Sonja Swanevelder

Interaction of fumonisin B1 and aflatoxin B1 in a short-term carcinogenesis model in rat liver

The co-existence of the fumonisin and aflatoxin mycotoxins in corn merited studies to investigate their possible synergistic toxicological and carcinogenic effects. When utilising a short-term carcinogenesis model in rat liver, both the compounds exhibited slow cancer initiating potency as monitored by the induction of foci and nodules stained positively for the placental form of gluthatione-S-transferase (GSTP(+)). However, when rats were treated in a sequential manner with AFB(1) and FB(1) the number and size of GSTP(+) lesions significantly increased as compared to the separate treatments. Histopathological analyses indicated that the individual treatments showed far less toxic effects, including occasional hepatocytes with dysplastic nuclei, oval cell proliferation and, in the case of FB(1), a few apoptotic bodies in the central vein regions. The sequential treatment regimen induced numerous foci and dysplastic hepatocyte nodules, and with oval cells extending from the periportal regions into the centrilobular regions. This would imply that, in addition to the cancer promoting activity of FB(1) of AFB(1)-initiated hepatocytes, the AFB(1) pre-treatment enhanced the FB(1) initiating potency, presumably by rendering the liver more susceptible to the toxic effects of FB(1). The co-occurrence of AFB(1) and FB(1) in corn consumed as a staple diet could pose an increased risk and should be included in establishing risk assessment parameters in humans.

Adjunctive Thalidomide Therapy for Childhood Tuberculous Meningitis: Results of a Randomized Study

Childhood tuberculous meningitis is associated with serious long-term sequelae, including mental retardation, behavior disturbances, and motor handicap. Brain damage in tuberculous meningitis results from a cytokine-mediated inflammatory response, which causes vasculitis and obstructive hydrocephalus. Thalidomide, a potent tumor necrosis factor α inhibitor, was well tolerated and possibly showed some clinical benefit in children with tuberculous meningitis during a pilot study. The purpose of the present study was to assess the effect of adjunctive thalidomide in addition to standard antituberculosis and corticosteroid therapy on the outcome of tuberculous meningitis. Thalidomide (24 mg/kg/day orally) or placebo was administered in a double-blind randomized fashion for 1 month to patients with stage 2 or 3 tuberculous meningitis. The study was terminated early because all adverse events and deaths occurred in one arm of the study (thalidomide group). Thirty of the 47 children enrolled received adjunctive thalidomide, of whom 6 (20%) developed a skin rash, 8 (26%) hepatitis, and 2 (6%) neutropenia or thrombocytopenia. Four deaths (13%) occurred in patients with very severe neurologic compromise at baseline; two deaths were associated with a rash. Motor outcome after 6 months of antituberculosis therapy was similar in the two groups, even though the thalidomide group showed greater neurologic compromise on admission. In addition, the mean IQ of the two treatment groups did not differ significantly (mean IQ thalidomide group 57.8 versus mean IQ control group 67.5; P = 16). These results do not support the use of adjunctive high-dose thalidomide therapy in the treatment of tuberculous meningitis. (J Child Neurol 2004;19:250-257).

Chemoprotective properties of rooibos (Aspalathus linearis), honeybush (Cyclopia intermedia) herbal and green and black (Camellia sinensis) teas against cancer promotion induced by fumonisin B1 in rat liver.

The chemoprotective properties of unfermented and fermented rooibos (Aspalathus linearis) and honeybush (Cyclopia intermedia) herbal teas, and green and black teas (Camellia sinensis) were investigated against fumonisin B1 (FB1) promotion in rat liver utilizing diethylnitrosamine (DEN) as cancer initiator. The various teas differently affected the clinical chemical parameters associated with liver and kidney damage associated with FB1 suggesting specific FB1/iron/polyphenolic interactions. Green tea enhanced (P<0.05) the FB1-induced reduction of the oxygen radical absorbance capacity, while fermented herbal teas and unfermented honeybush significantly (P<0.05) decreased FB1-induced lipid peroxidation in the liver. The teas exhibited varying effects on FB1-induced changes in the activities of catalase, glutathione peroxidase (GPx) glutathione reductase (GR) as well as the glutathione (GSH) status. Unfermented rooibos and honeybush significantly (P<0.05) to marginally (P<0.1) reduced the total number of foci (>10microm), respectively, while all the teas reduced the relative amount of the larger foci. Fermentation seems to reduce the protective effect of the herbal teas. Differences in the major polyphenolic components and certain FB1/polyphenolic/tissue interactions may explain the varying effects of the different teas on the oxidative parameters, hepatotoxic effects and cancer promotion in rat liver.

