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A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand In fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, 2003 / Dietary iron (Fe) overload, originally referred to as Bantu Visceral Siderosis, is an Reloading condition that is still prevalent in rural populations of sub-Saharan Africa. The better known Fe loading disease, hereditary haemochromatosis (HFI) is frequently complicated by hepatocellular carcinoma (HCC) and, in rare instances this occurs in the absence of cirrhosis. The latter, together with recent evidence that dietary Fe overload in the Black African carries an increased risk for HCC, suggests that excessive hepatic iron may itself be carcinogenic. The aim of the study was to determine if Fe alone could induce HCC in experimental rat models and, if so, to investigate possible mechanisms of hepatocarcinogenesis. 360 Wistar albino rats (Rattus norvegicus) were divided into 6 groups. The first group, the control animals, was designated C group. Groups 2-6 were Fe-fed alone or in combination with other chemicals: group 2 Fe alone (Fe group), group 3 (Fe + V) vitamins A & E supplementation [50 mg all trans-retinol (vitamin A) and 500 mg a-tocopherol (vitamin E) per kg diet], group 4 (Fe - V) received a diet totally devoid of vitamins A & E, group 5 (Fe + ASA) received 20 mg aspirin (ASA) per day, group 6 (Fe + Cu) received 300 mg/kg diet of copper sulphate (CuS04) supplementation for 12 months followed by 3% copper hydroxide carbonate [CuC03»Cu(0H)2] / IT2018

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand In fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, 2003 / Dietary iron (Fe) overload, originally referred to as Bantu Visceral Siderosis, is an Fe- loading condition that is still prevalent in rural populations of sub-Saharan Africa. The better known Fe loading disease, hereditary haemochromatosis (HH) is frequently complicated by hepatocellular carcinoma (HCC) and, in rare instances this occurs in the absence of cirrhosis. The latter, together with recent evidence that dietary Fe overload in the Black African carries an increased risk for HCC, suggests that excessive hepatic iron may itself be carcinogenic. The aim of the study was to determine if Fe alone could induce HCC in experimental rat models and, if so, to investigate possible mechanisms of hepatocarcinogenesis. 360 Wistar albino rats (Rattus norvegicus) were divided into 6 groups. The first group, the control animals, was designated C group. Groups 2 - 6 were Fe-fed alone or in combination with other chemicals: group 2 Fe alone (Fe group), group 3 (Fe + V) vitamins A & E supplementation [50 mg all trans-retinol (vitamin A) and 500 mg a-tocopherol (vitamin E) per kg diet], group 4 (Fe - V) received a diet totally devoid of vitamins A & E, group 5 (Fe + ASA) received 20 mg aspirin (ASA) per day, group 6 (Fe + Cu) received 300 mg/kg diet of copper sulphate (CuS04) supplementation for 12 months / IT2018

ArtinM é uma lectina ligante a carboidrato D-manose que se interage a receptores de células fagocíticas induzindo a produção de mediadores pró- inflamatórios relacionados à resposta imune antitumoral. Aflatoxinas são micotoxinas produzidas por fungos do gênero Aspergillus. A Aflatoxina B1 (AFB1) é a toxina sintetizada mais abundantemente e a que apresenta o maior poder toxigênico, sendo capaz de induzir carcinoma hepatocelular (CHC) em humanos. O objetivo deste estudo foi investigar o papel da lectina ArtinM na hepatocarcinogênese induzida pela AFB1 em ratos. Setenta e dois ratos recém-desmamados foram divididos em três grupos: Controle - animais tratados com veículo; AFB1 - animais intoxicados com AFB1; AFB1+ArtinM - animais tratados com AFB1 e ArtinM. Ratos Wistar foram intoxicados por gavagem com 400 ?g de AFB1 por quilograma de ração ingerida durante três meses, enquanto o grupo AFB1+ArtinM recebeu adicionalmente três doses da lectina por via subcutânea (50 ?g por quilograma de peso do animal por dose) nos 45, 60 e 75 após inicio do experimento. Animais foram eutanasiados 3 e 12 meses após início das gavagens. A expressão hepática de proteínas relacionadas à hepatocarcinogênese foi avaliada por técnicas de imunohistoquímica, Western blotting e PCR em tempo real nos animais eutanasiados após 3 meses de intoxicação. A incidência de lesões pré-neoplásicas e de tumores hepáticos foi mensurada 3 e 12 meses após início das gavagens, respectivamente. Os animais tratados com ArtinM apresentaram maior expressão hepática de proteínas supressoras tumorais além de redução