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.

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.

Noninvasive identification of interindividual variation in xenobiotic-metabolizing enzymes: implications for cancer epidemiology and biomarker studies.

In this study, DNA extracted from frozen urine was used in the analysis of polymorphisms in genes coding for xenobiotic-metabolizing enzymes (XMEs). These included single-nucleotide polymorphisms (SNP) in microsomal epoxide hydrolase (mEH), that is, substitutions of tyrosine by histidine in codon 113 (Y113H) and histidine by arginine in codon 139 (H139R), and deletion polymorphisms in glutathione S-transferase (GST) M1 and T1 genes. The concentration of DNA extracted from urine of a Ghanaian population (n = 91) exposed to aflatoxins in their diet ranged from 82.5 to 573 ng/ml urine. Polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) procedures were used for the characterization of mEH polymorphisms, whereas a multiplex PCR method was utilized to identify GST deletion polymorphisms. In total, 91% and 94% of 91 samples were genotyped for mEH exon 3 and exon 4 polymorphisms, respectively. In the multiplex analysis of GST polymorphisms, 94% and 91% of 91 individuals were genotyped for GSTM1 and GSTT1 polymorphisms, respectively. The polymorphisms in the mEH exon 4, GSTM1 and GSTT1, were not in Hardy-Weinberg equilibrium (HWE) except for mEH exon 3. Representative genotypes identified by PCR-RFLP were cloned and sequenced, then confirmed by comparison with reference sequences of human DNA published in the GenBank BLAST database. These results demonstrate that XMEs can be genotyped from urine with reliable accuracy and may be useful in cancer and molecular epidemiology studies.

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.

Aflatoxin-induced toxicity and depletion of hepatic vitamin A in young broiler chicks: protection of chicks in the presence of low levels of NovaSil PLUS in the diet.

Aflatoxin contamination of foods and livestock feeds is an ongoing problem. In this research, NovaSil PLUS (NSP), a calcium montmorillonite clay that is commonly used as an anticaking agent in feeds, was evaluated for its ability to bind aflatoxin B1 (AfB1) in vitro and to prevent the onset of aflatoxicosis and vitamin A depletion in broiler chicks in vivo. Isothermal analyses were conducted with NSP and AfB1 to quantitate and characterize critical sorption parameters at equilibrium, i.e., ligand saturation capacities, affinity constants, and thermodynamics of the sorption process. In vitro results indicated that AfB1 was tightly sorbed onto the surface of NSP, which provided a high capacity and high affinity for the ligand. Thermodynamics favored sorption of AfB1 to NSP. The process was exothermic and spontaneous with a mean heat of sorption equal to approximately -50 kJ/mol, suggesting chemisorption (or tight binding). In addition to the in vitro studies, the effectiveness of NSP as an aflatoxin enterosorbent to attenuate the onset of aflatoxicosis in broiler chicks was determined at 3 different inclusion levels in the diet (0.5, 0.25, and 0.125%). NSP alone was not toxic to chicks at a level as high as 0.5% in the total diets (based on body and organ weights, feed intake, and hepatic vitamin A levels). NSP in the diet significantly protected chicks from the effects of highlevel exposure to aflatoxins (i.e., 5 mg/kg) and preserved hepatic vitamin A levels, even at lower dietary intake of clay.