Aflatoxins and fumonisins contamination of home-made food (weanimix) from cereal-legume blends for children.

BACKGROUND: Weanimix is an important food for children in Ghana. Mothers are trained to prepare homemade weanimix from beans, groundnuts and maize for their infants. Groundnuts and maize are prone to aflatoxin contamination while fumonisin contaminates maize. Aflatoxin, is produced by the Asperguillus fungi while fumonisin, is produced by Fusarium fungi. These mycotoxins occur in tropical areas worldwide due to favorable climate for their growth. OBJECTIVE: The objective of the study was to determine the levels of aflatoxin and fumonisin in homemade weanimix in the Ejura-Sekyedumase district in the Ashanti Region of Ghana. METHODS: Thirty six homemade weanimix samples (50g each) were collected from households. Aflatoxin and fumonisin were measured using a fluorometric procedure described by the Association of Official Analytical Chemist (AOAC official method 993.31, V1 series 4). RESULTS: Aflatoxin and fumonisin were detected in all 36 samples, range 7.9-500ppb. Fumonisin levels range: 0.74-11.0ppm). Thirty (83.3%) of the thirty six samples were over the action limit of 20ppb for aflatoxin with an overall mean of 145.2 ppb whiles 58.3% of the samples had fumonisins above the action limit of 4 ppm with an overall mean of 4.7 ppm. CONCLUSION: There were significant aflatoxin and fumonisin contamination of homemade weanimix. Children fed on this nutritional food were being exposed to unacceptable levels of aflatoxin and fumonisin. Therefore there is a critical need to educate mothers on the dangers of mycotoxin exposure and to develop strategies to eliminate exposure of children fed homemade weanimix to aflatoxin and fumonisin.

Calcium montmorillonite clay reduces urinary biomarkers of fumonisin B1 exposure in rats and humans.

Fumonisin B1 (FB1) is often a co-contaminant with aflatoxin (AF) in grains and may enhance AF's carcinogenicity by acting as a cancer promoter. Calcium montmorillonite (i.e. NovaSil, NS) is a possible dietary intervention to help decrease chronic aflatoxin exposure where populations are at risk. Previous studies show that an oral dose of NS clay was able to reduce AF exposure in a Ghanaian population. In vitro analyses from our laboratory indicated that FB1 (like aflatoxin) could also be sorbed onto the surfaces of NS. Hence, our objectives were to evaluate the efficacy of NS clay to reduce urinary FB1 in a rodent model and then in a human population highly exposed to AF. In the rodent model, male Fisher rats were randomly assigned to either FB1 control, FB1 + 2% NS or absolute control group. FB1 alone or with clay was given as a single dose by gavage. For the human trial, participants received NS (1.5 or 3 g day⁻¹) or placebo (1.5 g day⁻¹) for 3 months. Urines from weeks 8 and 10 were collected from the study participants for analysis. In rats, NS significantly reduced urinary FB1 biomarker by 20% in 24 h and 50% after 48 h compared to controls. In the humans, 56% of the urine samples analysed (n = 186) had detectable levels of FB1. Median urinary FB1 levels were significantly (p < 0.05) decreased by >90% in the high dose NS group (3 g day⁻¹) compared to the placebo. This work indicates that our study participants in Ghana were exposed to FB1 (in addition to AFs) from the diet. Moreover, earlier studies have shown conclusively that NS reduces the bioavailability of AF and the findings from this study suggest that NS clay also reduces the bioavailability FB1. This is important since AF is a proven dietary risk factor for hepatocellular carcinoma (HCC) in humans and FB1 is suspected to be a dietary risk factor for HCC and oesophageal cancer in humans.

Aflatoxin-albumin adducts and correlation with decreased serum levels of vitamins A and E in an adult Ghanaian population.

