L. van der Westhuizen

Exposure assessment for fumonisins in the former Transkei region of South Africa

The fumonisins are mycotoxins produced mainly by Fusarium verticillioides and F. proliferatum in maize, the predominant cereal staple for subsistence farming communities in southern Africa. In order to assess exposure to these mycotoxins in the Bizana (now known as Mbizana) and Centane magisterial areas of the former Transkei region of the Eastern Cape Province of South Africa, the actual maize consumption by different age groups in these communities was measured. In the groups 1–9 years (n = 215) and 10–17 (n = 240) years, mean consumption (±standard error) was 246 ± 10.8 and 368 ± 10.3 g per person day−1, respectively, with no significant difference (p > 0.05) between the magisterial areas. For adults (18–65 years) mean maize consumption in Bizana (n = 229) and Centane (n = 178) were significantly different (p < 0.05) at 379 ± 10.5 and 456 ± 11.9 g per person day−1, respectively. An exposure assessment was performed by combining the maize consumption distribution with previously determined levels of total fumonisin (fumonisins B1 and B2 combined) contamination in home-grown maize in these two areas. Assuming an individual adult body weight of 60 kg, fumonisin exposure in Bizana, an area of relatively low oesophageal cancer incidence, was 3.43 ± 0.15 µg kg−1 body weight day−1, which was significantly lower (p < 0.05) than that in Centane (8.67 ± 0.18 µg kg−1 body weight day−1), an area of high oesophageal cancer incidence. Mean fumonisin exposures in all age groups in both Bizana and Centane were above the provisional maximum tolerable daily intake (PMTDI) of 2 µg kg−1 body weight day−1 set by the Joint FAO/WHO Expert Committee on Food Additives.

Biomarkers of exposure to fumonisin mycotoxins: A review

The investigation of adverse health effects associated with fungal mycotoxins requires the measurement of human exposure. Most frequently, this exposure is estimated from contamination levels of raw foodstuffs, which are the primary source of toxin exposure, and data on food consumption patterns. However, variations in food preparation methods, food intake, contamination level, intestinal absorption, toxin distribution and excretion lead to individual variations in toxin exposure that are more readily measured with a biomarker. Fumonisin biomarkers have been sought in the measurement of levels of the toxin in physiological samples such as serum, urine, faeces, hair and nails. However, due to the low bioavailability of fumonisin, these samples pose a variety of analytical challenges and also still require validation as biomarkers. The most widely researched fumonisin biomarkers have been those related to the disruption of de novo sphingolipid biosynthesis, namely elevated levels of the sphingoid base, sphinganine, or of its ratio with sphingosine. Elevation of these parameters in humans would potentially provide a biomarker of biochemical effect. A number of investigations into the possible elevation of sphinganine (or its ratio with sphingosine) in human blood and urine have generally failed to correlate with estimates of fumonisin exposure. The sphingoid bases occur naturally in human blood and urine such that their levels have normal ranges, which can be influenced by dietary factors other than fumonisin ingestion. The lower exposures from human diets, as compared with doses in experimental animals, have made detection of changes in these sphingoid biomarkers problematic.

Sphinganine/sphingosine ratio in plasma and urine as a possible biomarker for fumonisin exposure in humans in rural areas of Africa.

This study was conducted in the Transkei region of the Eastern Cape and KwaZulu-Natal province, South Africa and in the Bomet district, western Kenya. The sphinganine (Sa)/sphingosine (So) ratios in the plasma and urine of male and female volunteers consuming a staple diet of home-grown maize in Transkei, were 0.34 +/- 0.36 (mean +/- standard deviation) (n = 154) and 0.41 +/- 0.72 (n = 153), respectively and in plasma samples from KwaZulu-Natal it was 0.44 +/- 0.23 (n = 26). In Kenya, the ratios in plasma and urine were 0.28 +/- 0.07 (n = 29) and 0.34 +/- 0.20 (n = 27), respectively. Mean total fumonisin level in home-grown maize, randomly collected in Transkei from the same region where the human volunteers lived, was 580 ng/g (n = 40), as compared to the KwaZulu-Natal province, where no fumonisin (n = 17) were detected (< 10 ng/g) in the home-grown maize. In Kenya, only one of seven samples was contaminated with 60 ng/g fumonisins. No significant differences were found in the Sa/So ratios between males and females within the regions nor between the different regions (P > 0.05). It is possible that the ratio is not sensitive enough to act as a biomarker for fumonisin exposure in humans at these levels of contamination in maize. This is the first report on Sa/So ratios determined in rural populations in Africa consuming home-grown maize as their staple diet.

Effect of fumonisin B1 on protein and lipid synthesis in primary rat hepatocytes.

