Reduction in exposure to carcinogenic aflatoxins by postharvest intervention measures in west Africa: a community-based intervention study

Background Aflatoxins are fungal metabolites that frequently contaminate staple foods in much of sub-Saharan Africa, and are associated with increased risk of liver cancer and impaired growth in young children. We aimed to assess whether postharvest measures to restrict aflatoxin contamination of groundnut crops could reduce exposure in west African villages.

Methods We undertook an intervention study at subsistence farms in the lower Kindia region of Guinea. Farms from 20 villages were included, ten of which implemented a package of postharvest measures to restrict aflatoxin contamination of the groundnut crop; ten controls followed usual postharvest practices. We measured the concentrations of blood aflatoxin-albumin adducts from 600 people immediately after harvest and at 3 months and 5 months postharvest to monitor the effectiveness of the intervention.

Findings In control villages mean aflatoxin-albumin concentration increased postharvest (from 5.5 pg/mg [95% CI 4.7-6.1] immediately after harvest to 18.7 pg/mg [17.0-20.6] 5 months later). By contrast, mean aflatoxin-albumin concentration in intervention villages after 5 months of groundnut storage was much the same as that immediately postharvest (7.2 pg/mg [6.2-8.4] vs 8.0 pg/mg [7.0-9.2]). At 5 months, mean adduct concentration in intervention villages was less than 50% of that in control villages (8.0 pg/mg [7.2-9.2] vs 18.7 pg/mg [17.0-20.6], p<0.0001). About a third of the number of people had non-detectable aflatoxin-albumin concentrations at harvest. At 5 months, five (2%) people in the control villages had non-detectable adduct concentrations compared with 47 (20%) of those in the intervention group (p<0.0001). Mean concentrations of aflatoxin B1 in groundnuts in household stores in intervention and control villages were consistent with measurements of aflatoxin-albumin adducts.

Interpretation Use of low-technology approaches at the subsistence-farm level in sub-Saharan Africa could substantially reduce the disease burden caused by aflatoxin exposure.

Co-exposures of aflatoxins with deoxynivalenol and fumonisins from maize based complementary foods in Rombo, Northern Tanzania

Children consuming maize based foods in Tanzania may be exposed to multiple mycotoxins. We estimated co-exposures of aflatoxins with Deoxynivalenol (DON) and fumonisins for children in rural Tanzania. Food consumption by the children was estimated by twice administering a 24 h dietary recall questionnaire to mothers of 18-24 months old children in Kikelelwa village. Each mother also; provided a sample of maize based flour used for feeding her child in the previous day. Each child's body weight (bw) was measured by following standard procedures. Aflatoxins, DON and fumonisins were determined in each sample using validated HPLC methods. Exposures for a mycotoxin were estimated by multiplying flour consumption (g/child/kgbw/day) by its contamination (mu g/kg). Complete data were obtained for 41 children. Maize flour consumption ranged from 16 to 254 g/child/day. Thirteen (32%) of the 41 children consumed flour with detectable aflatoxin levels (range, 0.11-386 mu g/kg), resulting in exposures from 1 to 786 ng/kg bw/day. All these children exceeded the aflatoxins exposure of concern (0.017 ng/kg bw/day). Eighteen (44%) of the children consumed flour with detectable DON levels (57-825 mu g/kg) and 34(83%), detectable fumonisins levels (63-2284 mu g/kg), resulting in respective exposure ranges of 0.38-8.87 mu g/kg bw/day and 0.19-26.37 mu g/kg bw/day. Twelve (66%) of the DON exposed children and 56% of the fumonisins exposed children exceeded the respective provisional tolerable daily intakes of 1 mu g/kg bw and 2 ng/kg bw. Co-exposures for aflatoxins with both DON and fumonsins were determined in 10% of the 41 children. Co-exposures of aflatoxins with fumonisins alone were found in 29% and of fumonisins with DON alone in 41% of the children. The study showed that children consuming maize based complementary foods in Northern Tanzania are at a risk of exposure to multiple mycotoxins. We recommend adoption of appropriate measures to minimize exposures of multiple mycotoxins in Tanzania. (C) 2014 Elsevier Ltd. All rights reserved.