A study of aflatoxin (AF) exposure and the levels of vitamins A and E was carried out with a group of 507 Ghanaian participants. AFB(1)-albumin adducts (AFB-AA) were measured by radioimmunoassay and vitamins A and E were measured by high-performance liquid chromatography (HPLC). The average level of serum AFB-AA was 0.94 +/- 0.64 (range = 0.1-4.44) pmol mg(-1) albumin. Mean levels of vitamins A and E were 1.32 +/- 0.48 (range = 0.41-4.85) micromol l(-1) and 15.68 +/- 4.12 (range = 6.35-30.40) micromol l(-1), respectively. A significantly negative correlation was found between serum AFB-AA and vitamin A levels (r = -0.110, p = 0.013). An even stronger, significant negative, correlation was found between serum AFB-AA and vitamin E levels (r = -0.149, p < 0.001). Serum AFB-AA levels were statistically higher (median = 0.985 pmol mg(-1) albumin) in subjects who had low levels of both vitamins A and E as compared with the levels (median = 0.741 pmol mg(-1) albumin) subjects who had high vitamins A and E levels (p(trend) = 0.001). To verify these findings, blood samples were again collected from 165 of the 507 people 3 months after the initial collection. Significantly negative correlations were confirmed between levels of serum AFB-AA and both vitamins A (r = -0.232, p = 0.003) and E (r = -0.178, p = 0.023). Again, high serum AFB-AA concentrations (median = 1.578 pmol mg(-1) albumin) were found in subjects with low levels of vitamins A and E compared with the concentrations (median = 1.381 pmol mg(-1) albumin) in subjects with high levels of vitamins A and E (p(trend) = 0.002). These data show that AF exposure was associated with decreased levels of serum vitamins A and E in high-risk human populations, which may significantly influence the incidence of AF-related adverse health effects.

NovaSil clay does not affect the concentrations of vitamins A and E and nutrient minerals in serum samples from Ghanaians at high risk for aflatoxicosis.

To assess the potential interference of NovaSil (NS) clay with micronutrients in humans, vitamins A and E and minerals (15 nutrient and 15 non-nutrient minerals) were measured in serum samples from a 3-month intervention trial with NS. Participants (n = 177) were randomly divided into three groups that received 3.0 g NS day(-1) (high dose, HD), 1.5 g NS day(-1) (low dose, LD), or placebo (PL). Levels of vitamins A and E in serum were comparable among the three study groups at baseline, 1 month and 3 months of NS intervention. Gender-stratified non-parametric mixed-effect model analysis showed no significant effects of dose and dose-time interaction for levels of vitamins A and E. A significant time effect was detected; however, it was limited to an increase in vitamin E in the male participants over the course of the study. No significant differences were found in levels of the nutrient and non-nutrient minerals between the HD and PL groups at baseline and 3 months of NS intervention, except for strontium levels. Strontium was significantly increased (p < 0.001) in the HD group (male = 113.65 +/- 28.00 microg l(-1); female = 116.40 +/- 24.26 microg l(-1)) compared with the PL group (male = 83.55 +/- 39.90 microg l(-1); female = 90.47 +/- 25.68 microg l(-1)) following the 3-month intervention with NS. These results, combined with safety and efficacy data, confirm that NS clay is highly effective in reducing aflatoxin exposure and acts as a selective enterosorbent that does not affect the serum concentrations of important vitamins and nutrient minerals in humans.

NovaSil clay intervention in Ghanaians at high risk for aflatoxicosis: II. Reduction in biomarkers of aflatoxin exposure in blood and urine.

The efficacy of NovaSil clay (NS) to reduce aflatoxin (AF) biomarkers of exposure was evaluated in 656 blood samples and 624 urine samples collected from study participants during a 3-month phase IIa clinical intervention trial in Ghana. NS was delivered before meals via capsules. Serum AFB (1)-albumin adduct was measured by radioimmunoassay and urinary AFM (1) metabolites were quantified by immunoaffinity-high-performance liquid chromatography (HPLC)-fluorescence methods. Levels of AFB (1) -albumin adduct in serum samples collected at baseline and at 1 month were similar (p = 0.2354 and p = 0.3645, respectively) among the placebo (PL), low dose (LD, 1.5 g NS day (-1)), and high dose (HD, 3.0 g NS day (-1)) groups. However, the levels of AFB (1)-albumin adduct at 3 months were significantly decreased in both the LD group (p < 0.0001) and the HD group (p < 0.0001) compared with levels in the PL group. Levels of AFM(1) in urine samples collected at baseline and at 1 month were not statistically different among the three study groups. However, a significant decrease (up to 58%) in the median level of AFM (1) in samples collected at 3 months was found in the HD group when compared with the median level in the PL group (p < 0.0391). In addition, significant effects were found for dose, time, and dose-time interaction with serum AFB(1)-albumin adduct and dose-time interaction with urinary AFM (1) metabolites. The results suggest that capsules containing NS clay can be used to reduce effectively the bioavailability of dietary AF based on a reduction of AF-specific biomarkers.