The effect of fumonisin B1 (FB1) on protein and lipid synthesis was evaluated in primary rat hepatocytes. FB1 did not affect incorporation of [3H]leucine into hepatocytes at either non-toxic (150 microM) or cytotoxic (500 microM) concentrations indicating that protein synthesis was not affected. However, FB1 significantly (P < 0.01 to P < 0.0001) inhibited incorporation of [14C]palmitic acid into hepatocyte cultures implying that lipid synthesis was altered. Incorporation of the radiolabel was significantly (P < 0.05 to P < 0.0001) lowered in triacylglycerol (TAG) and sphingomyelin fractions and increased in phosphatidylcholine (PC) and phosphatidylethanolamine (PEA) in both FB1 concentrations. The incorporation pattern of [14C]palmitic acid closely resembles the changes in phospholipid levels in the treated cells. The sphingolipid, sphinganine (Sa), was significantly (P < 0.0001) increased in treated cells but there was no significant difference between the toxic and non-toxic dose levels implying that the increased Sa level alone is not responsible for the in vitro toxicity. FB1 significantly (P < 0.01 to P < 0.001) decreased the level of free cholesterol within the cell, resulting in an increased PC:cholesterol ratio suggesting a more rigid membrane structure. Subsequent studies on the fatty acid (FA) profiles in PC and the neutral lipid, TAG, indicated that FB1 significantly (P < 0.05 to P < 0.0001) increased the levels of the polyunsaturated FAs C18:2n-6 and C20:4n-6 at both concentrations. The FB1-induced changes to cellular membranes, specifically those related to FA changes in the major membrane phospholipids, and the altered FA content of the hepatocytes are likely to be key events in explaining the cytotoxic effects and altered growth responses induced by fumonisins in primary hepatocytes.

Individual fumonisin exposure and sphingoid base levels in rural populations consuming maize in South Africa

Low and high oesophageal cancer incidence areas of the former Transkei region of South Africa have been associated with corresponding low and high levels of fumonisin contaminated home-grown maize. This is the first study in South Africa assessing fumonisin B (FB) mycotoxin exposure by quantifying individual maize consumption with weighed food records and FB levels from maize in each participants household and concurrently evaluating sphinganine (Sa), sphingosine (So) and Sa/So ratios in plasma and urine of these participants as possible biomarkers of FB exposure. The high consumption of maize in Bizana (n=36) and Centane (n=30) of 0.41+/-0.21 and 0.39+/-0.19 kg/day, respectively, confirms the reliance on maize as the dietary staple. Mean total FB (FB(1)+FB(2)+FB(3)) levels in home-grown maize were 0.495+0.880 and 0.665+0.660 mg/kg in Bizana and Centane, respectively. Mean fumonisin exposure based on individual consumption was 3.9+/-7.3 and 4.1+/-7.6 microg/kg body weight/day, respectively, for Bizana and Centane. The mean combined sphinganine/sphingosine ratios in Bizana and Centane were similar and ranged from 0.10-0.55 in plasma (n=41) and urine (n=62). There was no association between sphingoid base levels and/or Sa/So ratios in the plasma and urine and individual fumonisin exposure, negating the sphingoid bases as potential biomarkers of fumonisin exposure in humans.

Effect of fumonisin B1 on the levels and fatty acid composition of selected lipids in rat liver in vivo

The modulating role of fumonisin B1 (FB1) on lipid biosynthesis was evaluated in a short-term (21 day) experiment using male Fischer rats fed high dietary levels (50, 100 and 250 mg FB1/kg) and in a long-term (2 yr) experiment using male BD IX rats fed low dietary levels (1, 10 and 25 mg FB1/kg) of FB1. The total serum and liver cholesterol was significantly (P < 0.01) increased in the rats fed 250 mg FB1/kg diet for 21 days, while the liver phospholipids, sphingomyelin and phosphatidylethanolamine (PE) were significantly decreased (P < 0.01) and increased (P < 0.05), respectively. In the long-term study, only PE was significantly (P < 0.05) increased in all the FB1-treated animals. Fatty acid (FA) analysis of PE indicated that C18:2n-6 was significantly increased (P < 0.05 to P < 0.01) in the FB1-treated rats of the short-term study, while it was markedly (not significantly) increased in phosphatidylcholine (PC). The same pattern was observed in the PC and PE fractions of the liver of the FB1-treated rats from the long-term studies, but the changes were not significant due to the small number (three rats per group) of rats analysed. The levels of C22:5n-6 and C22:6n-3 were also markedly decreased and increased respectively in the 10 and 25 mg FB1/kg-treated groups. When the FAs were determined in the total lipids in a larger number of rats (four to six animals per group) the level of C18:2n-6 was significantly increased in the 10 (P < 0.01) and 25 (P < 0.05) mg FB1/kg-treated groups. Similar effects were noticed in plasma PC with respect to the C18:5n-6 and C22:55n-6 in both the long- and short-term treated groups, except that C20:4n-6 was also lower in both cases. The total n-6 FAs and polyunsaturated FAs were significantly (P < 0.01) and markedly reduced in PC and PE, respectively, of the rats fed the 250 mg FB1/kg diet. In the long-term experiment the n-6/n-3 ratio was significantly (P < 0.01) decreased in PE and markedly lowered in PC due to a significant (P < 0.05) increase in the n-3 FAs of both phospholipid fractions. The sphinganine/sphingosine ratio was significantly (P < 0.05) altered in the liver of the rats fed the 100 and 250 mg FB1/kg diets for 21 days, while in the long-term study no significant changes were noticed in either the liver or sera. The present data indicate that FB1 affects lipid biosynthesis in rat liver and plasma differently, depending on the dietary level and duration of treatment. Alterations to the n-3 and n-6 FA biosynthetic pathways, detected in rats fed relatively low dietary levels of FB1, are likely to be important mediators for FB1-induced effects on hepatocyte cell proliferation.