Fast and sensitive aflatoxin B1 and total aflatoxins ELISAs for analysis of peanuts, maize and feed ingredients

Aflatoxins are a group of carcinogenic compounds produced by Aspergillus fungi that can grow on different agricultural crops. Both acute and chronic exposure to these mycotoxins can cause serious illness. Due to the high occurrence of aflatoxins in crops worldwide fast and cost-effective analytical methods are required for the identification of contaminated agricultural commodities before they are processed into final products and placed on the market. In order to provide new tools for aflatoxin screening two prototype fast ELISA methods: one for the detection of aflatoxin B₁ and the other for total aflatoxins were developed. Seven monoclonal antibodies with unique high sensitivity and at the same time good cross-reactivity profiles were produced. The monoclonal antibodies were characterized and two antibodies showing IC₅₀ of 0.037 ng/mL and 0.031 ng/mL for aflatoxin B₁ were applied in simple and fast direct competitive ELISA tests. The methods were validated for peanut matrix as this crop is one of the most affected by aflatoxin contamination. The detection capabilities of aflatoxin B₁ and total aflatoxins ELISAs were 0.4 μg/kg and 0.3 μg/kg for aflatoxin B₁, respectively, which are one of the lowest reported values. Total aflatoxins ELISA was also validated for the detection of aflatoxins B₂, G₁ and G₂. The application of the developed tests was demonstrated by screening 32 peanut samples collected from the UK retailers. Total aflatoxins ELISA was further applied to analyse naturally contaminated maize porridge and distiller's dried grain with solubles samples and the results were correlated with these obtained by UHPLC-MS/MS method.

Mycotoxins and human disease: a largely ignored global health issue

Aflatoxins and fumonisins (FB) are mycotoxins contaminating a large fraction of the world's food, including maize, cereals, groundnuts and tree nuts. The toxins frequently co-occur in maize. Where these commodities are dietary staples, for example, in parts of Africa, Asia and Latin America, the contamination translates to high-level chronic exposure. This is particularly true in subsistence farming communities where regulations to control exposure are either non-existent or practically unenforceable. Aflatoxins are hepatocarcinogenic in humans, particularly in conjunction with chronic hepatitis B virus infection, and cause aflatoxicosis in episodic poisoning outbreaks. In animals, these toxins also impair growth and are immunosuppressive; the latter effects are of increasing interest in human populations. FB have been reported to induce liver and kidney tumours in rodents and are classified as Group 2B 'possibly carcinogenic to humans', with ecological studies implying a possible link to increased oesophageal cancer. Recent studies also suggest that the FB may cause neural tube defects in some maize-consuming populations. There is a plausible mechanism for this effect via a disruption of ceramide synthase and sphingolipid biosynthesis. Notwithstanding the need for a better evidence-base on mycotoxins and human health, supported by better biomarkers of exposure and effect in epidemiological studies, the existing data are sufficient to prioritize exposure reduction in vulnerable populations. For both toxins, there are a number of practical primary and secondary prevention strategies which could be beneficial if the political will and financial investment can be applied to what remains a largely and rather shamefully ignored global health issue.

TP53 R249S Mutations, Exposure to Aflatoxin, and Occurrence of Hepatocellular Carcinoma in a Cohort of Chronic Hepatitis B Virus Carriers from Qidong, China