Reducing human exposure to aflatoxin through the use of clay: a review.

Innovative sorption strategies for the detoxification of aflatoxins have been developed. NovaSil clay (NS) has been shown to prevent aflatoxicosis in a variety of animals when included in their diet. Results have shown that NS clay binds aflatoxins with high affinity and high capacity in the gastrointestinal tract, resulting in a notable reduction in the bioavailability of these toxins without interfering with the utilization of vitamins and other micronutrients. This strategy is being evaluated as a potential remedy for acute aflatoxicosis, and as a sustainable human intervention for aflatoxins via the diet. Phase I and II clinical trials confirmed the apparent safety of NS for further study in humans. A recent study in Ghanaians at high risk for aflatoxicosis has indicated that NS (at a dose level of 0.25%) is effective in decreasing biomarkers of aflatoxin exposure and does not interfere with the levels of serum vitamins A and E, and iron and zinc. In summary, enterosorption strategies/therapies based on NS clay are promising for the management of aflatoxins and as a sustainable public health intervention. The NS clay remedy is novel, inexpensive and easily disseminated. Based on the present research, aflatoxin sequestering clays should be rigorously evaluated in vitro and in vivo, and should meet the following criteria: (1) favourable thermodynamic characteristics of mycotoxin sorption, (2) tolerable levels of priority metals, dioxins/furans and other hazardous contaminants, (3) safety and efficacy in multiple animal species, (4) safety and efficacy in long-term studies, and (5) negligible interactions with vitamins, iron and zinc and other micronutrients.

NovaSil clay intervention in Ghanaians at high risk for aflatoxicosis. I. Study design and clinical outcomes.

A 3-month double-blind and placebo-controlled, phase IIa clinical trial was conducted in Ghana to investigate the safety, tolerance and aflatoxin-sorption efficacy of dietary NovaSil (NS). Volunteers (507 subjects) were clinically screened to evaluate their general health, pregnancy status and blood AFB(1)-albumin adduct levels. Of these subjects, 177 were randomly assigned to three groups: high-dose (HD), low-dose (LD) and placebo-control (PL) groups receiving 3.0, 1.5 and 0 g NS day(-1) in capsules. Trained study-monitors supervised NS capsule administration to participants and recorded side-effects daily. Physical examinations were performed monthly. Blood and urine samples were collected for laboratory analysis. Approximately 92% of the participants (162 of 177) completed the study and compliance rate was over 97%. Overall, 99.5% of person x time reported no side-effects throughout the study. Mild to moderate health events ( approximately 0.5% of person x time) were recorded in some participants. Symptoms included nausea, diarrhea, heartburn and dizziness. These side-effects were statistically similar among all three groups. No significant differences were shown in hematology, liver and kidney function or electrolytes in the three groups. These findings demonstrate that NS clay is apparently safe and practical for the protection of humans against aflatoxins in populations at high risk for aflatoxicosis.

Haptoglobin phenotypes in HIV-1-seropositive patients in Ghana: decreased risk for Hp0 individuals.

Plasma haptoglobin phenotypes were determined by polyacrylamide gel electrophoresis, followed by benzidine staining for 58 HIV-1 seropositive Ghanaians and 79 randomly selected age-matched controls. Hp0 was present in only 14% of HIV-1 seropositive individuals compared with more than 40% of the controls. The Hp0 individuals showed a highly significant reduced risk for HIV-1 infection (OR = 0. 21, 95% CI = 0.09-0.51, p = 0.0002). Hp0 may have a protective effect in HIV-1 infection.

Haptoglobin 1-1 is associated with susceptibility to severe Plasmodium falciparum malaria.

