Nicole J. Mitchell

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.

Calcium montmorillonite clay reduces AFB1 and FB1 biomarkers in rats exposed to single and co‐exposures of aflatoxin and fumonisin

Aflatoxins (AFs) and fumonisins (FBs) can co‐contaminate foodstuffs and have been associated with hepatocellular and esophageal carcinomas in humans at high risk for exposure. One strategy to reduce exposure (and toxicity) from contaminated foodstuffs is the dietary inclusion of a montmorillonite clay (UPSN) that binds AFs and FBs in the gastrointestinal tract. In this study, the binding capacity of UPSN was evaluated for AFB1, FB1 and a combination thereof in Fischer 344 rats. Rats were pre‐treated with different dietary levels of UPSN (0.25% or 2%) for 1 week. Rats were gavaged with a single dose of either 0.125 mg AFB1 or 25 mg FB1 per kg body weight and a combination thereof in the presence and absence of an aqueous solution of UPSN. The kinetics of mycotoxin excretion were monitored by analyzing serum AFB1‐albumin, urinary AF (AFM1) and FB1 biomarkers over a period of 72 h. UPSN decreased AFM1 excretion by 88–97%, indicating highly effective binding. FB1 excretion was reduced, to a lesser extent, ranging from 45% to 85%. When in combination, both AFB1 and FB1 binding occurred, but capacity was decreased by almost half. In the absence of UPSN, the combined AFB1 and FB1 treatment decreased the urinary biomarkers by 67% and 45% respectively, but increased levels of AFB1‐albumin, presumably by modulating its cytochrome metabolism. UPSN significantly reduced bioavailability of both AFB1 and FB1 when in combination; suggesting that it can be utilized to reduce levels below their respective thresholds for affecting adverse biological effects. Copyright

Modified Hydra Bioassay to Evaluate the Toxicity of Multiple Mycotoxins and Predict the Detoxification Efficacy of a Clay-Based Sorbent

Food shortages and a lack of food supply regulation in developing countries often leads to chronic exposure of vulnerable populations to hazardous mixtures of mycotoxins, including aflatoxin B1 (AFB1) and fumonisin B1 (FB1). A refined calcium montmorillonite clay [i.e. uniform particle size NovaSil (UPSN)] has been reported to tightly bind these toxins, thereby decreasing bioavailability in humans and animals. Hence, our objectives in the present study were to examine the ability of UPSN to bind mixtures of AFB1 and FB1 at gastrointestinally relevant pH in vitro, and to utilize a rapid in vivo bioassay to evaluate AFB1 and FB1 toxicity and UPSN efficacy. Isothermal sorption data indicated tight AFB1 binding to UPSN surfaces at both pH 2.0 and 6.5, but substantially more FB1 bound at pH 2.0 than 6.5. Site‐specific competition occurred between the toxins when exposed to UPSN in combination. Importantly, treatment with UPSN resulted in significant protection to mycotoxin‐exposed hydra maintained at pH 6.9–7.0. Hydra were exposed to FB1, AFB1 and FB1/AFB1 combinations with and without UPSN. A toxic response over 92 h was rated based on morphology and mortality. Hydra assay results indicated a minimum effective concentration (MEC) of 20 µg ml–1 for AFB1, whereas the MEC for FB1 was not reached. The MEC for co‐exposure was 400 µg ml–1 FB1 + 10 µg ml–1 AFB1. This study demonstrates that UPSN sorbs both mycotoxins tightly at physiologically relevant pH levels, resulting in decreased bioavailability, and that a modified hydra bioassay can be used as an initial screen in vivo to predict efficacy of toxin‐binding agents. Copyright

Sequential dietary exposure to aflatoxin B1 and fumonisin B1 in F344 rats increases liver preneoplastic changes indicative of a synergistic interaction