Hepatocellular carcinoma (HCC) has a high mortality in East Asia and Sub-Saharan Africa, two regions where the main etiologic factors are chronic infections with hepatitis B vir-us and dietary exposure to aflatoxin. A single base substitution at the third nucleotide of codon 249 of TP53 (R249S) is common in HCC in these regions and has been associated with aflatoxin-DNA adducts. To determine whether R249S may be detected in plasma DNA before HCC diagnosis, we conducted a case-control study nested in a cohort of adult chronic hepatitis B virus carriers from Qidong County, People's Republic of China. Of the 234 plasma specimens that yielded adequate DNA, only 2 (0.9%) were positive for R249S by restriction fragment length polymorphisms, and both of them were controls. Of the 249 subjects tested for aflatoxin-albumin adducts, 168 (67%) were positive, with equal distribution between cases and controls. Aflatoxin-albumin adduct levels were low in the study, suggesting an overall low ongoing exposure to aflatoxin in this cohort. The R249S mutation was detected in 11 of 18 (61%) available tumor tissues. To assess whether low levels of mutant DNA were detectable in pre-diagnosis plasma, 14 plasma specimens from these patients were analyzed by short oligonucleotide mass analysis. Nine of them (64%) were found to be positive. Overall, these results suggest that HCC containing R249S can occur in the absence of significant recent exposure to aflatoxins. The use of short oligonucleotide mass analysis in the context of low ongoing aflatoxin exposure may allow the detection of R249S in plasma several months ahead of clinical diagnosis. (Cancer Epidemiol Biomarkers Prev 2009;18(5):1638-43)

Human aflatoxin albumin adducts quantitatively compared by ELISA, HPLC with fluorescence detection, and HPLC with isotope dilution mass spectrometry

Essential to the conduct of epidemiologic studies examining aflatoxin exposure and the risk of heptocellular carcinoma, impaired growth, and acute toxicity has been the development of quantitative biomarkers of exposure to aflatoxins, particularly aflatoxin B-1. In this study, identical serum sample sets were analyzed for aflatoxin-albumin adducts by ELISA, high-performance liquid chromatography (HPLC) with fluorescence detection (HPLC-f), and HPLC with isotope dilution mass spectrometry (IDMS). The human samples analyzed were from an acute aflatoxicosis outbreak in Kenya in 2004 (n = 102) and the measured values ranged from 0.018 to 67.0, nondetectable to 13.6, and 0.002 to 17.7 ng/mg albumin for the respective methods. The Deming regression slopes for the HPLC-f and ELISA concentrations as a function of the IDMS concentrations were 0.71 (r(2) = 0.95) and 3.3 (r(2) = 0.96), respectively. When the samples were classified as cases or controls, based on clinical diagnosis, all methods were predictive of outcome (P < 0.01). Further, to evaluate assay precision, duplicate samples were prepared at three levels by dilution of an exposed human sample and were analyzed on three separate days. Excluding one assay value by ELISA and one assay by HPLC-f, the overall relative SD were 8.7%, 10.5%, and 9.4% for IDMS, HPLC-f, and ELISA, respectively. IDMS was the most sensitive technique and HPLC-f was the least sensitive method. Overall, this study shows an excellent correlation between three independent methodologies conducted in different laboratories and supports the validation of these technologies for assessment of human exposure to this environmental toxin and carcinogen.

Study of aflatoxin exposure in a Brazilian population using an aflatoxin - albumin biomarker

The evaluation of exposure to aflatoxins (AF) by measurement of the level of contamination in food is hampered due to the heterogeneous distribution of AF in food. Therefore, an alternative is to estimate the exposure using specific biological markers (biomarkers) based on an understanding of the metabolism of the compound. For AF, these include aflatoxin-N-7-guanine in the urine, or AFB(1)-albumin (AF-alb) in the blood. This study assessed the level of exposure to AF in Brazilian individuals using a biomarker approach, i.e. the AF-alb adducts. Blood samples were collected from urban residents (n=50; aged 18-52) in June 1999, at the Blood Center of Antonio Carlos de Camargo Hospital, Sao Paulo, Brazil. AF-alb adduct levels were determined, by ELISA following serum albumin extraction and digestion. AF-alb adducts were detected in 31/50 (62%) samples [range 0-57.3 pg AFB(1)-lys adducts/mg of blood albumin (pg/mg)]. The mean level of positives was 14.9 pg/mg and males had the two highest levels measured (57.1 and 57.3 pg/mg). There was no correlation with age or profession. This is the first study of Brazilian, or indeed South American, individuals that has determined exposure to AF at the individual level using a biomarker approach. These levels are similar to those observed in the Philippines. These data warrant further investigation of both the sources and consequences of exposure to this potent toxin in Brazil.

