S. Abel

Hepatotoxicity and -Carcinogenicity of the Fumonisins in Rats

Cancer induction by the non-genotoxic mycotoxin, fumonisin B1, has been investigated by studying the mechanisms involved during cancer initiation and promotion in rat liver. Cancer initiation is effected through a toxic-proliferative response while the inhibitory effect on hepatocyte cell proliferation appears to be a key aspect determining cancer promotion. Dose-response effects of the fumonisins on the induction of early neoplastic lesions in both long- and short-term animal experiments have been established. The biphasic response of FB1 on hepatocyte proliferation will be discussed in relation to the known mechanisms of cancer induction by the genotoxic hepatocarcinogens. Recent investigations regarding the effect of the fumonisins on lipid biosynthesis and its inhibitory effect on hepatocyte growth stimulatory responses in vitro will be highlighted. Integration of our current knowledge regarding the carcinogenic potential of the fumonisins in setting a realistic and applicable risk assessment model for this non-genotoxic carcinogen will finally be addressed.

Inhibition of sphingolipid biosynthesis in rat primary hepatocyte cultures by fumonisin B1 and other structurally related compounds

The fumonisins and toxins produced by Alternaria alternata f. sp. lycopersici (AAL toxins) are structurally related mycotoxins that disrupt sphingolipid biosynthesis by inhibiting the rate-limiting enzyme, ceramide synthase. Rat primary hepatocytes were exposed to fumonisin B1 (FB1), its N-acetyl analogue, FA1, its fully hydrolysed analogue, AP1 and the AAL toxins (TA and TB) at concentrations of 1 microM for 40 hr in culture. The extent to which these compounds disrupt sphingolipid biosynthesis in hepatocytes in vitro was investigated by analysing the sphingosine (So) and sphinganine (Sa) levels by HPLC. The inhibition of ceramide synthase was irreversible as the Sa:So ratio was maximally increased by FB1 after 24 hr of exposure and the subsequent removal of FB1 had no effect on the ratio as compared with the 40-hr incubation period in the presence of FB1. The Sa concentration was significantly (P < 0.01) increased in all the cultures treated with the different structurally related compounds, while only AP1 increased the So concentration significantly (P < 0.05) above the control. As AP1 was found to be less effective in disrupting sphingolipid biosynthesis it would appear that the tricarballylic (TCA) moiety is required for maximal inhibition of ceramide synthase. The presence of an amino group appears not to be a requisite for activity, since FA1 increased the Sa:So ratio to the same extent as FB1. The AAL toxins TA and TB increased the Sa concentration significantly (P < 0.01) above that of FB1 and FA1, while the Sa:So ratios were altered to the same extent. The structural requirements for the induction of cytotoxicity differ from those required for ceramide synthase inhibition as TA and TB were significantly (P < 0.05 to P < 0.01) less toxic to primary hepatocytes than FB1 at all the concentrations tested.

Cancer initiation by fumonisin B1 in rat liver – role of cell proliferation

Fumonisin B(1) (FB(1)), a carcinogenic mycotoxin produced by the fungus Fusarium verticillioides in corn, causes cancer initiation in rat liver in a similar manner to genotoxic carcinogens although apparently with different kinetics. The present experiment was designed to evaluate the role of regenerative cell proliferation, effected by partial hepatectomy (PH) and carbontetrachloride (CCl(4)) and direct mitogen-induced hyperplasia, induced by lead nitrate (PbNO(3)), on FB(1)-induced cancer initiation. Initiation was effected over a period of 14 days by gavage administration of FB(1) at different daily doses ranging from 0.14 to 3.5 mg FB(1)/100 g body weight while the stimuli for cell proliferation were introduced 7 days after the start of the FB(1) treatment. Based on the proliferative stimulus used, cancer promotion was effected 3 weeks after completion of the initiating treatment by 2-acetylaminofluorene (2-AAF) treatment followed by PH or carbon tetrachloride CCl(4) on day 4. Cancer initiation by FB(1) was associated with a hepatotoxic effect and an increase in lipid peroxidation. In contrast to compensatory liver cell proliferation induced by PH and CCl(4), mitogen-induced hyperplasia (PbNO(3)) failed to enhance the cancer initiating potential of FB(1) suggesting that cancer induction by a non-genotoxic carcinogen is supported by regenerative cell proliferation. Cognizance of the enhancing role of cell proliferation during cancer initiation by FB(1) is required in assessing the risks posed by this mycotoxin to humans.

Dietary PUFA and cancer.

