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CHAPTER 1 Introduction
1.1 THE DIET-CANCER RELATIONSHIP
Food is one of the ultimate complex mixtures to which man is exposed. Research indicates that dietary variables, anti-carcinogenic substances, and exposure to carcinogens that occur in foods or are induced during its' preparation, are relevant determinants in the diet-cancer relationship (Institute of Food Technologists, 1993). Given the popular misconception that the natural human diet is "safe", and that such risks are due to contamination by pesticides, xenobiotics and other "chemical" residues, there is a need for continual evaluation of the avenues open to investigators to reduce disease incidence. Well-structured studies would ultimately wield meaningful results enabling medical and scientifically based advice to be given to the public with regard to improving dietary habits.
Numerous hypotheses on cancer causation exist. Their diversity and variety often reflect the paucity or conflicting, often over-abundance of experimental and epidemiological evidence on which they are based. Correlation is not necessarily causation, and hypotheses, however plausible, require unbiased evaluation. Although the complete web of cancer is complex, medical science is concerned with effective action, and this firstly requires no more than the identification of the relevant proximate influence, which, if removed, would lead to a substantial reduction in the risk of disease. On this empirical basis, what is diagnosed as a causal relation may turn out to be a predisposing, carcinogenic or co-carcinogenic factor. These factors may be several degrees away from those immediately responsible for carcinogenesis, but their discovery would help disclose the chain of causality, whose precise links could then be ascertained (Oettle', 1965).
1.2 MYCOTOXINS
Food-borne illnesses are of global importance. Among the factors that may induce these illnesses, mycotoxins that are defined as secondary metabolites synthesised by fungi under certain environmental conditions, have received considerable attention (International Agency for Research on Cancer, 1993a). Mycotoxins are potentially
carcinogenic compounds produced by fungi mainly in the genera Aspergillus, Penicillium and Fusarium.
Mycotoxlns of importance in human health include the aflatoxins, ochratoxin A, fumonisins, trichothecenes and zearalenone (Pitt et aI., 2000). The interest they generate stems from their high toxicity and potency in producing tumours in laboratory animals (Institute of Food Technologists, 1993). Consumers of foods contaminated by fungi are vulnerable to acute toxicity and to chronic long-term mutagenic and immunosuppressive effects of mycotoxins, which exacerbate primary infections or interact with inter-current diseases. The present study focuses on the Fusarium mycotoxins fusaric acid (FA), moniliformin (MON), zearalenone (ZEA), deoxynivalenol (DON), and T -2 toxin (T2), with specific emphasis being placed on Fumonisin BI (FBI).
1.3 EXPOSURE TO Fusarium FUNGI AND FUMONISINS IN SOUTH AFRICA
Exposure to FBI and other Fusarium metabolites is a public health hazard where mass food production and improper storage conditions are conducive to mould spoilage and subsequent mycotoxin production. Between 1984 to 1993, over 1600 samples of agricultural commodities comprising maize, compound animal feeds, oil seeds, soy bean, fish meal and forage in KwaZulu-Natal (KZN), South Africa (SA) were examined using a multi-mycotoxin screen. Aflatoxin had the highest incidence with over 14% of all samples examined followed by trichothecenes at 10%, and ZEA at 4%. Since 1989, selected maize samples with high levels of Fusarium species were examined for FBI and of these (n=20), 90% were positive in 1993. As a consequence of these results and high incidence of Fusarium species (32%) in maize and maize containing feeds, which was higher than either Aspergillus species (27%) or Penicillium species (12%), concern was expressed with regard to the potential presence and exposure to fumonisin in the South African food chain (Dutton and Kinsey, 1996).
As the potential for daily dietary exposure to these compounds via the ingestion of contaminated foods and feeds exists, their importance in food safety and as causes of human illness has increased. The diseases produced as a result of the consumption of contaminated foods however, remains poorly understood at the clinical level.
