained triterpenes with hypoglycaemic properties. Solvent extraction and fractionation of EAS yielded mixtures of oleanolic acid/ursolic acid (OA/UA) and methyl maslinate/methyl corosolate. Recrystallisation of the OA/UA mixture using ethanol yielded OA, the structure of which was confirmed by NMR spectroscopy ('H & 13C). Oral glucose tolerance test (OGTT) responses to various doses of OA (40, 80 and 120 mg/kg) were monitored in separate groups of non-diabetic and STZ-induced diabetic rats given a glucose load (0.86 g/kg, p.o.) after an 18-h fast. Rats treated with deionized water (3 ml/kg p.o.), or standard drugs, (insulin, 200 Hg/kg, s.c; metformin, 500 mg/kg, p.o. and glibenclamide, 500 f^g/kg, p.o.) acted as untreated and treated positive controls, respectively. To investigate the possible interaction between OA and standard drugs in lowering blood glucose, OGTT responses were studied in separate groups of animals simultaneously treated with OA at either 40 or 80 mg/kg and insulin (100 or 200 ug/kg, s.c), metformin, (250 or 500 mg/kg, p.o.) or glibenclamide (250 or 500 mg/kg, p.o.). Blood glucose was monitored at 15-min intervals for the first hour, and hourly thereafter for 3 h. Plasma insulin concentrations were measured in separate parallel groups of rats prepared as for OGTT studies to examine whether there was an association between OA treatment and pancreatic insulin secretion. Acute effects of OA on kidney function and mean arterial blood pressure (MAP) were investigated in anaesthetized rats challenged with hypotonic saline after a 3'/2-h equilibration for 4 h consisting of 1 h control, 1XA h treatment and 1 Vi h recovery periods. OA was added to the infusate during the treatment period. Short-term effects of OA were studied in individually-caged rats treated twice daily with OA (80 mg/kg, p.o.) for 5 weeks. Results OA decreased blood glucose concentrations of both non-diabetic and diabetic rats, as did some standard drugs except glibenclamide which did not exhibit any effects in STZ-induced diabetic animals. The blood glucose lowering effects were most potent in STZ-induced rats treated with combined OA and insulin by comparison with all other treatments. Short-term treatment of non-diabetic and STZ-induced diabetic rats with OA alone for 5 weeks decreased blood glucose concentrations, but the reduction in non-diabetic rats was to values that did not achieve statistical significance. Except for non-diabetic rats treated with insulin alone or in combination with OA, plasma insulin concentrations were not altered by treatment in non-diabetic and STZ-induced diabetic animals. Hepatic glycogen concentrations of non-diabetic and STZ-induced diabetic rats were significantly increased by all treatments at the end of 5 weeks. Acute intravenous infusion of OA in anaesthetized rats significantly increased Na+ excretion outputs of non-diabetic and STZ-induced diabetic rats without affecting urine flow, K+ or CI" excretion rates. Similarly, daily OA treatment (80 mg/kg, p.o.) significantly increased Na+ excretion rates of non-diabetic and STZ-induced diabetic rats throughout the 5 week experimental period without affecting urine flow, K+ or CI" excretion rates. By comparison with respective control animals, Short-term administrations of OA significantly (p<0.05) increased GFR of non-diabetic (2.88±0.14 vs 3.71±0.30 ml/min) and STZ-diabetic rats (1.81± 0.32 vs 3.07±0.16 ml/min, n=6 in all groups) with concomitant reduction of plasma creatinine concentrations. Acute and Short-term administrations of OA non-diabetic and STZinduced diabetic rats reduced mean arterial blood pressure by comparison with respective control animals. Discussion The results suggest that S. cordatum leaf derived OA not only has the potential to lower blood glucose in diabetes, but also has beneficial effects on kidney function