ALTERED LIPID METABOLISM AS A POSSIBLE MECHANISM IN FUMONISIN- INDUCED HEPATOCARCINOGENESIS IN RATS AND INVESTIGATIONS INTO RISK ASSESSMENT IN HUMANS Hester Maria Burger Dissertation presented for the degree of DOCTOR OF PHILOSOPHY (BIOCHEMISTRY) In the Faculty of Science University of Stellenbosch PROMOTOR: PROF WCA GELDERBLOM CO-PROMOTORs: PROF P SWART DECEMBER 2013 Stellenbosch University http://scholar.sun.ac.za DECELRATION By submitting this dissertation, I declare that the entirity of the work contained therein is my own, original work, that I am the sole author thereof (save to the extent explicitly otherwise stated), that reproduction and publication thereof by Stellenbosch University will not infringe any third party rights and that I have not previously in its entirity or in part submitted it for obtaining any qualification. ___________________ ________________ Hester Maria Burger Date Copyright  2013 Stellenbosch University All rights reserved. i Stellenbosch University http://scholar.sun.ac.za SUMMARY 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 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 B (FB ) mycotoxin induced hepatocarcinogenesis via the disruption of the 1 1 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 B affects the integrity of biological membranes by altering key lipid and 1 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 FB 1 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 FB induce 1 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 FB which is 1 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. ii Stellenbosch University http://scholar.sun.ac.za 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. iii Stellenbosch University http://scholar.sun.ac.za 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 B (FB ) mikotoksien 1 1 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 B beïnvloed die integriteit van biologiese membrane deur die modulasie 1 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 FB versterk die ingewikkelde 1, 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 FB 1 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 FB versoorsaak hou ten nouste verband met 1 iv Stellenbosch University http://scholar.sun.ac.za 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 etniese- verbandskap. 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. v Stellenbosch University http://scholar.sun.ac.za Dedicated to my family: Albert, Johanna and Hendrik Burger Immeasurable grace from my Heavenly Father vi Stellenbosch University http://scholar.sun.ac.za In gratitude towards my Saviour and Lord, Christ Jesus “Therefore, behold, I will allure her and bring her into the wilderness, and I will speak tenderly and to her heart. There I will give her, her vineyards and make the Valley of Achor (Valley of trouble) to be for her a door of hope and expectation. And she shall sing there and respond as in the days of her youth and as at the time when she came up out of the land of Egypt. And it shall be in that day, says the Lord, that you will call Me Ishi (my Husband), and you shall no more call Me Baali (my Master)”. “And I will betroth you to Me forever; yes, I will betroth you to Me in righteousness and justice, in steadfast love, and in mercy. I will even betroth you to Me in stability and in faithfulness, and you shall know the Lord.” Hosea 2: 14-16 & 19-20, Scripture quotations taken from the Amplified® Bible. Copyright © 1954, 1958, 1962, 1964, 1965, 1987 by The Lockman Foundation. Used by permission. (www.Lockman.org) vii Stellenbosch University http://scholar.sun.ac.za TABLE OF CONTENTS PAGE Declaration i Summary ii Opsomming iv Dedication vi Abbreviations ix List of Tables xvii List of Figures xx List of Addenda xxiii CHAPTER 1 General Introduction 1 CHAPTER 2 Literature Review 9 CHAPTER 3 Altered lipid parameters in hepatic subcellular membrane fractions induced by 117 fumonisin B 1 CHAPTER 4 Fumonisin B -induced changes to the lipid composition of membrane rafts in 146 1 primary rat hepatocytes CHAPTER 5 The mycotoxin distribution in maize milling fractions under experimental 177 conditions CHAPTER 6 Development and evaluation of two sensitive mycotoxin risk assessment 204 models (MYCORAMs) CHAPTER 7 Dietary fumonisin exposure in a rural population of South Africa 233 CHAPTER 8 General Discussion and Conclusions 255 ACKNOWLEDGEMENTS 276 Addendum A 278 Addendum B 279 Addendum C 280 Addendum D 281 Addendum E 282 Addendum F 283 viii Stellenbosch University http://scholar.sun.ac.za ABBREVIATIONS % Percentage  Alpha  Omega  Beta  Gamma ˚C Degrees Celsius µ Micro µg Microgram AA Arachidonic acid ACP Acyl-carrier protein ADI Acceptable daily intake AF Aflatoxin AIN American Institute of Nutrition Akt Protein kinase B ALA Alpha- () linoleic acid ATP Adenosine triphosphate BHT Butylated hydroxytoluene bw Body weight C Carbon CaCl Calcium chloride 2 CANSA Cancer Association of South Africa Cat Catalogue CDase Ceramidase CDP Cytidine 5’-diphosphocholine citocoline CHOL Cholesterol ix