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Aflatoxin exposure during the first 1000 days of life in rural South Asia assessed by aflatoxin B1-lysine albumin biomarkers
Food and Chemical Toxicology 74 (2014) 184–189
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Food and Chemical Toxicology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f o o d c h e m t o x
Aflatoxin exposure during the first 1000 days of life in rural South Asia assessed by aflatoxin B1-lysine albumin biomarkers John D. Groopman 1,2,*, Patricia A. Egner 1, Kerry J. Schulze 2, Lee S.-F. Wu 2, Rebecca Merrill 2, Sucheta Mehra 2, Abu A. Shamim 2, Hasmot Ali 2, Saijuddin Shaikh 2, Alison Gernand 2 , Subarna K. Khatry 2 , Steven C. LeClerq 2 , Keith P. West Jr 2 , Parul Christian 2 1 2
Department of Environmental Health Sciences, Department of International Health, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA Center for Human Nutrition, Department of International Health, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA
A R T I C L E
I N F O
Article history: Received 11 August 2014 Accepted 22 September 2014 Available online 13 October 2014 Keywords: Aflatoxin Aflatoxin B1-lysine albumin biomarkers Child health Maternal exposure
A B S T R A C T
Aflatoxin B1 is a potent carcinogen, occurring from mold growth that contaminates staple grains in hot, humid environments. In this investigation, aflatoxin B1-lysine albumin biomarkers were measured by mass spectrometry in rural South Asian women, during the first and third trimester of pregnancy, and their children at birth and at two years of age. These subjects participated in randomized community trials of antenatal micronutrient supplementation in Sarlahi District, southern Nepal and Gaibandha District in northwestern Bangladesh. Findings from the Nepal samples demonstrated exposure to aflatoxin, with 94% detectable samples ranging from 0.45 to 2939.30 pg aflatoxin B1-lysine/mg albumin during pregnancy. In the Bangladesh samples the range was 1.56 to 63.22 pg aflatoxin B1-lysine/mg albumin in the first trimester, 3.37 to 72.8 pg aflatoxin B1-lysine/mg albumin in the third trimester, 4.62 to 76.69 pg aflatoxin B1-lysine/mg albumin at birth and 3.88 to 81.44 pg aflatoxin B1-lysine/mg albumin at age two years. Aflatoxin B1-lysine adducts in cord blood samples demonstrated that the fetus had the capacity to convert aflatoxin into toxicologically active compounds and the detection in the same 2-year-old children illustrates exposure over the first 1000 days of life. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Nearly 200,000,000 children worldwide suffer from impaired growth and development and nearly 2 million die each year before age 5 (Black et al., 2013; Smith et al., 2012). Many of these children are from South Asia and these long-term health deficits place an enormous burden on these economically developing countries. The overall contributing factors driving these health problems still needs to be completely identified and better intervention strategies developed (Bhutta et al., 2013). Aflatoxin B1 is a very potent toxin and carcinogen for humans and this naturally occurring mycotoxin occurs as a consequence of specific mold growth that permeates staple grains, legumes and nuts consumed by people, especially in hot, humid environments in lowincome countries (Wogan et al., 2012). Aflatoxin induces many
Abbreviation: AFB1, aflatoxin B1. * Corresponding author. Dr. John D. Groopman, Johns Hopkins University, Department of Environmental Health Sciences, Bloomberg School of Public Health, Baltimore, MD 21205, United States, Tel.: + 4109553900; fax: + 4109550617. E-mail address: [email protected] (J.D. Groopman). http://dx.doi.org/10.1016/j.fct.2014.09.016 0278-6915/© 2014 Elsevier Ltd. All rights reserved.
adverse local and systemic effects that impair normal organ and tissue function resulting in growth retardation, inflammation and immune suppression, all of which contribute in multiple ways to poor health (Khlangwiset et al., 2011; Wild and Gong, 2010). Chronic exposure to aflatoxin also significantly enhances excess risk for liver cancer in many human populations (Kensler et al., 2011; Wild and Gong, 2010). Similar to most environmental toxicants, aflatoxin requires metabolic activation by intrinsic cytochrome P450 enzymes, mainly localized in the liver, to exert its toxicological effects. These metabolic activation pathways have been well described by several research teams (Guengerich, 2008; Wogan et al., 2012). One of these metabolic derivatives, the aflatoxin B1-lysine adduct, that is isolated from serum albumin, is a validated biomarker of exposure and risk (Chen et al., 2013; Sabbioni et al., 1990; Turner et al., 2005; Wang et al., 1996; Wild et al., 1992). A number of studies have found strong associations between aflatoxin exposure, as measured by aflatoxin– albumin biomarkers, and significant reductions in growth in children as assessed by height for age and weight for age (Gong et al., 2002, 2003, 2004; Turner et al., 2007). Aflatoxin B1 contamination has been detected in food, feed and a limited set of blood and urine samples collected in Nepal and at least one third of the foodstuffs examined contained elevated levels
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of this mycotoxin (Koirala et al., 2005; Okumura et al., 1993). Contamination of staple grains and foodstuffs were also found in Bangladesh (Begum and Samajpati, 2000; Dawlatana et al., 2002). Earlier literature in India had related aflatoxin exposure to childhood cirrhosis and acute aflatoxicosis (Krishnamachari et al., 1977; Tandon et al., 1978; Yadgiri et al., 1970). The major goal of this current study was to quantitatively measure aflatoxin exposure using validated biomarkers in pregnancy, early infancy and at ~24 months of age, spanning a critical developmental period referred to as “the first 1000 days of life”, in rural South Asia (Katz et al., 2013). Samples for this exploratory analysis were drawn from an extant collection of biospecimens collected from large samples of pregnant women and infants living in rural settings in the southern plains, or Terai, of Nepal and flood plains of northern Bangladesh. Both study areas, situated along the northern and eastern flanks of the greater Gangetic floodplain that has over 750 million people, are exposed to strong seasonality, high heat, humidity and a general lack of adequate food storage facilities that makes aflatoxin contamination much more likely. In this investigation, women’s serum or plasma samples from the first and third trimester of pregnancy in both country settings and, additionally in Bangladesh, cord blood at birth and serum samples from the same children at ~24 months of age were studied. A state-of-the-art isotope dilution quantitative mass spectrometry method was used for measuring aflatoxin B1-lysine adducts in albumin in these samples as biomarkers of aflatoxin exposure. Thus, the findings reported here represent an initial attempt to characterize the exposure to aflatoxin during pregnancy and early life of the children. These results reveal almost universal excessive exposure to this potent, naturally occurring mycotoxin.
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Fig. 1. Aflatoxin B1-lysine adduct exposures in 30 Nepal pregnant women track between 1st and 3rd trimesters.