do número de focos pré-neoplásicos e de tumores hepáticos em relação aos animais que receberam apenas a micotoxina. Conclui-se, portanto, que ArtinM possui efeito protetor durante o processo de hepatocarcinogênese induzida por AFB1. / ArtinM is a D-mannose carbohydrate-binding lectin that interacts with phagocytic cell receptors inducing the production of pro-inflammatory mediators related to the antitumor immune response. Aflatoxins are mycotoxins produced by fungi of the genus Aspergillus. Aflatoxin B1 (AFB1) is the toxin most abundantly synthesized and the one with the highest toxigenic power, being able to induce hepatocellular carcinoma (HCC) in humans. The aim of this study was to investigate the role of ArtinM lectin in AFB1-induced hepatocarcinogenesis in rats. Seventy-two newly weaned rats were divided into three groups: Control - vehicle-treated animals; AFB1 - animals poisoned with AFB1; AFB1 + ArtinM - animals treated with AFB1 and ArtinM. Wistar rats were gavage-poisoned with 400 ?g AFB1 per kilogram of ration fed for three months, while the AFB1 + ArtinM group received three subcutaneous doses of the lectin (50 ?g per kilogram of animal weight per dose) 45, 60 and 75 days after the start of the experiment. Animals were euthanized 3 and 12 months after initiation of treatments. Hepatic expression of hepatocarcinogenesis-related proteins was assessed by immunohistochemistry, Western blotting, and realtime PCR in euthanized animals after three months of intoxication. The incidence of pre-neoplastic lesions and liver tumors was measured 3 and 12 months after the start of treatments, respectively. Animals treated with ArtinM had fewer pre-neoplastic foci and hepatic tumors than the animals that receiving mycotoxin alone, as well as showing greater hepatic expression of tumor suppressor proteins. It is concluded, therefore, that ArtinM has a protective effect during the process of hepatocarcinogenesis induced by AFB1.

ArtinM é uma lectina ligante a carboidrato D-manose que se interage a receptores de células fagocíticas induzindo a produção de mediadores pró- inflamatórios relacionados à resposta imune antitumoral. Aflatoxinas são micotoxinas produzidas por fungos do gênero Aspergillus. A Aflatoxina B1 (AFB1) é a toxina sintetizada mais abundantemente e a que apresenta o maior poder toxigênico, sendo capaz de induzir carcinoma hepatocelular (CHC) em humanos. O objetivo deste estudo foi investigar o papel da lectina ArtinM na hepatocarcinogênese induzida pela AFB1 em ratos. Setenta e dois ratos recém-desmamados foram divididos em três grupos: Controle - animais tratados com veículo; AFB1 - animais intoxicados com AFB1; AFB1+ArtinM - animais tratados com AFB1 e ArtinM. Ratos Wistar foram intoxicados por gavagem com 400 ?g de AFB1 por quilograma de ração ingerida durante três meses, enquanto o grupo AFB1+ArtinM recebeu adicionalmente três doses da lectina por via subcutânea (50 ?g por quilograma de peso do animal por dose) nos 45, 60 e 75 após inicio do experimento. Animais foram eutanasiados 3 e 12 meses após início das gavagens. A expressão hepática de proteínas relacionadas à hepatocarcinogênese foi avaliada por técnicas de imunohistoquímica, Western blotting e PCR em tempo real nos animais eutanasiados após 3 meses de intoxicação. A incidência de lesões pré-neoplásicas e de tumores hepáticos foi mensurada 3 e 12 meses após início das gavagens, respectivamente. Os animais tratados com ArtinM apresentaram maior expressão hepática de proteínas supressoras tumorais além de redução do número de focos pré-neoplásicos e de tumores hepáticos em relação aos animais que receberam apenas a micotoxina. Conclui-se, portanto, que ArtinM possui efeito protetor durante o processo de hepatocarcinogênese induzida por AFB1. / ArtinM is a D-mannose carbohydrate-binding lectin that interacts with phagocytic cell receptors inducing the production of pro-inflammatory mediators related to the antitumor immune response. Aflatoxins are mycotoxins produced by fungi of the genus Aspergillus. Aflatoxin B1 (AFB1) is the toxin most abundantly synthesized and the one with the highest toxigenic power, being able to induce hepatocellular carcinoma (HCC) in humans. The aim of this study was to investigate the role of ArtinM lectin in AFB1-induced hepatocarcinogenesis in rats. Seventy-two newly weaned rats were divided into three groups: Control - vehicle-treated animals; AFB1 - animals poisoned with AFB1; AFB1 + ArtinM - animals treated with AFB1 and ArtinM. Wistar rats were gavage-poisoned with 400 ?g AFB1 per kilogram of ration