Fusarium fungi have been implicated in haemorrhagic, oestrogenic, emetic, and feed refusal syndromes in animals. There is considerable evidence that the central nervous system (eNS) is a potential site of action for FBI and is exemplified in horses by the disease equine leukoencephalomalacia (ELEM). This disease is characterised by marked neurotoxicity and liquefactive necrotic lesions in the white matter of the cerebral hemispheres of the brain of horses and other equine species (Marasas et al., 1988a;
Kellerman et al., 1990; Wilson et al., 1992; Ross et al., 1993).
Studies on the pathophysiology of fumonisin toxicoses are essential in disease diagnosis, establishment of safety parameters, and development of control and prevention procedures.
As the potential for dietary exposure to Fusarium mycotoxins exists, the human eNS is at risk of exposure to their biochemical effects and potential carcinogenicity. The data that exists on the adverse health effects of fumonisins in animals serves as the basis for concern for their effects on human health. However difficulty is inherent in the extrapolation of risk from animals to humans, and in the determination of what a significant biological exposure constitutes, due to differences in species-specific responses to a compound.
Fusarium mycotoxins have been implicated in alimentary toxic aleukia, Drov or Kashin-Beck disease, Akakabi-byo or scabby grain intoxication, and oesophageal cancer in humans (Marasas et al., 1979a; 1988b; Rheeder et al., 1992). However, these published studies demonstrate inconclusive associations of fumonisins with human disease. In addition, minimal work has been channelled into investigating directly the possible implication of Fusarium mycotoxins, especially FBI, in the aetiology of human brain cancer, or in relation to isolated cells of the eNS and their lesions.
The selection of a single agent for investigation does not deny the complex aetiology of cancer, nor does it exclude the possibility of other factors contributing to disease causation.
Over simplification in an effort to shed clarity of understanding to a problem for proper elucidation is sometimes considered better than taking the dichotomous route of complication beyond recognition. This holds true especially if the latter were to become no more than a bewildered acknowledgment of complexity that explains away anomalies and effectively hinders logical analysis.
1.4 BRAIN CANCER IN SOUTH AFRICA
The National Cancer Registry (NCR) collates information on cancer diagnosed via a network of private and public pathology laboratories in SA. The most recent available report by Sitas et al. (1998), provides statistics for the time-frame between 1993 to 1995, that includes frequencies by population, sex, age-group, cancer site, age standardized, cumulative incidence rates and lifetime risks of developing cancer. These statistics (Table 1) indicate that a total of 387 females and 494 males were diagnosed with brain or CNS tumours between 1993 and 1995. The Asian and White race groups seemed more susceptible to development of tumours as indicated by the calculated lifetime risk of getting these tumours, with males across all race groups at greater risk than women (Table 1). Figures Al.l and Al.2 (Appendix 1) graphically illustrate the frequency of histologically diagnosed brain and CNS cancer, and the age specific cancer rates per 100 000 in SA between 1993 and 1995 (Sitas et aI., 1998).
Table 1:
93
Total 154
female
Total male 192
Asian 10
Female
Asian 6
Male
Black 45
Female
Blacl< 63 Male
Coloured 10 Female
Coloured 8 Male
White 69
Female
White 87
Male
Race 20
unknown Female
Race 28
unlmown male
Summary statistics for cancer in the brain and central nervous system in South Africa, 1993-1995 (Sitas et a/., 1998).
94 95 93 % No of cases/ Age Cumulative Lifetime
to of population, standardised incidence risk of
95 all Per incidence rate developing
cancers 100000 rate per (0-74 yrs) a cancer 100000
135 98 387 0.57 0.72 0.85 0.09 1111
142 160 494 0.69 0.94 1.1923 0.11 909
7 3 20 1.21 1.75 1.58 0.12 833
5 11 22 1. 91 2.03 2.34 0.24 417
31 29 105 0.51 0.29 0.31 0.03 3333
28 38 129 0.69 0.38 0.43 0.04 2500
7 3 20 0.86 0.53 0.56 0.04 2500
6 5 19 0.90 0.53 0.79 0.07 1429
58 38 165 0.56 7.80 2.62 0.26 385
62 65 214 0.61 3.78 3.57 0.34 294
32 25 67
41 41 110
The increased incidence of brain cancer in adults and children is suspected of having a strong environmental influence (Curan and Jones, 1991). The cancers most prevalent amongst South African children follows a worldwide trend, according to the Cancer Association of South Africa (CANSA) with brain cancer ranking second amongst the most common cancers, and accounting for 21% of all cancers diagnosed. Neuroblastomas, a cancer of the sympathetic nervous system, also ranked fifth amongst most common childhood cancers (Cancer Information Services, 1998).