The aim of the present paper is to give a brief overview on the role of dietary fat in carcinogenesis and as possible anticancer agents. Dietary fat is an essential nutrient and important source for the essential fatty acids (FA), linoleic and α-linolenic acids, which contribute to proper growth and development. However, dietary fat has been associated with the development of colorectal, breast, prostate, endometrial and ovarian cancers, with the type and quality of fat playing an underlying role. Tumour growth is the disruption of the homoeostatic balance regulating cell differentiation, proliferation and apoptosis and is associated with altered lipid metabolism. Animal cancer models and human cancer biopsy tissue demonstrate that a characteristic lipid profile is associated with the growth and development of neoplastic lesions. This entails alterations in membrane cholesterol, phospholipid and PUFA metabolism. Particularly, alterations in cell membrane FA metabolism involving the n-6 and n-3 PUFA, are associated with changes in membrane structure, function, cellular oxidative status, activity of enzymes and signalling pathways. These events are a driving force in sustaining the altered growth of cancerous lesions and provide unique targets for intervention/cancer modulation. Challenges in utilising FA in cancer modulation exist regarding intake and effect on cell structure and biochemical interactions within the cell in the prevention of cancer development. Therefore, utilising dietary PUFA in a specific n-6:n-3 ratio may be an important chemopreventive tool in altering the growth characteristics of cancer cells.

Thresholds and kinetics of fatty acid replacement in different cellular compartments in rat liver as a function of dietary n-6/n-3 fatty acid content

The kinetics of fatty acid (FA) replacement in different membrane compartments in the rat liver were investigated using diets with varying n-6/n-3 FA ratios. Rats at different stages of growth, i.e. after weaning and at 150 g body weight, were either fed a modified AIN 76A diet containing sunflower oil as fat source or the same diet containing sunflower oil and fish oil to achieve n-6/n-3 FA ratios of 12:1 and 6:1 (diets A and B, respectively). In the adult rats, fed diet A for 8 weeks, C18:2n-6 increased significantly at week 2 in the phosphatidylcholine (PC) fraction of the plasma membranes, microsomes and plasma but not in phosphatidylethanolamine (PE), C20:3n-6 increased significantly at week 2 in the plasma membrane and microsomal PC, but did only increased in PE of both compartments by week 8. C20:4n-6 and the n-3 FAs significantly decreased and increased, respectively, at week 2 in PC and PE of both membrane compartments and plasma PC. The experimental diets led to a change in the plasma membrane fluidity but not in the microsomes. The FA changes in the weaned rats followed a similar pattern as in the adult rats although the changes were greater, depending on the phospholipid fraction and specific FA. The decrease in C20:4n-6 was significantly greater in the microsomal PC and PE and plasma PC but not in the plasma membrane PC and PE. The n-3 FAs increased significantly above the adult levels in the plasma membrane PC and PE respectively but not in the microsomal phospholipid fractions. A plateau for maximal n-3 and n-6 FA incorporation was achieved in the adult rats fed diet A in the microsomes after 2 weeks with no further alterations occurring with diet B. In the plasma PC and plasma membranes most of the n-3 FAs achieved a threshold incorporation after 2 weeks on diet A, except for C22:6n-3 in the plasma membranal PE and certain n-6 FAs in the plasma membrane PC and PE. The present data shows that differences exist in the kinetics of FA incorporation and replacement depending on the specific phospholipid fraction, membrane compartment, age and to a certain extent the dietary n-6/n-3 FA ratio.

Differential modulation of the lipid metabolism as a model for cellular resistance to fumonisin B1–induced cytotoxic effects in vitro

Differential sensitivity of primary hepatocytes and Chang cells to the cancer promoter fumonisin B1 (FB1)-induced cytotoxic effects were investigated in relation to changes in membrane lipid distribution. In contrast to primary hepatocytes, Chang cells were resistant to FB1-induced cytotoxic effects. This was associated with a high cholesterol (Chol) and sphingomyelin (SM) and low phosphatidylcholine (PC) content, resulting in a significant (P<0.05) decrease in phosphatidylethanolamine (PE)/PC ratio, increased Chol/total phosphoglyceride (TPG) ratios and low total polyunsaturated fatty acids (PUFA) content in PC and PE, suggesting a more rigid membrane structure. High levels of C18:1 and reduced polyunsaturated fatty acid (PUFA) levels are likely to provide selective resistance to FB1-induced oxidative stress. FB1-associated lipid changes included decreases in SM and Chol, increases in sphinganine (Sa) and PE with the increases in key saturated, monounsaturated, and PUFAs in PE as key role players in the differential responses to FB1-induced cell growth responses in cells.