fed for three months, while the AFB1 + ArtinM group received three subcutaneous doses of the lectin (50 ?g per kilogram of animal weight per dose) 45, 60 and 75 days after the start of the experiment. Animals were euthanized 3 and 12 months after initiation of treatments. Hepatic expression of hepatocarcinogenesis-related proteins was assessed by immunohistochemistry, Western blotting, and realtime PCR in euthanized animals after three months of intoxication. The incidence of pre-neoplastic lesions and liver tumors was measured 3 and 12 months after the start of treatments, respectively. Animals treated with ArtinM had fewer pre-neoplastic foci and hepatic tumors than the animals that receiving mycotoxin alone, as well as showing greater hepatic expression of tumor suppressor proteins. It is concluded, therefore, that ArtinM has a protective effect during the process of hepatocarcinogenesis induced by AFB1.

Hepatocarcinoma cancer (HCC) is one the most common visceral malignancies in Taiwan, which has a very high incidence and a devastatingly poor prognosis. BMP4, belonging to the TGF-£] super-family of proteins is a multifunctional cytokine, known to exert its biological effects through SMAD and non-SMAD dependent pathways and is also known to be involved in human carcinogenesis. However, the effects of the BMP4 signaling in liver carcinogenesis are not yet clearly defined. In this study, we first demonstrate that BMP4 and its receptor, BMPR1A, are over-expressed in a majority of primary HCC and promote the growth and migration of HCC cell lines in vitro. We also further identify that BMP4 can induce HCC CDK1 and cyclinB1 up-regulation to accelerate cell cycle progression. Our study indicates that the induction of HCC cell proliferation is independent on the SMAD signaling pathway, since Smad4 knockdown of BMP4 induced HCC cell lines still leads to the up-regulation of CDK1 and cyclinB1 expression in HCC. Using MEK kinase selective inhibitors, the induction of CDK1 and cyclinB1 mRNA and protein were shown to be dependent on the activation of MEK/ERK signaling. In vivo xenograft studies confirmed that the BMPR1A- knockdown cells were significantly less tumorigenic than control groups. Taken together, our findings show that the up-regulation of BMP4 and BMPR1A in HCC promote the proliferation and metastasis of HCC cells and that CDK1 and cyclinB1 are important, SMAD-independent molecular targets in BMP4 signaling pathways during the HCC tumorigenesis. We propose here that BMP4 signaling pathways may have potential as new therapeutic targets, in HCC treatment.

Cell proliferation plays an important role in multistage chemical carcinogenesis. Again, several reports demonstrated that upregulation of metallothionein (MT) expression is associated with increased cell proliferation that may contribute to the pathogenesis of preneoplastic phenotype to frank malignancy. In this study, we evaluated the roles of early DNA damage, altered expressions of liver MT and Ki-67 nuclear antigen, and altered hepatic levels of zinc (Zn) and copper (Cu) on cell proliferation and the progression of hepatocarcinogenesis through premalignant, late premalignant and malignant transformation phases in male Sprague-Dawley rats. We have further studied the association between MT expression and cell proliferation in hepatocarcinogenesis. There was substantial induction of DNA single-strand breaks (SSBs) (P < 0.001) and development of hepatocellular premalignant lesions along with significant decrease in hepatic levels of Zn and increase in Cu content following a single, necrogenic, intraperitoneal (i.p.) injection (200 mg/Kg body weight) of diethylnitrosamine (DEN) at week 4 of the experimental protocol. Moreover, DEN + phenobarbital (PB)-treatment significantly elevated MT-, Ki-67-, and BrdU-immunoexpressions along with their immunolabeling indices. Furthermore, positive correlations between MT- and Ki-67- labeling (P = 0.0006) at various time intervals, as well as, between MT immunoreactivity and 5'-bromo-2'-deoxyuridine-labeling index (BrdU-LI) (P = 0.0007) indicate that, MT expression might be associated with Ki-67 expression and cell proliferation thereby. The study suggests that DEN treatment may lead to alteration of Zn and Cu levels resulting in early DNA damage along with elevation of MT expression that may ultimately lead to hepatic cell proliferation. The results thus provide evidence in support of the role of MT as a potential positive regulator of cell growth during the early stages of hepatocellular transformation in rats.

