Evans Afriyie-Gyawu

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: 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 AFB1–albumin adduct was measured by radioimmunoassay and urinary AFM1 metabolites were quantified by immunoaffinity-high-performance liquid chromatography (HPLC)-fluorescence methods. Levels of AFB1–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 AFB1–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 AFM1 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 AFM1 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 AFB1–albumin adduct and dose–time interaction with urinary AFM1 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.

Short-term safety evaluation of processed calcium montmorillonite clay (NovaSil) in humans

NovaSil clay (NS) provides significant protection from the adverse effects of aflatoxins (AFs) in multiple animal species by decreasing bioavailability from the gastrointestinal tract. It is postulated that NS clay can be safely added to human diets to diminish exposure and health risks from AF contaminated food. To determine the safety and tolerance of NS in humans and establish dosimetry protocols for long-term efficacy studies, a randomized and double-blinded phase I clinical trial was conducted. Volunteers (20–45 yr in age), were clinically screened for confirmation of their health status. Fifty subjects (23 males and 27 females) were randomly divided into two groups: The low-dose group received nine capsules containing 1.5 g/day, and the high-dose group received nine capsules containing 3.0 g/day for a period of 2 wk. NS capsules were manufactured in the same color and size and were distributed to each participant three times a day at designated sites where follow-up was taken to record any side effects and complaints. Blood and urine samples were collected before and after the study for laboratory analysis. All participants completed the trial and compliance was 99.1%. Mild GI effects were reported in some participants. Symptoms included abdominal pain (6%, 3/50), bloating (4%, 2/50), constipation (2%, 1/50), diarrhea (2%, 1/50), and flatulence (8%, 4/50). No statistical significance was found between the two groups for these adverse effects (p > 0.25). No significant differences were shown in hematology, liver and kidney function, electrolytes, vitamins A and E, and minerals in either group. These results demonstrate the relative safety of NS clay in human subjects and will serve as a basis for long-term human trials in populations at high risk for aflatoxicosis.

Chronic Toxicological Evaluation of Dietary NovaSil Clay in Sprague-Dawley Rats

NovaSil (NS) clay, a common anti-caking agent in animal feeds, has been shown to sorb aflatoxins in the GI tract and diminish their bioavailability and adverse effects in short-term animal studies. Based on this evidence, it is hypothesized that clay-based enterosorption of aflatoxins may be a useful strategy for the prevention of aflatoxicosis in human populations. However, the potential toxicity of long-term dietary exposure to NS has not been determined. In this research, 5–6-week-old male and female Sprague-Dawley rats were fed rations containing 0, 0.25, 0.5, 1.0, or 2.0% (w/w) levels of NS for 28 weeks. Analysis of the NS showed negligible levels of dioxin and furan contaminants. Total feed consumption, cumulative feed consumption, body weight, total body weight gain, feed conversion efficiency, cumulative feed conversion efficiency, and relative organ weights were unaffected in either sex at the doses tested. No NS-dependent differences in relative organ weights or gross or histopathological changes were observed. Analysis of hematological parameters, clinical chemistry, and selected vitamin and mineral levels revealed isolated significant differences between some treatments and control groups (mean corpuscular hemoglobin, serum Ca, serum vitamin A, and serum Fe). However, the differences observed in each case were not dose-dependent. These results suggest that dietary inclusion of NS at levels as high as 2.0% (w/w) does not result in overt toxicity. These findings (as well as others) support the use of NS clay for dietary intervention studies in human populations at high risk for aflatoxicosis.

Toxicological evaluation and metal bioavailability in pregnant rats following exposure to clay minerals in the diet.

Silicate clays are frequently added to animal feeds to bind and reduce the bioavailability of mycotoxins in the gastrointestinal tract. However, the bioavailability of trace metals in these clay feed additives has not been thoroughly investigated. Clays that act nonselectively may interact with nutrients, minerals, and other feedborne chemicals to pose significant hidden risks. In this study, a calcium montmorillonite clay (Novasil Plus, NSP) commonly used as an enterosorbent for aflatoxins and a sodium montmorillonite clay (Swy-2) (Source Clay Minerals Repository, Columbia, MO) were examined. Clays were supplemented in the balanced diet of Sprague-Dawley rats during pregnancy at a level of 2% (w/w). Evaluations of toxicity were performed on gestation d 16 and included maternal body weights, maternal feed intakes, litter weights, and embryonic resorptions. Liver, kidneys, tibia, brain, uterus, pooled placentas, and pooled embryonic mass were collected and weighed. Tissues were lyophilized and neutron activation analysis (NAA) was performed. Elements considered by NAA included Al, Ba, Br, Ca, Ce, Co, Cr, Cs, Cu, Dy, Eu, Fe, Hf, K, La, Lu, Mg, Mn, Na, Nd, Ni, Rb, S, Sb, Sc, Se, Sm, Sr, Ta, Tb, Te, Th, Ti, Tl, U, V, Yb, Zn, and Zr. Inductively coupled plasma–mass spectroscopy further confirmed that Al was below detection limits (<0.5 ppm) in the brain. Animals supplemented with either NSP or Swy-2 were similar to controls with respect to toxicity evaluations and metal analysis, with the exception of decreased brain Rb following clay supplementation. Overall, the results of this study suggest that neither NSP nor Swy-2, at relatively high dietary concentrations, influences mineral uptake or utilization in the pregnant rat.

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 AFB1–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 × time reported no side-effects throughout the study. Mild to moderate health events (∼0.5% of person × 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.

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 AFs 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.

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 µg l−1; female = 116.40 ± 24.26 µg l−1) compared with the PL group (male = 83.55 ± 39.90 µg l−1; female = 90.47 ± 25.68 µg 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.

Determinants of the Variability of Aflatoxin–Albumin Adduct Levels in Ghanaians

Hepatocellular carcinoma (HCC) is a multifactorial disease with various host and environmental factors involved in its etiology. Of these, aflatoxin exposure has been established as an important risk factor in the development of HCC; the presence of aflatoxin–albumin (AA) adducts in the blood serves as a valuable biomarker of human exposure. In this study, the relationship between a variety of different HCC host factors and the incidence of AA adduct levels was examined in a Ghanaian population at high risk for HCC. These factors included age, gender, hepatitis virus B (HVB) and hepatitis C virus (HCV) status, and genetic polymorphisms in both microsomal epoxide hydrolase (mEH) and glutathione S-transferases (GSTs). Blood samples were analyzed for AA adducts and HBV and HCV status. GSTM1 and GSTT1 deletion polymorphisms and mEH exon 3 and exon 4 single-nucleotide polymorphisms (SNPs) were determined from urine samples. In univariate analysis, age, HBV and HVC status, and GSTT1 and mEH exon 3 genotypes were not associated with AA adduct levels. However, mean adduct levels were significantly higher in both females and individuals typed heterozygous for mEH exon 4 (vs. wild types). Stratification analysis also showed that gender along with mEH exon 4 genotype and HBV status had a significant effect on adduct levels. Both females typed HBsAg+ and males with mEH exon 4 heterozygote genotypes showed significantly higher adduct levels as compared to the HBsAg– and wild types, respectively. Understanding the relationships between these host factors and the variability in aflatoxin-adduct levels may help in identifying susceptible populations in developing countries and for targeting specific public health interventions for the prevention of aflatoxicoses in populations with HCC and chronic liver diseases.

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. AFB1–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) µmol l−1 and 15.68 ± 4.12 (range = 6.35–30.40) µmol 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.