(n = 16, >1 g/L) or normal (n = 14, ≤1 g/L) AGP in early pregnancy and corresponding 1st and 3rd trimester samples, allowing us to examine aflatoxin exposure longitudinally through pregnancy and its association with inflammation in early pregnancy. A larger set of early pregnancy samples from the Nepal set was then randomly selected based on birth outcome (n = 27–30 women per group having had normal birth, miscarriage, stillbirth, preterm, or low birth weight for a total of n = 141 samples); data from this larger set were used to examine determinants of aflatoxin exposure, with natural log-transformations used for analysis of the aflatoxinadduct values by a variety of factors by ANOVA. Data presented as geometric means and standard deviations. From the Bangladesh study, a longitudinal cohort of maternal 1st trimester, 3rd trimester, and cord blood samples and samples from corresponding children at 24 months of age was assessed by examining all samples available at the intersection of the main sub-study, cord blood collection substudy, and child follow-up at 24 months of age (n = 63).
2. Methods 2.2. Aflatoxin B1-lysine albumin adduct assay by isotope dilution mass spectrometry 2.1. Study subjects, serum processing and sample selection Archived, serum or plasma specimens for pregnant women and their children were selected from a cohort of subjects who participated in multiple, randomized, antenatal micronutrient supplementation trials carried out in the southern rural plains of Nepal from 1999 to 2001 and more recently (2008–2012) in Bangladesh (West et al., 2013). Details of the design, major findings and biospecimen collection procedures for the trials have been previously reported (Christian et al., 2003a, 2003b). Each trial was approved by institutional review boards in their respective countries, the Nepal Health Research Council, Kathmandu, Nepal and the Bangladesh Medical Research Council, in Dhaka, Bangladesh, and the Institutional Review Board (IRB) of the Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA. The Nepal and Bangladesh trials enrolled n = 4926 and n = 44,567 women, respectively, with subsets of women based on geographic residence recruited for more intensive investigations that included blood draws in the home following enrollment and at approximately 32 weeks of gestation. To be eligible for participation, women were screened at 5-weekly intervals for menstrual status and given a urinebased pregnancy test if menstrual status since the previous visit was negative, allowing for enrollment into the studies prior to a gestational age of 11 weeks on average. In total, n = 1165 baseline (i.e. first trimester) samples and n = 779 third trimester samples were collected at the Nepal site, and n = 2070 first trimester and n = 1597 third trimester samples were collected at the Bangladesh site. Thirty (30) of the Nepal participants were randomly selected for the initial first and third trimester analysis. At the Bangladesh site, a smaller subset of women (n = 399) were additionally enrolled in a study that allowed for the collection of cord blood. Finally, children born to women at the Bangladesh site were followed to 24 months of age. A subset of these children (n = 177) was enrolled in a study that required blood sample collection to evaluate biochemical indicators related to growth. In this study, there were 63 mothers for the first and third trimester samples, 63 cord blood samples and the same 63 two-year olds at follow-up available and analyzed. At both sites, blood was processed to serum or plasma, which was subsequently shipped to The Johns Hopkins Center for Human Nutrition and stored at −80 oC to form a large repository of samples from which the samples selected for this series of investigations were drawn. Longitudinal (1st and 3rd trimester) samples from pregnant women from Nepal were randomly sampled from a larger set of samples in which alpha-1-acid glycoprotein (AGP), a marker of inflammation, had previously been assessed along with C-reactive protein (CRP) using methods as previously described (Christian et al., 1998). Approximately equal numbers of women were selected based on having elevated
All of the serum or plasma samples were analyzed using a minor variation of the method reported by McCoy and colleagues (McCoy et al., 2008). The sample (200 μL) was mixed with an internal standard (10 μL × 0.1 ng AFB1-D4-lys/mL) and Pronase solution (250 μL, 13 mg/mL PBS), and incubated for 18 h at 37 °C. Solidphase extraction–processed samples were analyzed by UPLC with mass spectrometric detection using a ThermoElectron TSQ Vantage operated in the positive electrospray ionization SRM mode. The internal standard parent molecular ion [(M + H)+, m/z 461.3] fragmented to yield an ion at m/z 398.2. The AFB1-lys molecular ion (m/z 457.2) fragmented to yield an ion at m/z 394.1. A 10-point isotopic dilution standard curve was generated by triplicate injection (10 μL) of AFB1-D4-lys (100 pg) mixed with varying amounts of AFB1-lys (0–0.4 ng) prepared via 2-fold serial dilutions. The data were fitted using the method of least-squares with a 1/× weighting factor. The isotope dilution standard curve was linear over the range of 0.2 pg to 0.4 ng AFB1-lys injected onto the column in 10 μL aliquots (R2 = 0.995). The limit of quantitation was 0.2 pg AFB1-lysine/mg albumin in these analyses.
3. Results This research was initiated by an analysis of aflatoxin B1-lysine albumin adducts in a pooled sample representing one thousand 6–7 year old children in Sarlahi, Nepal who were being analyzed for plasma proteome differences related to micronutrient status (Scholl et al., 2012). That analysis showed 56.5 pg aflatoxin B1-lysine/mg albumin, prompting the more detailed examination of aflatoxin exposure during pregnancy and early life described here. The aflatoxin B1-lysine albumin adduct biomarker levels in first and third trimester samples from thirty Nepalese pregnant women are shown in Fig. 1. This study examined the individual tracking of aflatoxin exposure between the first and third trimester. As seen in these results, exposures were quite consistent across these two measures obtained approximately 6 months apart, indicating a high exposure to both the fetus and the mother during pregnancy. Only two and four of the samples from the first and third trimester had non-detectable levels, respectively. These preliminary data were suggestive of limited seasonal variation in aflatoxin exposure.
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Table 1 Determinants of aflatoxin-albumin adducts (pg aflatoxin B1-lysine/mg albumin) among Nepalese women (n = 141) during early pregnancy. pg/mg1
Fig. 2. Aflatoxin B1-lysine adduct exposures in first trimester of pregnancy in 141 Nepal women associate with elevated inflammation biomarker, AGP.