Carcinogen-induced formation of DNA adducts and other types of DNA lesions are the critical molecular events in the initiation of chemical carcinogenesis and modulation of such events by chemopreventive agents could be an important step in limiting neoplastic transformation in vivo. Vanadium, a dietary micronutrient has been found to be effective in several types of cancers both in vivo and in vitro and also possesses profound anticarcinogenicity against rat models of mammary, colon and hepatocarcinogenesis. Presently, we report the chemopreventive potential of vanadium on diethylnitrosamine (DEN)-induced early DNA damages in rat liver. Hepatocarcinogenesis was induced in male Sprague-Dawley rats with a single, necrogenic, intraperitoneal (i.p.) injection of DEN (200 mg/kg body weight) at week 4. There was a significant induction of tissue-specific ethylguanines, steady elevation of modified DNA bases 8-hydroxy-2′-deoxyguanosines (8-OHdGs) (P < 0.0001; 89.93%) along with substantial increment of the extent of single-strand breaks (SSBs) (P < 0.0001) following DEN exposure. Supplementation of 0.5 ppm of vanadium throughout the experiment abated the formations of O6-ethylguanines and 7-ethylguanines (P < 0.0001; 48.71% and 67.54% respectively), 8-OHdGs (P < 0.0001; 81.37%), length:width (L:W) of DNA mass (P < 0.01; 62.12%) and the mean frequency of tailed DNA (P < 0.001; 53.58%), and hepatic nodulogenesis in preneoplastic rat liver. The study indicates that 0.5 ppm vanadium is potentially and optimally effective, as derived from dose-response studies, in limiting early molecular events and preneoplastic lesions, thereby modulating the initiation stage of hepatocarcinogenesis. Vanadium is chemopreventive against DEN-induced genotoxicity and resulting hepatocellular transformation in rats.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Exposure to food contaminates such as mycotoxins have been associated with a variety of animal and human diseases worldwide. In South Africa, maize is the most To further refine risk assessment in the socio-demographic heterogeneous population of South Africa, the development and evaluation of a sensitive and interactive model the Mycotoxin Risk Assessment Model (MYCORAM) proofed to be more sensitive compared to the classical probable daily intake (PDI). The development of the MYCORAM was based on mycotoxin distribution during dry milling of maize in milling fractions intended for human consumption which was superimposed on the maize intake profiles of the South African population. Although dry milling, including a degerming step, is an effective way to reduce mycotoxins, risk and exposure assessment are influenced by maize dietary intakes, gender and ethnicity. This became evident when considering FB dietary exposure in rural maize subsistence farming communities in the Eastern Cape Province, South Africa confirmed the vulnerability of this subpopulation to risk of fumonisin exposure. Specific maximum tolerated maximum levels (MTL) to safeguard these communities fall outside the international regulatory processes and need to be urgently addressed. With the complex nature of cancer development in mind, integration of basic science and nutritional epidemiology will be important to contribute to our understanding of the adverse effects of FB and to define relevant risk assessment parameters. important commercial grain crop not just economically but also as a local food commodity both commercially and in subsistence rural farming communities. In order to control and manage mycotoxin contamination in food, evidence-based risk assessment is needed that includes mechanistic and human exposure studies. From this perspective the current study was conducted and aimed in further unravelling fumonisin B1 (FB1) mycotoxin induced hepatocarcinogenesis via the disruption of the lipid metabolism. The study also critically evaluates aspects of human risk assessment due to its relevance and importance to food safety known to impact on food security. This entails mycotoxin distribution during maize dry milling and the assessment of mycotoxin exposure in the South African population and vulnerable rural communities at risk. Fumonisin