and blood pressure. We suggest that the hypoglycaemic effects of OA mimic those of metformin as evidenced by the fact that neither of these treatments altered plasma insulin concentration of non-diabetic rats. OA-evoked increases in urinary Na+ outputs of STZ-diabetic rats and elevation of GFR suggest up-regulation of renal function by the triterpene. The findings are of considerable importance because they suggest the hypoglycaemic, renal and hypotensive effects of OA in the management of diabetes mellitus. Conclusion The results demonstrated that the oleanolic acid extracted from S. cordatum leaf has blood glucose-lowering effects comparable to standard anti-diabetic drugs in STZ-induced diabetic rats. Furthermore, OA augmented the hypoglycaemic effects of insulin in STZ-induced diabetic rats. These findings suggest that OA may have beneficial effects on some of the processes that are associated with renal derangement in STZ-induced diabetic rats. The results introduce the first in vivo evidence that OA ameliorates kidney function in STZinduced diabetic rats. Keywords: Renal function; diabetes mellitus; triterpenoids; oleanolic acid, hypoglycaemia / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, 2008.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Bothropoides erythromelas à responsÃvel por muitos acidentes no Nordeste do Brasil. O veneno desta serpente induz insuficiÃncia renal aguda. Rins isolados de ratos Wistar, pesando 250 a 300g, foram perfundidos durante 120 min com soluÃÃo Krebs-Henseleit contendo 6g% de albumina bovina. O veneno total de Bothropoides erythromelas foi estudado anteriormente (SOUSA, 2004) e utilizado neste estudo para posterior comparaÃÃo com os grupos tratados com as fraÃÃes PLA2 Lys 49 e Asp 49 do veneno. O veneno total (10mg/mL) e as fraÃÃes PLA2 Lys 49 e Asp 49 (5mg/mL) de B. erythromelas foram adicionados ao sistema 30 min apÃs o inÃcio de cada experimento. Os parÃmetros estudados incluÃram pressÃo de perfusÃo (PP), resistÃncia vascular renal (RVR), ritmo de filtraÃÃo glomerular (RFG), fluxo urinÃrio (FU), percentual de transporte tubular de sÃdio, potÃssio e cloreto (%TNa+, %TK+ e %TCl-), percentual de transporte proximal de sÃdio, potÃssio e cloreto (%pTNa+, %pTK+ e %pTCl-), excreÃÃo de sÃdio, potÃssio e cloreto (ENa+, EK+ e ECl-) e clearance osmÃtico (Cosm) (p< 0,05*). O grupo controle perfundido com albumina foi funcionalmente estÃvel por todos os 120 min. A infusÃo do veneno causou um aumento significante no FU, ENa+, ECl- e Cosm e uma diminuiÃÃo na PP, RVR, %TNa+, %TK+, %TCl-, %pTNa+, %pTK+ e %pTCl-. O RFG e a EK+ diminuÃram aos 60 min e aumentaram aos 90 e 120 min quando comparado com o grupo controle. A infusÃo de Lys 49 causou um aumento significante na PP, FU, ENa+, EK+ e Cosm e diminuiu o RFG e o %TNa+ quando comparada com o grupo controle. Lys 49 nÃo modificou os outros parÃmetros funcionais renais. A infusÃo de Asp 49 modificou apenas os parÃmetros funcionais renais %pTK+ (diminuiÃÃo) e EK+ (aumento) quando comparada ao grupo controle. Lys 49 apresentou um efeito similar ao veneno total nos parÃmetros FU, %TNa+, ENa+, EK+ e Cosm e Asp 49 nos parÃmetros %pTK+ e EK+. A anÃlise histolÃgica mostrou uma quantidade moderada de material proteinÃceo nos glomÃrulos e tÃbulos de rins perfundidos com o veneno, Lys 49 e Asp 49, bem como regiÃes focais de apoptose/necrose em rins perfundidos com Lys 49 e Asp 49. CÃlulas MDCK foram cultivadas em meio de cultura RPMI 1640 suplementado com 10% vv de soro bovino fetal e entÃo avaliadas na presenÃa do veneno total, Lys 49 e Asp 49 de B. erythromelas nas concentraÃÃes (100; 50; 25; 12,5; 6,25 e 3,125mg/mL). A anÃlise dos efeitos citotÃxicos em cÃlulas MDCK foi executada pelo mÃtodo MTT. O veneno promoveu efeito citotÃxico nas concentraÃÃes de 50 e 100mg/mL (IC50 =93,31mg/mL). Lys 49 promoveu efeito citotÃxico nas