On the basis of racial classification, South Africans were previously subject to environments that determined their lifestyle, diet, socio-economic condition, residence, type of work and access to health care (Sitas and Pacella, 1994). These differences influenced reported cancer patterns observed among population groups. However, major socio-economic changes have occurred in SA over the past ten years that have impacted on these aspects. Were more recent statistics available, it would provide a more accurate representation of current cancer incidence across the racial groups due to increased reporting and increased access to health care for all races. The statistics presented by
~itas et al. (1998) are representative of cases reported within only the 1993 -1995 timeframe. At that time, in socially impoverished communities, limited access to medical facilities and treatment, lack of money and knowledge on disease treatment, may have prevented people from seeking and securing help. Nevertheless, this information was helpful in providing insight into past trends of reported cancer incidence in SA.
1.5 POTENTIAL FOR FUNGAL ASSOCIATION IN NEUROLOGICAL DISEASES
The overall goal of neurotoxicological investigations is to develop and validate quantitative biomarkers of neurotoxicity and to utilize these to elucidate toxicity mechanisms. This would increase the certainty of assumptions underlying risk assessment for neurotoxicants.
Advances in medical science have revolutionized understanding of brain tumours and shown that abnormal proliferation, inability of the cells to die and their potential to modify their tissue environment, result from accumulation of genetic aberrations. Understanding the potential initiators and chain of events responsible for brain tumour growth is a prerequisite for the development of effective therapeutic modalities leading to improved prognosis and cures.
In order to develop and implement methods of preventing intoxication and managIng symptoms of poisoning by Fusarium mycotoxins known to potentially affect the CNS, identification and understanding the mechanisms whereby these toxicants exert their effects is imperative. The selection of Fusarium toxins for study was on the basis of their incidence in analytical surveys of commercially available SA food and feeds (Dutton and Kinsey, 1995; 1996), as well as on the requirement for further research into their potential role in CNS-associated human disease. The significant contribution of maize and maize-based foods to the diet, and therefore their potential diet-toxicity interaction, warranted the present study to ascertain whether selected Fusarium mycotoxins may be associated with the pathophysiology of brain cancer in the population of KwaZulu-Natal, South Africa.
Diagnosis of neurological diseases caused by fungi can be difficult as clinical signs are often ambiguous or similar to those found in other diseases, and histological lesions may be non-specific or absent. Therefore, the diagnosis of diseases caused by neurotoxic mycotoxins depends not only on history, clinical signs or finding the mycotoxin in the appropriate food and feed material (Plumlee and Galey, 1994), but also on finding the specific mycotoxin directly within lesions, in infected tissues, and/or in physiological fluids.
1.6 OBJECTIVES
This study comprised of an in vitro experimental, as well as a in vivo clinical component. The objectives of the in vitro component were to assess the: -
• potential for selected Fusarium mycotoxins to induce cytotoxicity, apoptosis, growth arrest or necrosis in the N2a. mouse neuroblastoma cell line;
• structural and ultrastructural changes that occur as a result of exposure to Fusarium mycotoxins in N2a. mouse neuroblastoma cells using immunochemical techniques; and
• in vitro effects of FBI on sphinganine (Sa) and sphingosine (So) metabolism in the N2a.
cells.
The objectives of the clinical component of this study were to: -
• analyse sera obtained from non-cancer subjects and brain tumour patients for levels of FBI, Sa, So and Sa:So ratios in order to establish whether any correlation exists between these variables, and
• immunolocalise FBI within brain tumour tissues.