The aflatoxin biomarker levels from the first trimester samples (shown in Fig. 1) were then evaluated by inflammatory status as measured by alpha-1-acid glycoprotein (AGP). These findings are shown in Fig. 2 and reveal that the aflatoxin-albumin adduct levels were higher among women with increased levels of this circulating marker of inflammation. The findings in Fig. 2 are the first to associate elevated AGP with increasing aflatoxin exposure. Among the larger group of samples obtained in early pregnancy among Nepalese women, only nine of the n = 141 samples had a non-detectable level of aflatoxin-albumin adducts, and the range of detectable exposures was from 0.45 to 2939.30 pg aflatoxin B1-lysine/mg albumin. The median level of these samples was 22.45 pg aflatoxin B1-lysine/mg albumin, with a geometric mean of 25.28 pg aflatoxin B1-lysine/mg albumin, and the 25th to 75th percentile ranged from 7.42 to 77.94 pg aflatoxin B1-lysine/mg albumin. Determinants of aflatoxin exposure are further explored in Table 1. Although aflatoxin did not vary by AGP in these analyses, it was significantly higher when another marker of inflammation, C-reactive protein (CRP), was elevated. Further exploration of these data revealed differences in exposure related to local ethnicities. Aflatoxinalbumin adducts in samples from the ethnic Pahadi people (N = 61) were statistically significantly higher (p < .0001) than those in samples from the ethnic Madhesi (N = 80). The Pahadi live in the hill region of Nepal and traditionally have a higher maize consuming diet. The two highest values (2393.3 and 2939.3 pg aflatoxin B1lysine/mg albumin) were found in samples obtained during the June to August rainy season; seasonal variability was significant (p < 0.0001), with samples acquired in the September to November period having the highest level of adducts compared to the other three-month intervals during the year (Table 1). Depicted in Fig. 3 is aflatoxin exposure that occurred among women in Bangladesh across pregnancy from the first through the third trimester. The median levels of the aflatoxin biomarker in the first and third trimester were 18.08 and 25.35 pg aflatoxin B1-lysine/mg albumin, respectively (100% positive). The median level of aflatoxin–lysine albumin adducts in cord blood samples was 27.41 pg aflatoxin B1-lysine/mg albumin (100% positive). This demonstrated that the fetus had the biochemical capacity to convert aflatoxin that crossed the placenta into toxicologically active compounds. Further, the detection of the same biomarkers in 2-year-old children born to the mothers who were exposed to aflatoxins during pregnancy has a median level of 13.79 pg aflatoxin B1-lysine/mg albumin (100% positive). These findings illustrated the profound exposure for these children across their first 1000 days life.
Aflatoxin (total samples) AGP ≤1 g/L >1 g/L CRP ≤5 mg/L >5 mg/L Age 14–19 y 20–29 y ≥30 y Parity 0 1–2 ≥3 Pahadi No Yes Literate No Yes Ground floor construction No walls, thatch, grass, sticks or mud Wood or brick and cement Schooling None 1 or more years Season December–February March–May June–August September–November BMI <19 kg/m2 ≥19 kg/m2 1
p-Value
25.28 (5.81) 26.05 (5.75) 16.11 (7.17)
0.46
23.10 (5.64) 117.92 (5.42)
0.01
22.87 (6.75) 27.94 (6.11) 20.49 (4.31)
0.69
32.46 (6.75) 31.50 (5.53) 17.46 (5.31)
0.14
14.73 (5.16) 54.05 (5.05)
<0.0001
25.79 (5.93) 23.57 (5.53)
0.79
26.58 (5.99) 19.69 (4.85)
0.46
25.28 (5.99) 25.03 (5.26)
0.98
28.22 (4.95) 8.00 (3.53) 46.99 (5.87) 113.30 (3.32) 21.98 (6.30) 29.67 (5.31)
<0.0001
0.32
Data expressed as geometric mean (SD).
4. Discussion The greater Gangetic floodplain regions occupying southern Nepal and northwestern Bangladesh have hot and humid environments conducive to aflatoxin B1 contamination of staple grains (Begum and Samajpati, 2000; Dawlatana et al., 2002; Koirala et al., 2005). While substantial aflatoxin B1 contamination has been detected in a variety of foodstuffs, a much more limited data set exists relating these putative exposures to specific formation of biologically effective dose
Fig. 3. Bangladesh aflatoxin exposure studies: first, third trimester, cord blood and 2 year olds. There were 63 mothers for the first and third trimester samples, 63 cord blood samples and the same 63 two-year olds at follow-up.
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metrics, such as the aflatoxin B1-albumin biomarker, in people. To address this data gap, a quantitative isotope dilution mass spectrometry method was deployed to measure the aflatoxin B1-lysine albumin adduct at biologically critical times in mothers during pregnancy and in the first thousand days of life in their children. An initial analysis of aflatoxin-albumin adducts in a pooled sample from one thousand 6–7 year old children in Nepal found 56.5 pg aflatoxin B1lysine/mg albumin, a level comparable to that detected in very nutritionally compromised children in West Africa when the different method of analyses are considered (Gong et al., 2004; McCoy et al., 2008). This biomarker observation prompted a more detailed examination using samples already analyzed for its plasma proteome that had collected during the first and third trimester of pregnancy of women enrolled in a randomized micronutrient intervention clinical trial in Sarlahi, Nepal (Cole et al., 2013; Scholl et al., 2012). Aflatoxin B1 is a very potent toxic and carcinogenic agent for a variety of species including humans. Contamination of staple grains occurs both prior to harvest and during storage of these foodstuffs. Levels of contamination can vary widely resulting in human exposures that can range from nanograms per day to milligrams per day depending upon the specific staple grain and amount consumed (Kensler et al., 2011). While much of the international focus on this contamination has been framed within its carcinogenic properties, many of its other toxic effects can have both short and longterm health consequences. In South Asia, where both maternal and child health status is tremendously compromised, the potential effects of aflatoxin exposure are most likely manifested by these noncarcinogenic endpoints (Bhutta et al., 2013; Black et al., 2013). However, in the future, as early life health status improves across this region, these potential cancer endpoints could become much more consequential for these populations. Nonetheless, when aflatoxin exposure is determined to be substantial, these data should be viewed as being a sentinel for overall poor quality of staple grains and nutrition (CAST, 2003). The wide heterogeneity in aflatoxin contamination levels in grains results in a very difficult task to assess human exposure using dietary questionnaires and spot testing of staple foods. The development of mechanistically based biomarkers, such as the aflatoxin B1lysine albumin adduct, has helped to overcome the inherent difficulties of assessing aflatoxin exposure by measuring dietary constituents. These biomarkers have the added advantage of being able to integrate exposures over the course of many weeks or months due to the long half-lives of these biomarkers in plasma (McCoy et al., 2005, 2008). Research from our laboratory has also demonstrated the long-term stability of aflatoxin albumin biomarkers in stored samples where we have found these adducts to be stable for up to 25 years (Scholl and Groopman, 2008). The analytical technology for the measurement of these biomarkers has also substantially improved over the last several decades and comparative studies of paired samples have been done to relate measurements obtained across a number of analytical platforms. The correlation of aflatoxin albumin adducts measured by mass spectrometry, HPLC with fluorescence detection and immunoassays has been done and this permits composite comparisons of human data obtained from many diverse molecular epidemiologic studies across the globe (McCoy et al., 2008; Scholl et al., 2006). As a point of reference the values obtained in this comparative study revealed that the ELISA method used provided aflatoxin adduct levels 5-fold higher than mass spectrometry, but the correlation coefficient between the two techniques was 0.98 (McCoy et al., 2008). In the work reported in this study we have employed a quantitative isotope dilution mass spectrometry method that permits the measurement of aflatoxin lysine obtained from serum albumin with the use of an isotopically labeled internal standard. This method is highly sensitive and specific, permitting the quantitation of this biomarker in less than 100 μL of
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Fig. 4. Aflatoxin exposure levels vary widely across regions and time periods. The data for 1995 and 2012 in China are from our recent publication (Chen et al., 2013).