concentraÃÃes de 6,25; 12,5; 25; 50 e 100mg/mL (IC50 = 38,29mg/mL). Asp 49 promoveu efeito citotÃxico nas concentraÃÃes de 50 e 100mg/mL (IC50 = 158mg/mL). TambÃm foram mensurados os nÃveis de lactato desidrogenase (LDH) e nenhum aumento significante foi observado com veneno total e Asp 49. Lys 49 promoveu um aumento significante nos nÃveis de lactato desidrogenase apenas na concentraÃÃo de 100mg/mL. ApÃs o cultivo de cÃlulas MDCK com o veneno total, nas concentraÃÃes de 46,65 e 23,32mg/mL, foi realizada a reaÃÃo de polimerase em cadeia em tempo real para a avaliaÃÃo da expressÃo de genes prà (Caspase-3, Caspase-8 e Bax) e antiapoptÃticos (Bcl-XL e Mcl-1). NÃo foi realizada avaliaÃÃo da expressÃo de genes prà e antiapoptÃticos com Lys 49 e Asp 49. Na expressÃo de genes prÃ-apoptÃticos o veneno total promoveu um aumento da expressÃo de caspase-3 na concentraÃÃo de 23,32Âg/mL e de caspase-8 nas concentraÃÃes de 46,65 e 23,32Âg/mL, quando comparado com os controles positivo (DOXO) e negativo (PBS) e diminuiu a expressÃo de Bax em ambas as concentraÃÃes. Na expressÃo de genes antiapoptÃticos o veneno total promoveu induÃÃo significativa de Mcl-1 somente na concentraÃÃo de 46,65mg/mL e nÃo modificou a expressÃo de Bcl-XL, quando comparado com o controle negativo. O veneno e as fraÃÃes PLA2 Lys 49 e Asp 49 da serpente Bothropoides erythromelas à capaz de promover significativos efeitos sobre os parÃmetros de funÃÃo renal e sobre cÃlulas MDCK, com indicativo de morte celular por apoptose atravÃs da via extrÃnseca. / Bothropoides erythromelas is responsible for a great deal of snakebites in Northeastern from Brazil. The venom of this snake induces acute renal failure. Isolated kidneys from Wistar rats, weighting 250 to 300g, were perfused with Krebs-Henseleit solution containing 6g% of bovine serum albumin for 120 min. The whole venom of Bothropoides erythromelas been previously studied (SOUSA, 2004) and used in this study for comparison with the treated groups with the PLA2 fractions Lys 49 and Asp 49 of the venom. The whole venom (10mg/mL) and the fractions PLA2 Lys 49 and Asp 49 of B. erythromelas (5mg/mL) were added into the system 30 min after the beginning of each experiment. The parameters studied included perfusion pressure (PP), renal vascular resistance (RVR), glomerular filtration rate (GFR), urinary flow (UF), percent sodium, potassium and chloride tubular transport (%TNa+, %TK+ and %TCl-), percent sodium, potassium and chloride proximal transport (%pTNa+, %pTK+ and %pTCl-), sodium, potassium and chloride excretion (ENa+, EK+ e ECl-) and osmotic clearance (Cosm) (p< 0.05*). The control group perfused with albumin was functionally stable for over 120 min. The infusion of venom caused a significant increase in UF, ENa+, ECl- and Cosm and a decreased in PP, RVR, %TNa+, %TK+, %TCl-, %pTNa+, %pTK+ and %pTCl-. The GFR and the EK+ decreased at 60 min and increased at 90 and 120 min when compared with control group. The infusion of Lys 49 caused a significant increase in PP, UF, ENa+, EK+ and Cosm and decreased the GFR and the %TNa+ when compared with control group. Lys49 did not modify the others functional kidney parameters. The infusion of Asp 49 only modify the functional kidney parameters %pTK+ (decreased) and EK+ (increase) when compared with control group. Lys 49 showed a similar effect at whole venom in parameters UF, %TNa+, ENa+, EK+ and Cosm and Asp 49 in parameters %pTK+ and EK+. The histological analysis showed a mild amount of a proteinaceous substance in the renal tubules and glomeruli of kidneys perfused with the venom, Lys 49 and Asp 49, as well as focal areas of apoptosis/necrosis in perfused kidneys with Lys 49 e Asp 49. MDCK cells were cultured in RPMI 1640 medium supplemented with 10% vv fetal bovine serum and then assessed in the presence of the whole venom, Lys 49 and Asp 49 of