B1 affects the integrity of biological membranes by altering key lipid and fatty acid parameter in plasma, microsomal, mitochondrial and nuclear subcellular membrane fractions in rat liver. Changes in the major lipid constituents entailing an increase in cholesterol (CHOL) and phosphatidylethanolamine (PE) whilst sphingomyelin (SM) and phosphatidylcholine (PC) tended to decrease. Isolated plasma membrane lipid rafts, from rat primary hepatocytes exposed to FB1 augments the intricate effects exerted on the lipid metabolism regarding CHOL, SM and PE. The disruption of lipid and fatty acid constituents, such as arachidonic acid and ceramide, are likely to be key determinants affecting growth regulatory signaling pathways relevant to the critical balance between cell proliferation and apoptosis during cancer promotion. These changes provide further evidence that FB1 induce cancer promotion by differential inhibition and/or stimulation process whereby a few resistant “initiated” hepatocytes proliferate in an environment where the growth of normal cells is inhibited. A specific lipogenic phenotype is effected by FB1 which is closely associated with cancer development and considered to occur via an epigenetic-type of mechanism. These effects are not adequately addressed in defining risk assessment parameters. / AFRIKAANSE OPSOMMING: Die blootstelling aan voedsel-kontaminante soos mikotoksienes word wêreldwyd met ‘n verskeidenheid van dierlike en menslike siektes geassosiseer. In Suid-Afrika word mielies as ‘n belangrike graanoes beskou, nie net vir die ekonomie nie maar ook as ‘n plaaslike voedselproduk beide kommersieel en vir bestaansboere in landelike gemeenskappe. Ten einde mikotoksien-kontaminasie van voedsel te kan beheer en bestuur, vereis bewys-gebaseerde risiko-evaluering wat insluit meganistiese en menslike blootstelling studies. Vanuit hierdie perspektief is die huidige studie uitgevoer en gemik op die verdere ontleding van die fumonisin B1 (FB1) mikotoksien geïnduseerde lewer-karsinogenese deur die ontwrigting van die lipiedmetabolisme. Die studie ondersoek terselfdetyd aspekte van menslike risiko-evaluering ingevolge die relevansie en belangrikheid hiervan in voedselveiligheid wat ook ‘n impak op voedselsekerheid sal maak. Dit sluit in die verspreiding van mikotoksiene gedurende die droëmaalproses van mielies en mikotoksien blootstelling in Suid-Afrika asook onder kwesbare landelike gemeenskappe. Fumonisin B1 beïnvloed die integriteit van biologiese membrane deur die modulasie van die belangrike lipied en vetsuur samestelling van plasma, mikrosomale, mitochondriale en kern subsellulêre membraan-fraksies in rot lewer. Veranderinge in die belangrike lipiedbestanddele, insluitende ‘n verhoging in cholesterol (CHOL) en phosphatidylethanolamine (PE), terwyl sphingomyelin (SM) en phosphatidylcholine (PC) geneig was om te verlaag. Geïsoleerde plasma membraan lipied vlotte (lipid rafts), vanaf primêre rot hepatosiete blootgestel aan FB1, versterk die ingewikkelde gevolge wat uitgeoefen word op die lipiedmetabolisme insluitende die voorgestelde veranderings in CHOL, SM en PE vlakke. Die versteuring van lipiede en vetsure soos aragidoonsuur (arachidonic acid) en ceramied kan beskou word as belangrike determinante wat inmeng in groei-regulerende seinbane verwant aan die kritiese balans tussen selgroei en seldood. Die versteurings verskaf verdere bewyse dat FB1 kanker bevorder deur ‘n seleksie proses wat onderskeidelike die onderdrukking en\of die stimulasie van ‘n paar weerstandige of geneties veranderde hepatosiete laat vermeerder in ‘n omgewing waar die groei van normale selle geïnhibeer word. Die spesifieke lipogeniese fenotipe wat FB1 versoorsaak hou ten nouste verband met kankerontwikkeling en die voorkoms van epigenetiese-soort meganismes word voorgestel. Hierdie oorsake word tans nie voldoende aangespreek tydens die bepaling van risiko-evaluerings limiete nie. Om risiko-bepaling verder te verbeter in die sosio-demografies heterogene populasie van Suid-Afrika, was die ontwikkeling en evalueering van ‘n sensitiewe en interaktiewe model, die “Mycotoxin Risk Assessment Model” (MYCORAM) meer doeltreffend vergeleke met die gewone waarskynlike