plasma obtained in individuals residing in high exposure regions of the world. Despite the emphasis of many investigations on liver cancer consequent to aflatoxin exposure there have been a number of studies, primarily in West Africa, that have raised the potential etiologic contribution of this toxin to diminished child growth and development (Gong et al., 2002, 2003, 2004). Stunting is calculated to affect nearly 200,000,000 children worldwide primarily in sub-Saharan Africa and South Asia (Black et al., 2008; Smith et al., 2012). Stunting has profound associations with a variety of growth and development deficiencies in these children and to date the overall etiology of this problem remains obscure. Since aflatoxin B1 has major impacts on growth and development in a number of animal species that has been well characterized in the veterinary literature, it is reasonable hypothesis that similar growth and developmental effects can occur in people. The first step in the exploration of this hypothesis is the characterization of specific exposures in high-risk communities. The biologically effective dose biomarker, aflatoxin albumin adducts, provides an objective metric for characterizing these exposures for subsequent follow-up and potential intervention. In an attempt to place the extent of exposures in the populations reported here in Nepal and Bangladesh we compared these current data to those observed in other populations, experiencing acute toxic (Kenya) or chronic (China) exposures (Chen et al., 2013; McCoy et al., 2008), evaluated previously in our laboratory. The results depicted in Fig. 4 provide a composite perspective for the aflatoxin exposures detected in Nepal and Bangladesh in context with other documented exposures. There was an acute aflatoxicosis outbreak in Kenya in 2004 and again in 2005 in which nearly 150 people died after consuming aflatoxin-contaminated maize. The levels depicted, measured in our laboratory, were obtained from 107 people living in those affected Kenyan villages (McCoy et al., 2008; Strosnider et al., 2006). To provide perspective, the median adduct level was 10-times higher than the median for the 141 positive samples of 150 pregnant women in Nepal whose blood was collected in 1999. However, it is important to point out that many of the outlying samples in the Nepal data set overlap values of aflatoxin exposure in individuals from the Kenya toxicity event. The comparative values in Bangladesh (from Fig. 3) are shown next. Finally, three sets of data from our studies in eastern China, a region of very high liver cancer incidence, are shown in the far right of Fig. 4. Samples in China that were collected in 1983, 1995 and 2012 (100 samples for each time point) are depicted (Chen et al., 2013). While the levels found in 1983 are comparable to those measured more recently in Nepal and Bangladesh, the decline in China is seen by 1995 reflecting a transition in this population from a maize
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dominated diet to one in which rice quickly became the dominate staple. Maize is always more contaminated with aflatoxin compared to rice. By 2012, virtually all of the samples collected in Qidong, China were non-detectable for aflatoxin biomarkers. This outcome documents the reduction in aflatoxin exposures that can be obtained through primary prevention: in this case, improved food quality and diversity. Finally, we have found that this relatively recent reduction in aflatoxin exposure in Qidong, China, is associated with a nearly 50% reduction in liver cancer mortality among people under the age of 40 (but not older) in this population, suggesting strongly that reduction of early life exposure can have a major protective effect on cancer outcomes. Data from several published studies can be utilized to calculate approximations of daily aflatoxin exposure in adults and children. These data sets include information gleaned from micro-dosing experiments in humans and plate food-based analyses of exposure where aflatoxin–albumin adduct analysis was also determined (Cupid et al., 2004; Wild and Gong, 2010; Wild et al., 1992). Extrapolating previously published findings with the aflatoxin B1 albumin adduct levels reported in this work indicates that daily exposure in women and their children range from 1 to 20 μg per day. Assuming average body weight of 50 and 10 kg in women and children, respectively, the adult exposures range from 10 to 200 ng per kilogram body weight per day. The children’s exposure was substantially higher due to lower body mass resulting in an exposure range of 50 to 1000 ng per kilogram body weight per day. These exposure calculations provide a framework for determining targeted strategies to reduce dietary exposure to aflatoxin in these populations. In conclusion, in this study we have found that aflatoxin exposure is ubiquitous among at least some of the rural populations of Nepal and Bangladesh, as assessed by a robust mass spectrometrybased albumin adduct biomarker. High levels of aflatoxin B 1 lysine adducts are observed consistently across the first and third trimesters of pregnancy in both of these populations. Aflatoxin B1lysine albumin adducts in human cord blood samples demonstrate that the fetus has the capacity to metabolize aflatoxin to levels comparable to that of the mother. Substantial aflatoxin B1-lysine albumin adducts measured in the 2 year-old children, equivalent to adult levels, strongly suggests life-time exposures to this potent hepatotoxic and immunotoxic food contaminant occur. These data strongly suggest a need to further explore the sources of aflatoxin contamination in the staple grains to establish a process for intervention to lower these exposures. These data strongly indicate the need to develop interventions similar to those used in other highrisk high exposure settings. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgments Supported in part by the Bill and Melinda Gates Foundation (GH 614 and GCE 1046227, GCE 1046221, proteomics), Seattle, WA, USA and the US NIH P30 CA006973. References Begum, F., Samajpati, N., 2000. Mycotoxin production on rice, pulses and oilseeds. Naturwissenschaften 87, 275–277.