Dietary co-exposure to aflatoxin B1 (AFB1) and fumonisin B1 (FB1) and their interaction on hepatocellular carcinogenesis is of particular concern in toxicology and public health. In this study we evaluated the liver preneoplastic effects of single and sequential dietary exposure to AFB1 and FB1 in the F344 rat carcinogenesis model. Serum biochemical alterations, liver histopathological changes, and the formation of liver glutathione S transferase positive (GST-P+) foci were the major outcome parameters examined. Compared to the AFB1-only treatment, the FB1-only treatment induced less dysplasia, and more apoptosis and mitoses. Sequential AFB1 and FB1 treatment lead to increased numbers of dysplasia, apoptosis and foci of altered hepatocytes, as compared to either mycotoxin treatment alone. More importantly, sequential exposure to AFB1 and FB1 synergistically increased the numbers of liver GTP-P+ foci by approximately 7.3-and 12.9-fold and increased the mean sizes of GST-P+ foci by 6- and 7.5-fold, respectively, as compared to AFB1- or FB1-only treatment groups. In addition, liver ALT and AST levels were significantly increased after sequential treatment as compared to single treatment groups. The results demonstrate the interactive effect of dietary AFB1 and FB1 in inducing liver GST-P+ foci formation and provide information to model future intervention studies.

Reduction in the urinary aflatoxin M1 biomarker as an early indicator of the efficacy of dietary interventions to reduce exposure to aflatoxins.

Abstract Aflatoxin B1 is a persistent public health issue in Ghana. Assessment of AFB1 intervention efficacy is currently dependent on long-term biomarkers. This study was designed to determine whether daily AFM1 biomarker levels could be utilized as an early detection method for intervention efficacy. Participants were treated with a refined calcium montmorillonite clay (UPSN) or a placebo (calcium carbonate) in a crossover study. Urine samples were assessed for AFM1 levels daily. UPSN treatment reduced AFM1 biomarkers by 55% compared to the placebo. This is the first study to show that daily urinary AFM1 levels can be used as a biomarker of internal aflatoxin B1 exposure in short-term intervention trials to determine efficacy.

Integrative Toxicopathological Evaluation of Aflatoxin B1 Exposure in F344 Rats

In this study, male F344 rats were orally exposed to a single dose of aflatoxin B1 (AFB1) at 0, 50, 250, or 1,000 µg/kg body weight (BW) or repeated dose of 0, 5, 10, 25, or 75 µg/kg BW for up to 5 weeks. Biochemical and histological changes were assessed together with the formation of AFB1-lysine adduct (AFB-Lys) and liver foci positive for placental form glutathione S transferase (GST-P+). In single-dose protocol, serum aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP) showed dose-related elevation, with maximal changes observed (>100-fold) at day 3 after treatment. Animals that received 250 µg/kg AFB1 showed concurrent bile duct proliferation, necrosis, and GST-P+ hepatocytes at 3 day, followed by liver GST-P+ foci appearance at 1 week. In repeated-dose protocol, bile duct proliferation and liver GST-P+ foci co-occurred after 3-week exposure to 75 µg/kg AFB1, followed by proliferation foci formation after 4 week and dramatic ALT, AST, and CK elevations after 5 weeks. Liver GST-P+ foci were induced temporally and in a dose-related manner. Serum AFB-Lys increased temporally at low doses (5–25 µg/kg), and reached the maximum after 2-week exposure at 75 µg/kg. This integrative study demonstrated that liver GST-P+ cells and foci are sensitive biomarkers for AFB1 toxic effect and correlated with bile duct proliferation and biochemical alterations in F344 rats.