Aflatoxin exposure in utero causes growth faltering in Gambian infants

Background Growth faltering in West African children has previously been associated with dietary exposure to aflatoxins, particularly upon weaning. However, in animal studies in utero exposure to low levels of aflatoxin also results in growth faltering.

Objective This study investigated the effect of in utero aflatoxin exposure on infant growth in the first year of life in The Gambia.

Methods Height and weight were measured for 138 infants at birth and at regular monthly intervals for one year. Aflatoxin-albumin (AF-alb) adduct level was measured in maternal blood during pregnancy, in cord blood and in infants at age 16 weeks.

Results The geometric mean AF-alb levels were 40.4pg/mg (range 4.82-60.8pg/mg), 10.1pg/mg (range 5.01-89.6pg/mg) and 8.7pg/mg (range 5.0-30.2pg/mg) in maternal, cord and infant blood, respectively. AF-alb in maternal blood was a strong predictor of both weight (P = 0.012) and height (P = 0.044) gain, with lower gain in those with higher exposure. A reduction of maternal AF-alb from 110pg/mg to 10pg/mg would lead to a 0.8kg increase in weight and 2cm increase in height within the first year of life.

Conclusions This study shows a strong effect of maternal aflatoxin exposure during pregnancy on growth in the first year of life and thus extends earlier observations of an association between aflatoxin exposure during infancy and growth faltering. The findings imply value in targeting intervention strategies at early life exposures.

Dietary exposure to aflatoxin from maize and groundnut in young children from Benin and Togo, West Africa

Aflatoxins are a family of fungal toxins that are carcinogenic to man and cause immunosuppression, cancer and growth reduction in animals. We conducted a cross-sectional study among 480 children (age 9 months to 5 years) across 4 agroecological zones (SS, NGS, SGS and CS) in Benin and Togo to identify the effect of aflatoxin exposure on child growth and assess the pattern of exposure. Prior reports on this study [Gong, Y.Y., Cardwell, K., Hounsa, A., Egal, S., Turner, Hall, A.J., Wild, C.P., 2002. Dietary aflatoxin exposure and impaired growth in young children from Benin and Togo: cross sectional study. British Medical Journal 325, 20-21, Gong, Y.Y., Egal, S., Hounsa, A., Turner, P.C., Hall, A.J., Cardwell, K., Wild, C.P., 2003. Determinants of aflatoxin exposure in young children from Benin and Togo, West Africa: the critical role of weaning and weaning foods. International Journal of Epidemiology, 32, 556-562] showed that aflatoxin exposure among these children is widespread (99%) and that growth faltering is associated with high blood aflatoxinalbumin adducts (AF-alb adducts), a measure of recent past exposure. The present report demonstrates that consumption of maize is an important source of aflatoxin exposure for the survey population. Higher AF-alb adducts were correlated with higher A. flavus (CFU) infestation of maize (p=0.006), higher aflatoxin contamination (ppb) of maize (p<0.0001) and higher consumption frequencies of maize (p=0.053). The likelihood of aflatoxin exposure from maize was particularly high in agro-ecological zones where the frequency of maize consumption (SGS and CS), the presence of allatoxin in maize (SGS) or the presence of A. flavus on maize (NGS and SGS) was relatively high. Socio-economic background did not affect the presence of A. flavus and aflatoxin in maize, but better maternal education was associated with lower frequencies of maize consumption among children from the northernmost agro-ecological zone (SS) (p=0.001). The impact of groundinit consumption on aflatoxin exposure was limited in this population. High AF-alb adduct levels were correlated with high prevalence of A. flavus and aflatoxin in groundinit, but significance was weak after adjustment for weaning status, agro-ecological zone and maternal socio-economic status (resp. p = 0.091 and p = 0.083). Ingestion of A. flavus and aflatoxin was high in certain agro-ecological zones (SS and SGS) and among the higher socio-economic strata due to higher frequencies of groundnut consumption. Contamination of groundnuts was similar across socio-economic and agroecological boundaries.

In conclusion, dietary exposure to aflatoxin from groundnut was less than from maize in young children from Benin and Togo. Intervention strategies that aim to reduce dietary exposure in this population need to focus on maize consumption in particular, but they should not ignore consumption of groundnuts. (c) 2005 Elsevier B.V. All rights reserved.