Bhutta, Z.A., Das, J.K., Rizvi, A., Gaffey, M.F., Walker, N., Horton, S., et al., 2013. Evidence-based interventions for improvement of maternal and child nutrition: what can be done and at what cost? Lancet 382, 452–477. Black, R.E., Allen, L.H., Bhutta, Z.A., Caulfield, L.E., de Onis, M., Ezzati, M., et al., 2008. Maternal and child undernutrition: global and regional exposures and health consequences. Lancet 371, 243–260. Black, R.E., Victora, C.G., Walker, S.P., Bhutta, Z.A., Christian, P., de Onis, M., et al., 2013. Maternal and child undernutrition and overweight in low-income and middleincome countries. Lancet 382, 427–451. CAST, 2003. Mycotoxins: risk in plants, animals and human systems. Council for Agricultural Science and Technology, p. 217. Chen, J.G., Egner, P.A., Ng, D., Jacobson, L.P., Munoz, A., Zhu, Y.-R., et al., 2013. Reduced aflatoxin exposure presages decline in liver cancer mortality in an endemic region of China. Cancer Prev. Res. 6, 1038–1045. Christian, P., Schulze, K., Stoltzfus, R.J., West, K.P., Jr., 1998. Hyporetinolemia, illness symptoms, and acute phase protein response in pregnant women with and without night blindness. Am. J. Clin. Nutr. 67, 1237–1243. Christian, P., Khatry, S.K., Katz, J., Pradhan, E.K., LeClerq, S.C., Shrestha, S.R., et al., 2003a. Effects of alternative maternal micronutrient supplements on low birth weight in rural Nepal: double blind randomised community trial. BMJ 326, 571. Christian, P., West, K.P., Jr., Khatry, S.K., Leclerq, S.C., Pradhan, E.K., Katz, J., et al., 2003b. Effects of maternal micronutrient supplementation on fetal loss and infant mortality: a cluster-randomized trial in Nepal. Am. J. Clin. Nutr. 78, 1194– 1202. Cole, R.N., Ruczinski, I., Schulze, K., Christian, P., Herbrich, S., Wu, L., et al., 2013. The plasma proteome identifies expected and novel proteins correlated with micronutrient status in undernourished Nepalese children. J. Nutr. 143, 1540– 1548. doi: 1510.3945/jn.1113.175018. Epub 172013 Aug 175021. Cupid, B.C., Lightfoot, T.J., Russell, D., Gant, S.J., Turner, P.C., Dingley, K.H., et al., 2004. The formation of AFB 1-macromolecular adducts in rats and humans at dietary levels of exposure. Food Chem. Toxicol. 42, 559–569. Dawlatana, M., Coker, R.D., Nagler, M.J., Wild, C.P., Hassan, M.S., Blunden, G., 2002. The occurrence of mycotoxins in key commodities in Bangladesh: surveillance results from 1993 to 1995. J. Nat. Toxins 11, 379–386. Gong, Y., Hounsa, A., Egal, S., Turner, P.C., Sutcliffe, A.E., Hall, A.J., et al., 2004. Postweaning exposure to aflatoxin results in impaired child growth: a longitudinal study in Benin, West Africa. Environ. Health Perspect. 112, 1334–1338. Gong, Y.Y., Cardwell, K., Hounsa, A., Egal, S., Turner, P.C., Hall, A.J., et al., 2002. Dietary aflatoxin exposure and impaired growth in young children from Benin and Togo: cross sectional study. BMJ 325, 20–21. Gong, Y.Y., Egal, S., Hounsa, A., Turner, P.C., Hall, A.J., Cardwell, K.F., et al., 2003. Determinants of aflatoxin exposure in young children from Benin and Togo, West Africa: the critical role of weaning. Int. J. Epidemiol. 32, 556–562. Guengerich, F.P., 2008. Cytochrome p450 and chemical toxicology. Chem. Res. Toxicol. 21, 70–83. Katz, J., Lee, A.C., Kozuki, N., Lawn, J.E., Cousens, S., Blencowe, H., et al., 2013. Mortality risk in preterm and small-for-gestational-age infants in low-income and middle-income countries: a pooled country analysis. Lancet 382, 417–425. Kensler, T.W., Roebuck, B.D., Wogan, G.N., Groopman, J.D., 2011. Aflatoxin: a 50-year odyssey of mechanistic and translational toxicology. Toxicol. Sci. 120 (Suppl. 1), S28–S48. Khlangwiset, P., Shephard, G.S., Wu, F., 2011. Aflatoxins and growth impairment: a review. Crit. Rev. Toxicol. 41, 740–755. Koirala, P., Kumar, S., Yadav, B.K., Premarajan, K.C., 2005. Occurrence of aflatoxin in some of the food and feed in Nepal. Indian J. Med. Sci. 59, 331–336. Krishnamachari, K.A., Bhat, V.R., Nagarajan, V., Tilak, T.B., Tulpule, P.G., 1977. The problem of aflatoxic human disease in parts of India-epidemiological and ecological aspects. Ann. Nutr. Aliment. 31, 991–996. McCoy, L.F., Scholl, P.F., Schleicher, R.L., Groopman, J.D., Powers, C.D., Pfeiffer, C.M., 2005. Analysis of aflatoxin B1-lysine adduct in serum using isotope-dilution liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 19, 2203–2210. McCoy, L.F., Scholl, P.F., Sutcliffe, A.E., Kieszak, S.M., Powers, C.D., Rogers, H.S., et al., 2008. Human aflatoxin albumin adducts quantitatively compared by ELISA, HPLC with fluorescence detection, and HPLC with isotope dilution mass spectrometry. Cancer Epidemiol. Biomarkers Prev. 17, 1653–1657. Okumura, H., Kawamura, O., Kishimoto, S., Hasegawa, A., Shrestha, S.M., Okuda, K., et al., 1993. Aflatoxin M1 in Nepalese sera, quantified by combination of monoclonal antibody immunoaffinity chromatography and enzyme-linked immunosorbent assay. Carcinogenesis 14, 1233–1235. Sabbioni, G., Ambs, S., Wogan, G.N., Groopman, J.D., 1990. The aflatoxin B1-lysine adduct quantified by high-performance liquid chromatography from human serum albumin samples. Carcinogenesis 11, 2063–2066. Scholl, P.F., Groopman, J.D., 2008. Long-term stability of human aflatoxin B1 albumin adducts assessed by isotope dilution mass spectrometry and high-performance liquid chromatography-fluorescence. Cancer Epidemiol. Biomarkers Prev. 17, 1436–1439. Scholl, P.F., Turner, P.C., Sutcliffe, A.E., Sylla, A., Diallo, M.S., Friesen, M.D., et al., 2006. Quantitative comparison of aflatoxin B1 serum albumin adducts in humans by isotope dilution mass spectrometry and ELISA. Cancer Epidemiol. Biomarkers Prev. 15, 823–826. Scholl, P.F., Cole, R.N., Ruczinski, I., Gucek, M., Diez, R., Rennie, A., et al., 2012. Maternal serum proteome changes between the first and third trimester of pregnancy in rural southern Nepal. Placenta 33, 424–432. Smith, L.E., Stoltzfus, R.J., Prendergast, A., 2012. Food chain mycotoxin exposure, gut health, and impaired growth: a conceptual framework. Adv. Nutr. 3, 526–531.
J.D. Groopman et al./Food and Chemical Toxicology 74 (2014) 184–189
Strosnider, H., Azziz-Baumgartner, E., Banziger, M., Bhat, R.V., Breiman, R., Brune, M.N., et al., 2006. Workgroup report: public health strategies for reducing aflatoxin exposure in developing countries. Environ. Health Perspect. 114, 1898–1903. Tandon, H.D., Tandon, B.N., Ramalingaswami, V., 1978. Epidemic of toxic hepatitis in India of possible mycotoxic origin. Arch. Pathol. Lab. Med. 102, 372–376. Turner, P.C., Sylla, A., Gong, Y.Y., Diallo, M.S., Sutcliffe, A.E., Hall, A.J., et al., 2005. Reduction in exposure to carcinogenic aflatoxins by postharvest intervention measures in West Africa: a community-based intervention study. Lancet 365, 1950–1956. Turner, P.C., Collinson, A.C., Cheung, Y.B., Gong, Y., Hall, A.J., Prentice, A.M., et al., 2007. Aflatoxin exposure in utero causes growth faltering in Gambian infants. Int. J. Epidemiol. 36, 1119–1125. Wang, L.Y., Hatch, M., Chen, C.J., Levin, B., You, S.L., Lu, S.N., et al., 1996. Aflatoxin exposure and risk of hepatocellular carcinoma in Taiwan. Int. J. Cancer 67, 620–625.