Short-Term Safety and Efficacy of Calcium Montmorillonite Clay (UPSN) in Children

Recently, an association between childhood growth stunting and aflatoxin (AF) exposure has been identified. In Ghana, homemade nutritional supplements often consist of AF-prone commodities. In this study, children were enrolled in a clinical intervention trial to determine the safety and efficacy of Uniform Particle Size NovaSil (UPSN), a refined calcium montmorillonite known to be safe in adults. Participants ingested 0.75 or 1.5 g UPSN or 1.5 g calcium carbonate placebo per day for 14 days. Hematological and serum biochemistry parameters in the UPSN groups were not significantly different from the placebo-controlled group. Importantly, there were no adverse events attributable to UPSN treatment. A significant reduction in urinary metabolite (AFM1) was observed in the high-dose group compared with placebo. Results indicate that UPSN is safe for children at doses up to 1.5 g/day for a period of 2 weeks and can reduce exposure to AFs, resulting in increased quality and efficacy of contaminated foods.

Common African cooking processes do not affect the aflatoxin binding efficacy of refined calcium montmorillonite clay.

Aflatoxins are common contaminants of staple crops, such as corn and groundnuts, and a significant cause of concern for food safety and public health in developing countries. Aflatoxin B1 (AFB1) has been implicated in the etiology of acute and chronic disease in humans and animals, including growth stunting, liver cancer and death. Cost effective and culturally acceptable intervention strategies for the reduction of dietary AFB1 exposure are of critical need in populations at high risk for aflatoxicosis. Fermented gruels consisting of cornmeal are a common source for such exposure and are consumed by both children and adults in many countries with a history of frequent, high-level aflatoxin exposure. One proposed method to reduce aflatoxins in the diet is to include a selective enterosorbent, Uniform Particle Size NovaSil (UPSN), as a food additive in contaminated foods. For UPSN to be effective in this capacity, it must be stable in complex, acidic mixtures that are often exposed to heat during the process of fermented gruel preparation. Therefore, the objective of the present study was to test the ability of UPSN to sorb aflatoxin while common cooking conditions were applied. The influence of fermentation, heat treatment, acidity, and processing time were investigated with and without UPSN. Analyses were performed using the field-practical Vicam assay with HPLC verification of trends. Our findings demonstrated that UPSN significantly reduced aflatoxin levels (47-100%) in cornmeal, regardless of processing conditions. Upon comparison of each element tested, time appeared to be the primary factor influencing UPSN efficacy. The greatest decreases in AFB1 were reported in samples allowed to incubate (with or without fermentation) for 72 hrs. This data suggests that addition of UPSN to staple corn ingredients likely to contain aflatoxins would be a sustainable approach to reduce exposure.

In vivo Efficacy of Ferrihydrite as an Enterosorbent for Arsenic: Short-Term Evaluation in Rodents

The use of dietary adsorbents to reduce arsenic (As) exposure is innovative. Ferrihydrite successfully sorbs arsenite and asenate over a wide range of pH conditions and the As–ferrihydrite complexes are stable in gastrointestinal (GIT) models. Our objectives were to (1) compare structural characteristics (using x-ray diffraction and Fourier-transform infrared [FTIR] spectroscopy) and As binding affinities of industrially produced ferrihydrite (IDF) and lab-synthesized ferrihydrite and (2) evaluate the efficacy of the material displaying the best sorption capability as an As enterosorbent in a short-term mammalian model. Lab-synthesized ferrihydrite displayed superior binding affinity for both arsenate and arsenite in vitro, which led to its use in the in vivo portion of the study. Young Sprague-Dawley male rats were fed either a control diet or a 0.5% w/w ferrihydrite feed. After 1 wk of acclimation, rats were given 0.5 ml of 500 mg/L arsenate or arsenite via gavage with or without ferrihydrite. Rats were then transferred to metabolism cages, and urine collected after 24 and 48 h was analyzed for total As. Rats were evaluated daily for signs of morbidity and mortality for up to 1 wk. Ferrihydrite reduced mean urinary As levels by 74.9% and 43.6% after 24 h and 49.1% and 39.5% after 48 h for arsenite- and arsenate-treated groups, respectively. Importantly, treatment groups receiving ferrihydrite displayed no signs of As-related toxicity. All As reductions were statistically significant except for arsenate treatments at 24 h. Data suggest that, as an enterosorbent, ferrihydrite reduces bioavailability after As exposures.