Abstract B02: Disruption of lipid rafts constituents and implications for the cancer promotion properties of the carcinogenic mycotoxin, fumonisin B1

Substantial evidence links food as a source of natural occurring carcinogens with the incidence of cancer in humans. The carcinogenic group 2B mycotoxin, fumonisin B1 (FB1) is a natural occurring toxin, produced by different Fusarium species in maize. In South African rural maize-subsistence farming communities, the chronic exposure to high levels of fumonisin is epidemiologically linked to a high prevalence of oesophageal cancer (OC). Despite no direct association with the etiology of OC, FB1 has been identified as a liver cancer promoter involving the disruption of the lipid metabolism. Subsequently the disruption of the membrane integrity and function has been proposed as a possible mechanism for cancer promotion by fumonisin. The presence of highly specialized and functional lateral assemblies or microdomains on cell membranes surfaces has provided an additional platform to investigate the effect of FB1 on cancer promotion. Rat primary hepatocytes were exposed to FB1 (250 μM associated with moderate cytotoxicity) for 24hrs at 37°C, followed by lipid raft isolation using a non-detergent method. Characterization of major lipids such as cholesterol (CHOL), sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylethanolamine (PE) was determined. Raft CHOL isolated from treated hepatocytes, significantly (p Citation Format: Hester-Mari Burger, Stefan Abel, Wentzel CA Gelderblom. Disruption of lipid rafts constituents and implications for the cancer promotion properties of the carcinogenic mycotoxin, fumonisin B1 [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B02.

Abstract B20: Modulation of a cancer lipogenic phenotype by dietary n-6/n-3 fatty acids in rat liver and colon cancer models

Cancer development is a multistage process involving a breakdown of controlled processes such as cell differentiation, proliferation and apoptosis and regulatory feed-back mechanisms resulting in the characteristic rapid growth and metastases of cancer cells. The deregulation of cell growth and survival in cancer development has been associated with a distinct lipogenic phenotype involving cholesterol, phospholipids and fatty acids (FA). This phenotype is associated with changes in membrane structure and cellular oxidative status resulting in changes to membrane function, activity of enzymes and signal transduction pathways. Dietary components are associated with a protective effect against the development of certain cancers. Amongst these, dietary fat has been linked to either the promotion or prevention of cancer involving various biological mechanisms, depending on the type of fat and its FA content. The present study characterized the lipogenic phenotype in the liver and colon utilizing rat cancer models and the resultant modulation of membrane lipid components by dietary oils with specific fatty acid content. Sunflower oil (S) in combination with borage oil (B) and/or fish oil (F) was used to generate different n-6/n-3 FA ratios: SB (n-6/n-3: 38:1), SF (n-6/n-3: 13:1) and SBF (n-6/n-3: 10:1), while canola and olive oil were included as individual oil sources. In separate colon and liver in vivo cancer models, rats were fed the diets in respective groups and terminated whereupon liver and colon cancerous and respective adjacent normal tissue samples were collected for lipid and oxidative PCR micro-array analyses. The characteristic lipogenic phenotype associated with the growth and development of preneoplastic lesions involved a decrease in the phospholipid phosphatidylcholine / phosphatidylethanolamine (PC/PE) ratio, C20:4n-6 PC/PE ratio, n-3 polyunsaturated FA (PUFA) content and oxidative status. An interactive role of saturated FA, monounsaturated FA and C20:4n-6 appear to be a driving force sustaining altered growth characteristics in these lesions with a diet high in n-6 PUFA playing an underlying role. This characteristic phenotype was modulated by the dietary oils with varying n-6/n-3 FA ratios resulting in an increased n-3 PUFA tissue content, a higher lipid peroxidation level and altered growth of preneoplastic lesions. Depending on the specific stage of cancer development, dietary oil may either inhibit or stimulate the growth of preneoplastic lesions which appear to be related to differential changes in proliferative cell survival responses associated with an altered cellular redox status. Challenges in utilizing fatty acids in cancer modulation exist not only with regards to dietary sources and intake but also the effect on cell structure and modulation of signal transduction pathways governing altered growth responses in the prevention of cancer development. Therefore, the modulation of the lipid profile and oxidative status through dietary PUFA with a specific n-6/n-3 ratio may be an important chemopreventive tool in altering the growth characteristics of cancer cells. Citation Format: Stefan Abel, Celeste H. Abrahams, Maryna De Kock, Wentzel CA Gelderblom. Modulation of a cancer lipogenic phenotype by dietary n-6/n-3 fatty acids in rat liver and colon cancer models [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B20.