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.

The effect of a single gavage dose of fumonisin B1 on the sphinganine and sphingosine levels in vervet monkeys

This is the first report of sphinganine (Sa) and sphingosine (So) levels determined in serum and urine of vervet monkeys (Cercopithecus aethiops) dosed with pure fumonisin B(1) (FB(1)). Initially, experimental vervet monkeys were given a single gavage dose of either 1 or 10 mg FB(1) /kg body weight. Blood and urine were sampled daily and on day seven the monkeys were terminated and the kidneys and livers harvested. In a subsequent experiment, other vervet monkeys were similarly dosed and blood and urine samples were collected over a 50-day period. In the high-dose monkeys the serum Sa/So ratio, as well as levels of serum cholesterol and liver function enzymes, increased during the first week after dosing and remained elevated for several weeks thereafter. The urinary Sa/So ratio and the serum renal function indicators showed a more rapid response and a correspondingly more rapid return to pre-dosing levels. In the low-dose monkeys serum Sa and the Sa/So ratio were the only parameters to increase above the control levels. The Sa/So ratio in liver and kidney tissue showed an elevation over controls in a dose-dependent manner. The serum Sa/So ratio was exclusively elevated above the control levels in the low- and high-dose monkeys and seems more relevant as a marker for fumonisin exposure than any of the other indicators.

Liquid chromatographic determination of the sphinganine/sphingosine ratio in serum.

The fumonisin mycotoxins, which are worldwide contaminants of corn, inhibit de novo sphingolipid biosynthesis leading to elevation in the ratio of the sphingoid bases, sphinganine and sphingosine, in the serum of animals exposed to fumonisins. A new HPLC method for the determination of the ratio of these bases in serum has been developed involving lipid extraction, clean-up on a silica minicolumn and alkaline hydrolysis prior to precolumn o-phthaldialdehyde derivatisation and HPLC separation and quantification by fluorescence detection. Based on serum from both normal and fumonisin-exposed vervet monkeys, the method was shown to be reproducible (R.S.D.<10%).

Sphingoid base levels in humans consuming fumonisin-contaminated maize in rural areas of the former Transkei, South Africa: a cross-sectional study

High incidences of oesophageal cancer are associated with the consumption of subsistence-grown maize by rural populations in the former Transkei region of Eastern Cape Province, South Africa. This cross-sectional study was conducted in the north-eastern magisterial area of Bizana (a previously low oesophageal cancer incidence area) and the south-eastern area of Centane (a previously high incidence area). Plasma and urine samples of male and female participants were analysed for the sphingoid bases, sphinganine and sphingosine. Good home-grown and visibly mouldy maize samples, collected from the households of the participants, were analysed for fumonisin B1, B2 and B3. Plasma sphinganine/sphingosine ratios in males and females were significantly lower (p < 0.05) due to lower sphinganine levels in Bizana compared to Centane. In contrast, the urinary female and combined (males + females) sphinganine/sphingosine ratios were significantly higher (p < 0.05) in Bizana due to the significantly lower (p < 0.05) urinary sphingosine levels. Interestingly, urinary sphingoid base levels were significantly lower (p < 0.05) in males than females within each area. Based on the mean total fumonisin levels in good maize, the estimated mean probable daily intake (PDI) was 5.8 µg kg−1 body weight day−1 in Bizana during 2000 and 4.4 and 6.7 5.8 µg kg−1 body weight day−1 in Centane during 1997 and 2000, respectively, exceeding the maximum tolerable daily intake proposed by JECFA. However, there was no significant difference in the mean total fumonisin levels in the maize between the magisterial areas. The observed differences in plasma and urinary sphingoid base levels could not be ascribed as a biomarker of fumonisin exposure and further studies at an individual level are required.