daaglikse inname. Die ontwikkeling van die MYCORAM was gebaseer op die mikotoksien verspreiding tydens die droëmaalproses van mielies in fraksies wat vir menslike verbruik bedoel was tesame met mielie dieetinnames van die Suid-Afrikaanse populasie. Alhoewel, die droëmaalproses van mielies, insluitende die verwydering van die kiem doeltreffende maniere is om mikotoksienes te verminder, word risiko- en blootstellings evaluering beinvloed deur mielie dieetinnames, geslag en etnieseverbandskap. Hierdie was veral opmerklik gedurende blootstelling aan FB in die dieet van landelike mielie bestaansboer gemeenskappe in die Oos-Kaap van Suid- Afrika en bevestig hoe kwesbaar hierdie populasie is. Spesifieke maksimum toelaatbare vlakke om hierdie gemeenskappe te beskerm val buite die huidige internasionale regulatoriese prosesse en benodig dringende aandag. Met die ingewikkelde aard van kankerontwikkeling in gedagte, sal die integrasie van basiese wetenskappe en voedingsepidemiologie, ‘n belangrik bydrae lewer tot die kennis van die negatiewe eienskappe van FB om toepaslike risiko-evaluerings limiete te kan bepaal.

Thesis (DSc (Biochemistry))--Stellenbosch University, 2009. / The fumonisin mycotoxins are known to be the causative principle for several animal diseases and are associated with the development of liver and oesophagus cancer and neural tube defects in humans. The thesis focuses mainly on the characterisation of the compounds from maize cultures of the fungus Fusarium verticillioides, isolated from maize, the toxicological effects in animals, mechanism involved in hepato- and nephrocarcinogenicity and discussing the major differences and contradictions in the literature together with their impact on setting relevant risk assessment parameters to safeguard human health. Controversies include the importance of non-genotoxicity vs genotoxicity in the development of cancer, the role of threshold effects in carcinogenesis and the establishment of realistic risk assessment parameters that will also be applicable in developing countries. Recent approaches suggest that thresholds should also apply for genotoxic carcinogens as interaction with the DNA is only one event in the multi-step process of cancer development and therefore could not be taken as the basis for applying a no-effect threshold for genotoxins. It would appear that a carcinogen such as fumonisin, whether it is labeled genotoxic or non-genotoxic per se, exhibits some degree of risk at any level due to additive or synergistic interactions with other xenobiotics and/or dietary constituents. The underlying mechanisms of fumonisin-induced carcinogenicity includes the disruption of sphingolipid, phospholipids and fatty acid metabolism, which plays a major role in the modulation of apoptotic and cell proliferative pathways related to cancer development. Interactive responses between arachidonic acid and ceramide affect downstream cell signal transduction pathways and depending on the cell type the disruption of these pathways could either stimulate or inhibit cell proliferation which eventually will determine the induction of apoptosis and hence affect cell survival. The modulating roles of dietary constituents such as vitamins, protein and the South African herbal teas are also highlighted as they affected the outcome of toxicological assays, thus determining thresholds of the adverse effects in specific target organs that will impact risk assessment parameters. Regulation of the fumonisins in food and the associated risk are debated from many perspectives. In developing countries there is a lack of quality control implying that maize highly contaminated with mycotoxins may directly enter the food chain of adults and children as control of mycotoxins is difficult or in some cases totally absent. The interaction of politics, economy and technology will eventually determine the impact on health as the regulation of fumonisin in food differs between countries. Knowledge about the biological effects of the fumonisins is currently playing an important role in the development of simple and inexpensive methods to reduce the levels of the fumonisin in maize by targeting specific populations at risk.