B. erythromelas in the concentrations (100; 50; 25; 12.5; 6.25 and 3.125mg/mL). The analysis of cytotoxic effects on MDCK cells was performed by MTT method. The venom promoted cytotoxic effect in the concentrations of 50 and 100mg/mL (IC50 =93.31mg/mL). Lys 49 promoted cytotoxic effect in the concentrations of 6.25; 12.5; 25; 50 and 100 mg/mL (IC50 = 38.29mg/mL). Asp 49 promoted cytotoxic effect in the concentrations of 50 and 100 mg/mL (IC50 = 158mg/mL). Also the levels of lactic dehydrogenase (LDH) were measured and no significant increase was observed with whole venom and Asp 49. Lys 49 promoted a significant increase in the levels of LDH only in the concentration of 100mg/mL. After culture of MDCK cells with the whole venom, at concentrations of 46.65 and 23.32Âg/mL, was performed the real time polymerase chain reaction for evaluation of pro (Caspase-3, Caspase-8 and Bax) and antiapoptotic (Bcl-XL and Mcl-1) genes expression. The evaluation of pro and antiapoptotic genes expression with Lys 49 e Asp 49 did not realized. In the expression of pro-apoptotic genes the whole venom caused increase of caspase-3 at concentration of 23.32Âg/mL and of caspase-8 at concentrations of 46.65 and 23.32Âg/mL, when compared with negative (PBS) and positive (DOXO) controls and decreased the expression of Bax in both concentrations. In the expression of anti-apoptotic genes the whole venom caused significant induction of Mcl-1 only at a concentration of 46.65Âg/mL and did not modify the expression of Bcl-XL, when compared with the negative control. The venom and the fractions PLA2 Lys 49 e Asp 49 of Bothropoides erythromelas is able to promote significant effects on renal function parameters and on MDCK cells, with indications of cell death by apoptosis through the extrinsic pathway.

La biosurveillance humaine permet aujourd’hui d’évaluer notre exposition aux produits chimiques par la mesure soit des substances elles-mêmes, soit de leurs métabolites ou marqueurs d'effets sur la santé, à partir des fluides corporels ou des tissus. Les informations recueillies dans le cadre d’enquêtes épidémiologiques renseignent sur l'exposition humaine et constituent des bases précieuses dans la recherche des relations exposition-réponse chez les humains. Les travaux de cette thèse s’inscrivent dans le cadre de l’enquête transversale IMePoGe conduite entre 2008-2010 dans la région Nord–Pas-de-Calais (au nord de la France), incluant 2000 résidents adultes âgés de 20 à 59 ans, et visant à quantifier les niveaux d’imprégnation de la population à 14 métaux et métalloïdes (aluminium, antimoine, arsenic total, béryllium, cadmium, cobalt, chrome, mercure, manganèse, nickel, plomb, thallium, vanadium, zinc) choisis pour leurs effets toxiques et la fréquence de l’exposition professionnelle et environnementale. Les objectifs spécifiques de cette thèse étaient, tout en portant une attention particulière au plomb et au cadmium, deux métaux néphrotoxiques connus dans la littérature : i) d’établir la distribution de l’imprégnation aux métaux et métalloïdes dans la population du Nord de la France et de comparer le niveau régional d’imprégnation avec les données nationales et internationales ; ii) d’identifier les facteurs majeurs de variation de l’imprégnation et les sources d’exposition au plomb et au cadmium dans la population générale ; iii) d’étudier la relation entre la variation du débit de filtration glomérulaire et les niveaux d’imprégnation aux métaux. Globalement, les concentrations sanguines et urinaires de la plupart des métaux et métalloïdes étaient plus élevées que celles rapportées dans l’enquête nationale nutrition santé conduite sur la même période dans la population française, à l’exception du vanadium urinaire et du plomb sanguin. La plombémie moyenne régionale (moyenne géométrique) était de 18,8 μg/L. De nombreuses sources