Postweaning exposure to aflatoxin results in impaired child growth: A longitudinal study in Benin, west Africa

Aflatoxins are dietary contaminants that are hepatocarcinogenic and immunotoxic and cause growth retardation in animals, but there is little evidence concerning the latter two parameters in exposed human populations. Aflatoxin exposure of West African children is known to be high, so we conducted a longitudinal study over an 8-month period in Benin to assess the effects of exposure on growth. Two hundred children 16-37 months of age were recruited from four villages, two with high and two with low aflatoxin exposure (50 children per village). Serum aflatoxin-albumin (AF-alb) adducts, anthropometric parameters, information on food consumption, and various demographic data were measured at recruitment (February) and at two subsequent time points (June and October). Plasma levels of vitamin A and zinc were also measured. AF-alb adducts increased markedly between February and October in three of the four villages, with the largest increases in the villages with higher exposures. Children who were fully weaned at recruitment had higher AF-alb than did those still partially breast-fed (p < 0.0001); the major weaning food was a maize-based porridge. There was no association between AF-alb and micronutrient levels, suggesting that aflatoxin exposure was not accompanied by a general nutritional deficiency. There was, however, a strong negative correlation (p < 0.0001) between AF-alb and height increase over the 8-month follow-up after adjustment for age, sex, height at recruitment, socioeconomic status, village, and weaning status; the highest quartile of AF-alb was associated with a mean 1.7 cm reduction in growth over 8 months compared with the lowest quartile. This study emphasizes the association between aflatoxin and stunting, although the underlying mechanisms remain unclear. Aflatoxin exposure during the weaning period may be critical in terms of adverse health effects in West African children, and intervention measures to reduce exposure merit investigation.

Quantitative correlation of aflatoxin biomarker with dietary intake of aflatoxin in Tanzanian children

The association between aflatoxin intake from maize-based weaning food and aflatoxin albumin adducts (AF-alb) was investigated in 148 Tanzanian children aged between 12 and 22 months, at 2 visits 6 months apart. At the first visit (storage season) there was a significant correlation at the individual level between AF-alb (geometric mean 43.2 pg/mg albumin) and aflatoxin intake (geometric mean 81.7 ng/kg b.w./d) through maize-based weaning food (r = 0.51, p < 0.01). Overall, this correlation was r = 0.43 (p < 0.01). The AF-alb level in weaning-age children in Tanzania closely reflects aflatoxin intake from maize in weaning food. Exposure levels suggest children may be at risk from aflatoxin associated health effects.

A Prospective Study of Growth and Biomarkers of Exposure to Aflatoxin and Fumonisin during Early Childhood in Tanzania

BACKGROUND: Aflatoxin and fumonisin are toxic food contaminants. Knowledge about effects of their exposure and co-exposure on child growth is inadequate.

OBJECTIVE: To investigate the association between child growth and aflatoxin and fumonisin exposure in Tanzania.

METHODS: A total of 166 children were recruited at 6 to 14 months of age and studied at recruitment, and at the sixth and twelfth month following recruitment. Blood and urine samples were collected and analysed for plasma aflatoxin albumin adducts (AF-alb) using ELISA, and urinary fumonisin B1 (UFB1) using LC-MS, respectively. Anthropometric measurements were taken and growth index Z-scores were computed.

RESULTS: AF-alb geometric mean concentrations (95% confidence intervals) were 4.7 (3.9, 5.6), 12.9 (9.9, 16.7) and 23.5 (19.9, 27.7) pg/mg albumin at recruitment, six months, and 12 months from recruitment, respectively. At these respective sampling times, geometric mean UFB1 concentrations (95% CI) were 313.9 (257.4, 382.9), 167.3 (135.4, 206.7) and 569.5 (464.5, 698.2) pg/mL urine, and the prevalence of stunted children were 44%, 55% and 56%, respectively. UFB1 concentrations at recruitment were negatively associated with length for age Z-scores (LAZ) at six months (p = 0.016) and at 12 months from recruitment (p = 0.014). The mean UFB1 of the three sampling times (at recruitment, at six and 12 months from recruitment) in each child was negatively associated with LAZ (p < 0.001) and length velocity (p = 0.004) at 12 months from recruitment. The negative association between AF-alb and child growth did not reach statistical significance.