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West, K.P., Shamim, A.A., Labrique, A.B., Ali, H., Shaikh, S., Mehra, S., et al., 2013. Efficacy of antenatal multiple micronutrient (MM) vs iron-folic acid (ifa) supplementation in improving gestational and postnatal viability in rural Bangladesh: the JiVitA-3 Trial. FASEB J. 27. Wild, C.P., Gong, Y.Y., 2010. Mycotoxins and human disease: a largely ignored global health issue. Carcinogenesis 31, 71–82. Wild, C.P., Hudson, G.J., Sabbioni, G., Chapot, B., Hall, A.J., Wogan, G.N., et al., 1992. Dietary intake of aflatoxins and the level of albumin-bound aflatoxin in peripheral blood in The Gambia, West Africa. Cancer Epidemiol. Biomarkers Prev. 1, 229– 234. Wogan, G.N., Kensler, T.W., Groopman, J.D., 2012. Present and future directions of translational research on aflatoxin and hepatocellular carcinoma: a review. Food Addit. Contam Part A Chem. Anal. Control Expo. Risk Assess. 29, 249–257. Yadgiri, B., Reddy, V., Tulpule, P.G., Srikantia, S.G., Gopalan, C., 1970. Aflatoxin and Indian childhood cirrhosis. Am. J. Clin. Nutr. 23, 94–98.
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Food and Chemical Toxicology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f o o d c h e m t o x
Aflatoxin exposure during the first 1000 days of life in rural South Asia assessed by aflatoxin B1-lysine albumin biomarkers John D. Groopman 1,2,*, Patricia A. Egner 1, Kerry J. Schulze 2, Lee S.-F. Wu 2, Rebecca Merrill 2, Sucheta Mehra 2, Abu A. Shamim 2, Hasmot Ali 2, Saijuddin Shaikh 2, Alison Gernand 2 , Subarna K. Khatry 2 , Steven C. LeClerq 2 , Keith P. West Jr 2 , Parul Christian 2 1 2
Department of Environmental Health Sciences, Department of International Health, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA Center for Human Nutrition, Department of International Health, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA
A R T I C L E
I N F O
Article history: Received 11 August 2014 Accepted 22 September 2014 Available online 13 October 2014 Keywords: Aflatoxin Aflatoxin B1-lysine albumin biomarkers Child health Maternal exposure
A B S T R A C T
Aflatoxin B1 is a potent carcinogen, occurring from mold growth that contaminates staple grains in hot, humid environments. In this investigation, aflatoxin B1-lysine albumin biomarkers were measured by mass spectrometry in rural South Asian women, during the first and third trimester of pregnancy, and their children at birth and at two years of age. These subjects participated in randomized community trials of antenatal micronutrient supplementation in Sarlahi District, southern Nepal and Gaibandha District in northwestern Bangladesh. Findings from the Nepal samples demonstrated exposure to aflatoxin, with 94% detectable samples ranging from 0.45 to 2939.30 pg aflatoxin B1-lysine/mg albumin during pregnancy. In the Bangladesh samples the range was 1.56 to 63.22 pg aflatoxin B1-lysine/mg albumin in the first trimester, 3.37 to 72.8 pg aflatoxin B1-lysine/mg albumin in the third trimester, 4.62 to 76.69 pg aflatoxin B1-lysine/mg albumin at birth and 3.88 to 81.44 pg aflatoxin B1-lysine/mg albumin at age two years. Aflatoxin B1-lysine adducts in cord blood samples demonstrated that the fetus had the capacity to convert aflatoxin into toxicologically active compounds and the detection in the same 2-year-old children illustrates exposure over the first 1000 days of life. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Nearly 200,000,000 children worldwide suffer from impaired growth and development and nearly 2 million die each year before age 5 (Black et al., 2013; Smith et al., 2012). Many of these children are from South Asia and these long-term health deficits place an enormous burden on these economically developing countries. The overall contributing factors driving these health problems still needs to be completely identified and better intervention strategies developed (Bhutta et al., 2013). Aflatoxin B1 is a very potent toxin and carcinogen for humans and this naturally occurring mycotoxin occurs as a consequence of specific mold growth that permeates staple grains, legumes and nuts consumed by people, especially in hot, humid environments in lowincome countries (Wogan et al., 2012). Aflatoxin induces many
Abbreviation: AFB1, aflatoxin B1. * Corresponding author. Dr. John D. Groopman, Johns Hopkins University, Department of Environmental Health Sciences, Bloomberg School of Public Health, Baltimore, MD 21205, United States, Tel.: + 4109553900; fax: + 4109550617. E-mail address: [email protected] (J.D. Groopman). http://dx.doi.org/10.1016/j.fct.2014.09.016 0278-6915/© 2014 Elsevier Ltd. All rights reserved.
adverse local and systemic effects that impair normal organ and tissue function resulting in growth retardation, inflammation and immune suppression, all of which contribute in multiple ways to poor health (Khlangwiset et al., 2011; Wild and Gong, 2010). Chronic exposure to aflatoxin also significantly enhances excess risk for liver cancer in many human populations (Kensler et al., 2011; Wild and Gong, 2010). Similar to most environmental toxicants, aflatoxin requires metabolic activation by intrinsic cytochrome P450 enzymes, mainly localized in the liver, to exert its toxicological effects. These metabolic activation pathways have been well described by several research teams (Guengerich, 2008; Wogan et al., 2012). One of these metabolic derivatives, the aflatoxin B1-lysine adduct, that is isolated from serum albumin, is a validated biomarker of exposure and risk (Chen et al., 2013; Sabbioni et al., 1990; Turner et al., 2005; Wang et al., 1996; Wild et al., 1992). A number of studies have found strong associations between aflatoxin exposure, as measured by aflatoxin– albumin biomarkers, and significant reductions in growth in children as assessed by height for age and weight for age (Gong et al., 2002, 2003, 2004; Turner et al., 2007). Aflatoxin B1 contamination has been detected in food, feed and a limited set of blood and urine samples collected in Nepal and at least one third of the foodstuffs examined contained elevated levels
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of this mycotoxin (Koirala et al., 2005; Okumura et al., 1993). Contamination of staple grains and foodstuffs were also found in Bangladesh (Begum and Samajpati, 2000; Dawlatana et al., 2002). Earlier literature in India had related aflatoxin exposure to childhood cirrhosis and acute aflatoxicosis (Krishnamachari et al., 1977; Tandon et al., 1978; Yadgiri et al., 1970). The major goal of this current study was to quantitatively measure aflatoxin exposure using validated biomarkers in pregnancy, early infancy and at ~24 months of age, spanning a critical developmental period referred to as “the first 1000 days of life”, in rural South Asia (Katz et al., 2013). Samples for this exploratory analysis were drawn from an extant collection of biospecimens collected from large samples of pregnant women and infants living in rural settings in the southern plains, or Terai, of Nepal and flood plains of northern Bangladesh. Both study areas, situated along the northern and eastern flanks of the greater Gangetic floodplain that has over 750 million people, are exposed to strong seasonality, high heat, humidity and a general lack of adequate food storage facilities that makes aflatoxin contamination much more likely. In this investigation, women’s serum or plasma samples from the first and third trimester of pregnancy in both country settings and, additionally in Bangladesh, cord blood at birth and serum samples from the same children at ~24 months of age were studied. A state-of-the-art isotope dilution quantitative mass spectrometry method was used for measuring aflatoxin B1-lysine adducts in albumin in these samples as biomarkers of aflatoxin exposure. Thus, the findings reported here represent an initial attempt to characterize the exposure to aflatoxin during pregnancy and early life of the children. These results reveal almost universal excessive exposure to this potent, naturally occurring mycotoxin.