d’exposition au plomb existaient dans la population et étaient à la fois d’origine professionnelle, environnementale et alimentaire. Pour ce qui est du cadmium, le tabagisme se présentait comme la principale source d’exposition récente ou chronique au métal : la cadmiémie moyenne, reflet de l’exposition récente, était de 0,39 μg/L et passait de 0,26 μg/L chez les non-fumeurs à 0,84 μg/L chez les fumeurs ; la cadmiurie moyenne, reflet de l’exposition chronique, était de 0,37 μg/L (0,33 μg/g créatinine) et passait de 0,33 μg/L (0,29 μg/g créatinine) chez les non-fumeurs à 0,46 μg/L (0.37 μg/g créatinine) chez les fumeurs. Enfin, dans le cadre de l’étude de la relation entre l’imprégnation aux métaux et le débit de filtration glomérulaire, notre étude a montré que la prise en compte de la co-exposition à d’autres métaux et métalloïdes potentiellement néphrotoxiques bouleversait considérablement les associations antérieures jusqu’ici rapportées spécifiquement avec le plomb et le cadmium, dans le cadre de faibles niveaux d’imprégnation en population générale. / Human Biomonitoring allows us to evaluate our exposure to chemicals by measuring substances themselves or their metabolites or markers of health effects, from body fluids or tissues. The information collected through epidemiological surveys provide information on human exposure and are valuable databases in the research of exposure-response relationships in humans. This thesis is part of the cross-sectional IMePoGe survey conducted between 2008-2010 in the Nord–Pas-de-Calais region (in northern France), including 2,000 adult residents aged 20 to 59 years old, and aimed to quantify the impregnation levels of the population to 14 metals and metalloids (aluminum, antimony, total arsenic, beryllium, cadmium, cobalt, chromium, mercury, manganese, nickel, lead, thallium, vanadium, zinc) chosen for their toxic effects and the frequency of occupational and environmental exposure. The specific objectives of this thesis were, which a special interest for lead and cadmium, two nephrotoxic metals known in the literature: i) to establish the distribution of impregnation metals into the northern population of France and compare the exposure regional level to metals and metalloids with the national and international data; ii) to identify the major factors of variation of the impregnation and the sources of exposure to lead and cadmium in the general population; iii) to study the relationship between the change in glomerular filtration rate and the impregnation levels to metals. Overall, blood and urinary concentrations of most metals and metalloids were higher than those found in the national nutritional health survey conducted during the same period in the French population, with the exception of urinary vanadium and blood lead. The regional mean of blood lead level (geometric mean) was 18.8 μg/L. Several sources of lead exposure existed in the population and were link to the occupational, environmental and consumption parameters. Regarding cadmium, smoking was the main source of recent or chronic exposure to metal: the geometric mean of blood cadmium, reflecting a recent exposure, was 0.39 μg/L and increased from 0.26 μg/L in non-smokers to 0.84 μg/L in smokers; the geometric mean of urinary cadmium, reflecting the chronic exposure, was 0.37 μg/L (0.33 μg/g creatinine) and increased from 0.33 μg/L (0.29 μg/g creatinine) in non-smokers to 0.46 μg/L (0.37 μg/g creatinine) in smokers. Finally, as part of the study of the relationship between the metal levels and the glomerular filtration rate, our study showed that taking into account the multiple exposure to the other potentially nephrotoxic metals and metalloids upset considerably the previous associations specifically reported with lead and cadmium, in the context of low levels exposure in the general population.