The haptoglobin (Hp) phenotypes were determined by polyacrylamide-gel electrophoresis in plasma samples obtained in 1997 from 113 Plasmodium falciparum malaria patients (aged 1-12 years) with strictly defined cerebral malaria, severe malarial anaemia, or uncomplicated malaria and 42 age-matched healthy controls from the same area (coastal Ghana). Hp1-1 was significantly more prevalent among the patients (43%) than among healthy controls (7.1%), whereas Hp2-1 and Hp2-2 were underrepresented among the patients (11% and 2%, respectively) compared to the control donors (33% and 14%, respectively). No significant difference in frequency of Hp0 was observed between patients and controls. Among the malaria patients, the Hp1-1 phenotype was significantly more prevalent among patients with the complications of cerebral malaria and severe anaemia compared to patients with uncomplicated disease, whereas the reverse was seen with respect to Hp2-1 and Hp2-2. Our data suggest that the Hp1-1 phenotype is associated with susceptibility to P. falciparum malaria in general, and to the development of severe disease in particular.

Human breastmilk storage and the glutathione content.

Human breastmilk storage for use later in infant feeding is on the increase as a result of the economic activities of nursing mothers. This study investigated glutathione (GSH) status of stored human breastmilk due to its major antioxidant role and as a cofactor for enzymes in detoxification of carcinogens. In newborns, human breastmilk becomes an important source of dietary GSH since their GSH synthetic capacity may not be well developed. The results showed that the total GSH content of human breastmilk obtained from apparently healthy lactating mothers was 192.2 +/- 148.3 mumol/l (mean +/- SD). Early breastmilk (fed to infants up to 4 weeks old; GSH content of 252.5 +/- 173.9 mumol/l) was significantly higher (p < 0.05) when compared with their mature counterpart (milk from mothers with infants older than 1 month of age; GSH content 163.9 +/- 128.0 mumol/l). Substantial loss of GSH occurred when breastmilk was kept at either -20 degrees C, 4 degrees C or at room temperature for 2 h. When compared with fresh unstored breastmilk, the extent of the loss was 80.6, 79.1 and 73.0 per cent respectively. It is suggested that feeding infants on stored human milk could weaken the antioxidant and toxin refractory capacity of those in early childhood.

Haptoglobin polymorphism in human immunodeficiency virus infection: Hp0 phenotype limits depletion of CD4 cell counts in HIV-1-seropositive individuals.

Patients seropositive for human immunodeficiency virus (HIV) type 1 and seronegative control subjects were categorized by their haptoglobin phenotypes, which were determined by electrophoresis of hemoglobin-supplemented plasma samples followed by benzidine staining. The CD4 cell counts, determined by flow cytometry from peripheral blood mononuclear cells according to subject categories, were severely diminished in seropositive patients with the Hp2-2 phenotype (P<.025). In contrast, the CD4 cell counts for patients with the Hp0 phenotype remained relatively high (P<.025), compared with those of the controls. In seronegative patients, CD4 cell counts were generally high (P<.005), but they were more elevated in subjects with Hp2-2 and Hp1-1, although the differences were not significant. Thus, the Hp2-2 phenotype is associated with poor outcome in HIV-1 infection, whereas the Hp0 phenotype is associated with a better prognosis once the patient is infected with HIV-1. Haptoglobin polymorphism plays a significant role in HIV-1 infection and transmission.

Acute and subchronic evaluation of Indigofera arrecta: absence of both toxicity and modulation of selected cytochrome P450 isozymes in ddY mice.

Indigofera arrecta, an anti diabetic plant was investigated in ddY mice to determine its acute and subchronic effects, and whether it modulated hepatic cytochrome P450 (CYP) isozymes and glutathione (GSH). No mortality was observed in the acute (up to 10 g I. arrecta/kg body wt, p.o.) and subchronic (2 g I. arrecta/kg body wt, p.o. daily for 30 days) studies. The extract did not alter haematological indices, serum and tissue lipids and glutathione but lowered serum bile acids. The latter phenomenon is under further investigation. Neither the duration of pentobarbital (PB) and zoxazolamine (ZA) effects in vivo, nor CYP-dependent 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-depentylase (PROD) and p-nitrophenol hydroxylase (PNPH) activities in vitro were altered by I. arrecta. The extract was thus devoid of overt acute and subchronic toxic effects, and did not affect CYPs and GSH whose modulation may cause interactions of components in a multiple drug therapy.

Toxicity of low levels of methylglyoxal: depletion of blood glutathione and adverse effect on glucose tolerance in mice.