A randomised control trial in schoolchildren showed improvement in cognitive function after consuming a bread spread, containing fish flour from a marine source

BACKGROUND In humans, n-3 long-chain polyunsaturated fatty acids play a well-documented role in brain development and function. Docosahexaenoic acid and arachidonic acid are major structural components of the brain and a deficiency thereof may bring about changes in the behaviour domains of the brain. OBJECTIVE This trial investigated the effect of an experimental fish-flour bread spread rich in n-3 long-chain polyunsaturated fatty acids, on cognition of children (7-9yr). DESIGN Subjects (n=183) were randomly assigned to an experimental (n=91) and control group (n=92), receiving either the fish-flour spread or a placebo spread for 6 months in a single-blind study. Plasma and red blood cell phospholipid fatty acid composition and cognition were measured at baseline and post-intervention. RESULTS After the intervention, eicosapentaenoic and docosahexaenoic acid levels were significantly higher in the experimental group compared to the control group (p< 0.0001). Significant intervention effects were also observed for the Hopkins Verbal Learning Test Recognition (estimated effect size: 0.80; 95% confidence interval (CI): 0.15; 1.45) and Discrimination Index (estimated effect size: 1.10; 95% CI: 0.30; 1.91), as well as the Spelling test (estimated effect size: 2.81; 95% CI: 0.59; 5.02) by both per protocol and intention to treat analyses. A marginally significant (p=0.0646) effect was observed for the Reading test (estimated effect size: 2.21; 95% CI: -0.14; 4.56) only in the per protocol analysis. CONCLUSION This study suggests improvement of verbal learning ability and memory of children when supplemented with a fish-flour spread rich in n-3 long-chain polyunsaturated fatty acids.

Toxicological effects in rats chronically fed low dietary levels of fumonisin B1

The toxicity of low dietary levels of fumonisin B(1) (FB(1)), i.e. 1, 10 and 25 mg FB(1)/kg diet, were monitored in rats over a period of 24 months. No effects on the body weight gain and feed intake profiles were noticed, while the relative liver weight was significantly (P<0.05) reduced in the FB(1)-treated rats. Mild toxic effects, including single cell necrosis (apoptosis), proliferation of bile duct epithelial cells (DEC), and early signs of fibrosis, bile duct hyperplasia and in one case, adenofibrosis, were noticed in the liver of the rats fed the highest (25 mg/FB(1)/kg diet) dietary level. A significant (P<0.05) increase in the level of oxidative damage was also noticed in the liver of the rats of high dosage dietary group. The toxic effects were less severe in the 10 mg FB(1)/kg dietary group, whilst only a few ground glass foci were observed in the 1 mg FB(1)/kg dietary group. Hepatocyte nodules, staining positively for glutathione-S-transferase (placental form, PGST), were observed macroscopically in the 25 mg FB(1)/kg treated group and to a lesser extent in the 10 mg FB(1)/kg treated rats. The most prominent toxic lesions by FB(1) (10 and 25 mg FB(1)/kg dietary groups) in the kidneys were restricted to the tubular epithelium manifesting as granular cast, necrosis, apoptosis, calcification and the presence of regenerative foci in the proximal convoluted tubules. The existence of a cytotoxic/proliferative threshold with respect to cancer induction by FB(1) in rat liver became apparent, with a dietary level of <10-mg FB(1)/kg diet as a no effect threshold for the induction of hepatocyte nodules.

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.

Effect of home based HIV counselling and testing intervention in rural South Africa: cluster randomised trial.

Objective To assess the effect of home based HIV counselling and testing on the prevalence of HIV testing and reported behavioural changes in a rural subdistrict of South Africa. Design Cluster randomised controlled trial. Setting 16 communities (clusters) in uMzimkhulu subdistrict, KwaZulu-Natal province, South Africa. Participants 4154 people aged 14 years or more who participated in a community survey. Intervention Lay counsellors conducted door to door outreach and offered home based HIV counselling and testing to all consenting adults and adolescents aged 14-17 years with guardian consent. Control clusters received standard care, which consisted of HIV counselling and testing services at local clinics. Main outcome measures Primary outcome measure was prevalence of testing for HIV. Other outcomes were HIV awareness, stigma, sexual behaviour, vulnerability to violence, and access to care. Results Overall, 69% of participants in the home based HIV counselling and testing arm versus 47% in the control arm were tested for HIV during the study period (prevalence ratio 1.54, 95% confidence interval 1.32 to 1.81). More couples in the intervention arm had counselling and testing together than in the control arm (2.24, 1.49 to 3.03). The intervention had broader effects beyond HIV testing, with a 55% reduction in multiple partners (0.45, 0.33 to 0.62) and a stronger effect among those who had an HIV test (0.37, 0.24 to 0.58) and a 45% reduction in casual sexual partners (0.55, 0.42 to 0.73). Conclusions Home based HIV counselling and testing increased the prevalence of HIV testing in a rural setting with high levels of stigma. Benefits also included higher uptake of couple counselling and testing and reduced sexual risk behaviour. Trial registration Current Controlled Trials ISRCTN31271935.

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.