CONCLUSIONS: Exposure to fumonisin alone, or co-exposure with aflatoxins may contribute to child growth impairment.

Aflatoxin Exposure and Associated Human Health Effects, a Review of Epidemiological Studies

flatoxins are fungal toxins that possess acute life threatening toxicity, carcinogenic properties and other potential chronic adverse effects. Dietary exposure to aflatoxins is considered a major public health concern, especially for subsistence farming communities in sub-Saharan Africa and South Asia, where dietary staple food crops such as groundnuts and maize are often highly contaminated with aflatoxin due to hot and humid climates and poor storage, together with low awareness of risk and lack of enforcement of regulatory limits. Biomarkers have been developed and applied in many epidemiological studies assessing aflatoxin exposure and the associated health effects in these high-risk population groups. This review discusses the recent epidemiological evidence for aflatoxin exposure, co-exposure with other mycotoxins and associated health effects in order to provide evidence on risk assessment, and highlight areas where further research is necessary. Aflatoxin exposure can occur at any stage of life and is a major risk factor for hepatocellular carcinoma, especially when hepatitis B infection is present. Recent evidence suggests that aflatoxin may be an underlying determinant of stunted child growth, and may lower cell-mediated immunity, thereby increasing disease susceptibility. However, a causal relationship between aflatoxin exposure and these latter adverse health outcomes has not been established, and the biological mechanisms for these have not been elucidated, prompting further research. Furthermore, there is a dearth of information regarding the health effects of co-exposure to aflatoxin with other mycotoxins. Recent developments of biomarkers provide opportunities for important future research in this area.

Evaluation of an alternative spectroscopic approach for aflatoxin analysis: Comparative analysis of food and feed samples with UPLC-MS/MS

Increasing research has highlighted the effects of changing climates on the occurrence and prevalence of toxigenic Aspergillus species producing aflatoxins. There is concern of the toxicological effects to human health and animal productivity following acute and chronic exposure that may affect the future ability to provide safe and sufficient food globally. Considerable research has focused on the detection of these toxins, based on the physicochemical and biochemical properties of the aflatoxin compounds, in agricultural products for human and animal consumption. As improvements in food security continue more regulations for acceptable levels of aflatoxins have arisen globally; the most stringent in Europe. These regulations are important for developing countries as aflatoxin occurrence is high significantly effecting international trade and the economy. In developed countries analytical approaches have become highly sophisticated, capable of attaining results with high precision and accuracy, suitable for regulatory laboratories. Regrettably, many countries that are affected by aflatoxin contamination do not have resources for high tech HPLC and MS instrumentation and require more affordable, yet robust equally accurate alternatives that may be used by producers, processors and traders in emerging economies. It is especially important that those companies wishing to exploit the opportunities offered by lucrative but highly regulated markets in the developed world, have access to analytical methods that will ensure that their exports meet their customers quality and safety requirements.

This work evaluates the ToxiMet system as an alternative approach to UPLC–MS/MS for the detection and determination of aflatoxins relative to current European regulatory standards. Four commodities: rice grain, maize cracked and flour, peanut paste and dried distillers grains were analysed for natural aflatoxin contamination. For B1 and total aflatoxins determination the qualitative correlation, above or below the regulatory limit, was good for all commodities with the exception of the dried distillers grain samples for B1 for which no calibration existed. For B1 the quantitative R² correlations were 0.92, 0.92, 0.88 (<250 μg/kg) and 0.7 for rice, maize, peanuts and dried distillers grain samples respectively whereas for total aflatoxins the quantitative correlation was 0.92, 0.94, 0.88 and 0.91. The ToxiMet system could be used as an alternative for aflatoxin analysis for current legislation but some consideration should be given to aflatoxin M1 regulatory levels for these commodities considering the high levels detected in this study especially for maize and peanuts