Methylglyoxal, a metabolic by-product of glycolysis is also formed during food processing and has serious toxicological effects when in excess. In this study, ddY mice were exposed to low levels of methylglyoxal (1% v/v) via drinking water while in utero continuing until 2 months of age when investigations on blood GSH status and selected GSH dependent functions in the blood, and glucose tolerance were carried out. The results showed that GSH content was significantly decreased in the blood of methylglyoxal exposed mice when compared with controls (mean, 0.756 mmol/l vs. 1.090 mmol/l, p < 0.001). The data showed significant (p < 0.001) decreases in blood GSH-S-transferase activity and red blood cell (rbc) capacity to refract oxidative stress. Impaired glucose tolerance was 5.3 times more prevalent in the methylglyoxal exposed mice when compared with the controls. The results indicate that chronic intake of methylglyoxal, at levels that could be attained in food, is toxic by depletion of blood GSH and could have adverse effect on some GSH dependent functions in vivo.

Biliary excretion in persons with low blood glutathione levels.

A study to investigate the association between blood glutathione (GSH) levels and biliary excretory status was conducted in apparently healthy Ghanaian subjects without frank biliary disease and anaemia. The results showed that, in adults (mean age: 38.5 years) and children (mean age: 13.0 years), plasma conjugated bilirubin is inversely correlated with blood GS (respective site r = -0.524, p < 0.011 and -0.395, p < 0.005). Persons with elevated plasma conjugated bilirubin compared to controls (mean: 6.0 versus 2.5 umol/L, p < 0.001) also exhibited low blood GSH values (3.5 versus 4.2 umol/gHb, p < 0.029). Malaria parasites with counts up to 2,453 parasites/ul blood had no effect on the obtained data. The results suggest that low blood GSH levels may be relevant to delays in biliary excretion of conjugated toxins from the liver, as exemplified by the rise in conjugated bilirubin levels in the plasma, and predispose liver cells to increased oxidant state and damage.

Lead exposure in urban and rural school children in Ghana.

Human exposure to lead in non industrial urban areas is commonly ascribed to vehicular combustion of leaded gasoline. This belief is based on results of studies in societies with high vehicular density which show emission of lead fumes into the air by automobiles that use gasoline with high lead content. To assess this view, blood lead levels were evaluated in 11 to 15-year old school children in urban and rural communities of the Greater Accra Region, Ghana. Blood lead levels was significantly higher in all the urban children studied (mean +/-SD: 8.3+/- 12.7g/dl) than in their rural counterparts (4.0+/-7.2g/dl) (P<0.002). The trend was the same when only those who tested positive for blood lead were considered (24.1+/- 9.2g/dl, urban compared with 14.6+/- 5.8g/dl, rural). The prevalence of lead exposure was, however, not significantly different between the two groups (34.3. percent, urban and 27.1 per cent, rural). The presence of anaemia and/or increased urine total protein levels was unrelated to the blood lead levels in the children form both communities. Although a set goal to achieve lead free gasoline is desirable, the closeness of the prevalence rate of lead exposure obtained in the study indicate that factors other than leaded gasoline may be important determinant in exposure to lead in the Ghanaian community.

Lead levels and related biochemical findings occurring in Ghanaian subjects occupationally exposed to lead.

Blood and urine lead levels in relation to blood delta-aminolevulinic acid dehydratase (ALAD) activity and also blood and renal status were evaluated in lead smelters, automobile mechanics and gasoline retailers in the city of Accra, Ghana. Relationship between high blood lead levels (mean: 108 ug/dl) and low ALAD activity (mean: 74.3 units) indicating lead over exposure was found in the lead smelters. Non-toxic lead exposure was, however, noted in the automobile mechanics and the gasoline retailers. Their respective mean blood lead levels were 27.8 ug/dl (mean blood ALAD activity 212.5 units) and 8.6 ug/dl (ALAD: 239.9 units). Personal habits at the work place appear to play a major role in facilitating exposure to lead among all the three groups of workers in addition to lack of control measures at the work place of the lead smelters to protect them against lead exposure. Anaemia was found in 48% of the lead smelters, 12.5% of the gasoline retailers but in none of the automobile mechanics. When compared with lead free subjects (mean blood ALAD activity: 270.9 units), urine microalbumin was significantly (p < 0.01) raised in all the lead smelters suggesting that they may be prone to renal glomerular damage. Plasma creatinine, BUN and uric acid were raised in only one of the lead smelters. The data supports the establishment of blood ALAD activity level at 100 units or less as indication of excessive body lead.