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Fig. 1. Aflatoxin B1-lysine adduct exposures in 30 Nepal pregnant women track between 1st and 3rd trimesters.
(n = 16, >1 g/L) or normal (n = 14, ≤1 g/L) AGP in early pregnancy and corresponding 1st and 3rd trimester samples, allowing us to examine aflatoxin exposure longitudinally through pregnancy and its association with inflammation in early pregnancy. A larger set of early pregnancy samples from the Nepal set was then randomly selected based on birth outcome (n = 27–30 women per group having had normal birth, miscarriage, stillbirth, preterm, or low birth weight for a total of n = 141 samples); data from this larger set were used to examine determinants of aflatoxin exposure, with natural log-transformations used for analysis of the aflatoxinadduct values by a variety of factors by ANOVA. Data presented as geometric means and standard deviations. From the Bangladesh study, a longitudinal cohort of maternal 1st trimester, 3rd trimester, and cord blood samples and samples from corresponding children at 24 months of age was assessed by examining all samples available at the intersection of the main sub-study, cord blood collection substudy, and child follow-up at 24 months of age (n = 63).
2. Methods 2.2. Aflatoxin B1-lysine albumin adduct assay by isotope dilution mass spectrometry 2.1. Study subjects, serum processing and sample selection Archived, serum or plasma specimens for pregnant women and their children were selected from a cohort of subjects who participated in multiple, randomized, antenatal micronutrient supplementation trials carried out in the southern rural plains of Nepal from 1999 to 2001 and more recently (2008–2012) in Bangladesh (West et al., 2013). Details of the design, major findings and biospecimen collection procedures for the trials have been previously reported (Christian et al., 2003a, 2003b). Each trial was approved by institutional review boards in their respective countries, the Nepal Health Research Council, Kathmandu, Nepal and the Bangladesh Medical Research Council, in Dhaka, Bangladesh, and the Institutional Review Board (IRB) of the Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA. The Nepal and Bangladesh trials enrolled n = 4926 and n = 44,567 women, respectively, with subsets of women based on geographic residence recruited for more intensive investigations that included blood draws in the home following enrollment and at approximately 32 weeks of gestation. To be eligible for participation, women were screened at 5-weekly intervals for menstrual status and given a urinebased pregnancy test if menstrual status since the previous visit was negative, allowing for enrollment into the studies prior to a gestational age of 11 weeks on average. In total, n = 1165 baseline (i.e. first trimester) samples and n = 779 third trimester samples were collected at the Nepal site, and n = 2070 first trimester and n = 1597 third trimester samples were collected at the Bangladesh site. Thirty (30) of the Nepal participants were randomly selected for the initial first and third trimester analysis. At the Bangladesh site, a smaller subset of women (n = 399) were additionally enrolled in a study that allowed for the collection of cord blood. Finally, children born to women at the Bangladesh site were followed to 24 months of age. A subset of these children (n = 177) was enrolled in a study that required blood sample collection to evaluate biochemical indicators related to growth. In this study, there were 63 mothers for the first and third trimester samples, 63 cord blood samples and the same 63 two-year olds at follow-up available and analyzed. At both sites, blood was processed to serum or plasma, which was subsequently shipped to The Johns Hopkins Center for Human Nutrition and stored at −80 oC to form a large repository of samples from which the samples selected for this series of investigations were drawn. Longitudinal (1st and 3rd trimester) samples from pregnant women from Nepal were randomly sampled from a larger set of samples in which alpha-1-acid glycoprotein (AGP), a marker of inflammation, had previously been assessed along with C-reactive protein (CRP) using methods as previously described (Christian et al., 1998). Approximately equal numbers of women were selected based on having elevated
All of the serum or plasma samples were analyzed using a minor variation of the method reported by McCoy and colleagues (McCoy et al., 2008). The sample (200 μL) was mixed with an internal standard (10 μL × 0.1 ng AFB1-D4-lys/mL) and Pronase solution (250 μL, 13 mg/mL PBS), and incubated for 18 h at 37 °C. Solidphase extraction–processed samples were analyzed by UPLC with mass spectrometric detection using a ThermoElectron TSQ Vantage operated in the positive electrospray ionization SRM mode. The internal standard parent molecular ion [(M + H)+, m/z 461.3] fragmented to yield an ion at m/z 398.2. The AFB1-lys molecular ion (m/z 457.2) fragmented to yield an ion at m/z 394.1. A 10-point isotopic dilution standard curve was generated by triplicate injection (10 μL) of AFB1-D4-lys (100 pg) mixed with varying amounts of AFB1-lys (0–0.4 ng) prepared via 2-fold serial dilutions. The data were fitted using the method of least-squares with a 1/× weighting factor. The isotope dilution standard curve was linear over the range of 0.2 pg to 0.4 ng AFB1-lys injected onto the column in 10 μL aliquots (R2 = 0.995). The limit of quantitation was 0.2 pg AFB1-lysine/mg albumin in these analyses.
3. Results This research was initiated by an analysis of aflatoxin B1-lysine albumin adducts in a pooled sample representing one thousand 6–7 year old children in Sarlahi, Nepal who were being analyzed for plasma proteome differences related to micronutrient status (Scholl et al., 2012). That analysis showed 56.5 pg aflatoxin B1-lysine/mg albumin, prompting the more detailed examination of aflatoxin exposure during pregnancy and early life described here. The aflatoxin B1-lysine albumin adduct biomarker levels in first and third trimester samples from thirty Nepalese pregnant women are shown in Fig. 1. This study examined the individual tracking of aflatoxin exposure between the first and third trimester. As seen in these results, exposures were quite consistent across these two measures obtained approximately 6 months apart, indicating a high exposure to both the fetus and the mother during pregnancy. Only two and four of the samples from the first and third trimester had non-detectable levels, respectively. These preliminary data were suggestive of limited seasonal variation in aflatoxin exposure.
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Table 1 Determinants of aflatoxin-albumin adducts (pg aflatoxin B1-lysine/mg albumin) among Nepalese women (n = 141) during early pregnancy. pg/mg1
Fig. 2. Aflatoxin B1-lysine adduct exposures in first trimester of pregnancy in 141 Nepal women associate with elevated inflammation biomarker, AGP.