Alteration of glucose tolerance in mice fed low levels of aflatoxins and with depressed glyoxalase-I activity.

Glyoxalase-I activity plays an important role in glucose metabolism and has been reported to be depressed in mice fed low levels of aflatoxin B1. In the present study examination of glyoxalase-I activity, glucose tolerance and pancreatic beta cell sensitivity was made in mice fed 0.045 ng aflatoxin B1 + 0.450 ng aflatoxin G1/g feed prenatally and for 6 mo after birth. After glucose challenge the ratios between 0-h and 2-h serum glucose levels were significantly higher than controls, indicating an increase in tolerance of glucose in the aflatoxin-fed mice with lower glyoxalase-I activity. Pancreatic beta cell sensitivity to stimulation by tolbutamide was similar in both groups. However, liver malonic dialdehyde was significantly higher in the aflatoxin-fed mice, suggesting that the altered tolerance for glucose in the aflatoxin-fed mice might be a consequence of aflatoxin mediated peroxidative actions in the liver.

Enhanced depletion of glutathione and increased liver oxidative damage in aflatoxin-fed mice infected with Plasmodium berghei.

The effect of dietary aflatoxins B1 and G1 and Plasmodium berghei infection on glutathione (GSH) levels and liver status in mice was investigated. Three days after intraperitoneal injection of 0.1 x 10(6) parasitized red blood cells into the mice, there was a significant fall in blood glutathione levels accompanied by a significant increase in serum cholinesterase and liver malonic dialdehyde levels in the mice fed aflatoxin compared with those in the control group. The results suggested that malaria parasites can enhance depletion of host glutathione and oxidative damage of the liver in mice fed low levels of aflatoxins.

Observations on aflatoxins and the liver status of Ghanaian subjects.

To find out if the presumed intake of dietary aflatoxins (AFB1 and AFG1) has adverse effect on the liver of Ghanaians, the toxins were measured in serum, urine and faecal specimens obtained from a group of apparently healthy Ghanaian adults. Liver status of the subjects was monitored with serum alphafeto protein (AFP), alpha-l-antitrypsin (AAT) and direct: total bilirubin ratio. Aflatoxin G1, AFB1 and AFQ1, AFM1 (both metabolites of AFB1) were detected in one or more of the body specimens in 35% of the subjects (AFB1+ group). Sixty-five percent (26 out of 40) of the subjects had only AFG1 in their body specimens (AFB1- group). Serum levels of AFP (greater than 20.0 ng/ml, AAT (greater than 170.0 mg/dl) and direct: total bilirubin ratio (greater than 0.5) which indicate absence of predisposition to liver cancer in all the subjects but suggestive of liver inflammation were noted in both the AFB1+ and AFB1- subjects. The pattern of distribution of the aflatoxins in the subjects suggests that the suspected liver inflammation may involve other factors and may not only be due to the present intake levels of aflatoxins.

Evaluation of alpha-1-antitrypsin, alpha-1-acid glycoprotein and pre albumin in serum of Ghanaian subjects.

Serum alpha-1-antitrypsin (AAT) and alpha-1-acid glycoprotein (AAGp): pre albumin ratio, sensitive markers for liver damage were evaluated in 32 apparently healthy Ghanaian subjects. Eighteen of the subjects (Group 1) had Serum AAT values (mean +/- SD) of 151.9 +/- 18.6 mg/dl while 14 (Group 2) had 209.3 +/- 13.6 mg/dl. AAGp: pre albumin ratio were respectively 1.03 +/- 0.79 and 2.76 +/- 1.00. The difference between the two groups with respect to the AAT and AAGp: pre-albumin ratio is highly significant (p < 0.01). No correlation was seen between these proteins and serum gamma glutamyltransferase (GGT) or alanine aminotransferase (ALT) levels. The GGT and ALT levels were normal in the Group 1 subjects but elevated in some of those in Group 2. It is suggested that the obtained higher AAT values and AAGp: pre albumin ratio might be abnormal for Ghanaian subjects and that these proteins should be monitored to facilitate early detection of liver injury. This might be important for population groups living in geographic areas where environmental agents that cause liver damage are common.