The aflatoxin biomarker levels from the first trimester samples (shown in Fig. 1) were then evaluated by inflammatory status as measured by alpha-1-acid glycoprotein (AGP). These findings are shown in Fig. 2 and reveal that the aflatoxin-albumin adduct levels were higher among women with increased levels of this circulating marker of inflammation. The findings in Fig. 2 are the first to associate elevated AGP with increasing aflatoxin exposure. Among the larger group of samples obtained in early pregnancy among Nepalese women, only nine of the n = 141 samples had a non-detectable level of aflatoxin-albumin adducts, and the range of detectable exposures was from 0.45 to 2939.30 pg aflatoxin B1-lysine/mg albumin. The median level of these samples was 22.45 pg aflatoxin B1-lysine/mg albumin, with a geometric mean of 25.28 pg aflatoxin B1-lysine/mg albumin, and the 25th to 75th percentile ranged from 7.42 to 77.94 pg aflatoxin B1-lysine/mg albumin. Determinants of aflatoxin exposure are further explored in Table 1. Although aflatoxin did not vary by AGP in these analyses, it was significantly higher when another marker of inflammation, C-reactive protein (CRP), was elevated. Further exploration of these data revealed differences in exposure related to local ethnicities. Aflatoxinalbumin adducts in samples from the ethnic Pahadi people (N = 61) were statistically significantly higher (p < .0001) than those in samples from the ethnic Madhesi (N = 80). The Pahadi live in the hill region of Nepal and traditionally have a higher maize consuming diet. The two highest values (2393.3 and 2939.3 pg aflatoxin B1lysine/mg albumin) were found in samples obtained during the June to August rainy season; seasonal variability was significant (p < 0.0001), with samples acquired in the September to November period having the highest level of adducts compared to the other three-month intervals during the year (Table 1). Depicted in Fig. 3 is aflatoxin exposure that occurred among women in Bangladesh across pregnancy from the first through the third trimester. The median levels of the aflatoxin biomarker in the first and third trimester were 18.08 and 25.35 pg aflatoxin B1-lysine/mg albumin, respectively (100% positive). The median level of aflatoxin–lysine albumin adducts in cord blood samples was 27.41 pg aflatoxin B1-lysine/mg albumin (100% positive). This demonstrated that the fetus had the biochemical capacity to convert aflatoxin that crossed the placenta into toxicologically active compounds. Further, the detection of the same biomarkers in 2-year-old children born to the mothers who were exposed to aflatoxins during pregnancy has a median level of 13.79 pg aflatoxin B1-lysine/mg albumin (100% positive). These findings illustrated the profound exposure for these children across their first 1000 days life.
Aflatoxin (total samples) AGP ≤1 g/L >1 g/L CRP ≤5 mg/L >5 mg/L Age 14–19 y 20–29 y ≥30 y Parity 0 1–2 ≥3 Pahadi No Yes Literate No Yes Ground floor construction No walls, thatch, grass, sticks or mud Wood or brick and cement Schooling None 1 or more years Season December–February March–May June–August September–November BMI <19 kg/m2 ≥19 kg/m2 1
p-Value
25.28 (5.81) 26.05 (5.75) 16.11 (7.17)
0.46
23.10 (5.64) 117.92 (5.42)
0.01
22.87 (6.75) 27.94 (6.11) 20.49 (4.31)
0.69
32.46 (6.75) 31.50 (5.53) 17.46 (5.31)
0.14
14.73 (5.16) 54.05 (5.05)
<0.0001
25.79 (5.93) 23.57 (5.53)
0.79
26.58 (5.99) 19.69 (4.85)
0.46
25.28 (5.99) 25.03 (5.26)
0.98
28.22 (4.95) 8.00 (3.53) 46.99 (5.87) 113.30 (3.32) 21.98 (6.30) 29.67 (5.31)
<0.0001
0.32
Data expressed as geometric mean (SD).
4. Discussion The greater Gangetic floodplain regions occupying southern Nepal and northwestern Bangladesh have hot and humid environments conducive to aflatoxin B1 contamination of staple grains (Begum and Samajpati, 2000; Dawlatana et al., 2002; Koirala et al., 2005). While substantial aflatoxin B1 contamination has been detected in a variety of foodstuffs, a much more limited data set exists relating these putative exposures to specific formation of biologically effective dose
Fig. 3. Bangladesh aflatoxin exposure studies: first, third trimester, cord blood and 2 year olds. There were 63 mothers for the first and third trimester samples, 63 cord blood samples and the same 63 two-year olds at follow-up.
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metrics, such as the aflatoxin B1-albumin biomarker, in people. To address this data gap, a quantitative isotope dilution mass spectrometry method was deployed to measure the aflatoxin B1-lysine albumin adduct at biologically critical times in mothers during pregnancy and in the first thousand days of life in their children. An initial analysis of aflatoxin-albumin adducts in a pooled sample from one thousand 6–7 year old children in Nepal found 56.5 pg aflatoxin B1lysine/mg albumin, a level comparable to that detected in very nutritionally compromised children in West Africa when the different method of analyses are considered (Gong et al., 2004; McCoy et al., 2008). This biomarker observation prompted a more detailed examination using samples already analyzed for its plasma proteome that had collected during the first and third trimester of pregnancy of women enrolled in a randomized micronutrient intervention clinical trial in Sarlahi, Nepal (Cole et al., 2013; Scholl et al., 2012). Aflatoxin B1 is a very potent toxic and carcinogenic agent for a variety of species including humans. Contamination of staple grains occurs both prior to harvest and during storage of these foodstuffs. Levels of contamination can vary widely resulting in human exposures that can range from nanograms per day to milligrams per day depending upon the specific staple grain and amount consumed (Kensler et al., 2011). While much of the international focus on this contamination has been framed within its carcinogenic properties, many of its other toxic effects can have both short and longterm health consequences. In South Asia, where both maternal and child health status is tremendously compromised, the potential effects of aflatoxin exposure are most likely manifested by these noncarcinogenic endpoints (Bhutta et al., 2013; Black et al., 2013). However, in the future, as early life health status improves across this region, these potential cancer endpoints could become much more consequential for these populations. Nonetheless, when aflatoxin exposure is determined to be substantial, these data should be viewed as being a sentinel for overall poor quality of staple grains and nutrition (CAST, 2003). The wide heterogeneity in aflatoxin contamination levels in grains results in a very difficult task to assess human exposure using dietary questionnaires and spot testing of staple foods. The development of mechanistically based biomarkers, such as the aflatoxin B1lysine albumin adduct, has helped to overcome the inherent difficulties of assessing aflatoxin exposure by measuring dietary constituents. These biomarkers have the added advantage of being able to integrate exposures over the course of many weeks or months due to the long half-lives of these biomarkers in plasma (McCoy et al., 2005, 2008). Research from our laboratory has also demonstrated the long-term stability of aflatoxin albumin biomarkers in stored samples where we have found these adducts to be stable for up to 25 years (Scholl and Groopman, 2008). The analytical technology for the measurement of these biomarkers has also substantially improved over the last several decades and comparative studies of paired samples have been done to relate measurements obtained across a number of analytical platforms. The correlation of aflatoxin albumin adducts measured by mass spectrometry, HPLC with fluorescence detection and immunoassays has been done and this permits composite comparisons of human data obtained from many diverse molecular epidemiologic studies across the globe (McCoy et al., 2008; Scholl et al., 2006). As a point of reference the values obtained in this comparative study revealed that the ELISA method used provided aflatoxin adduct levels 5-fold higher than mass spectrometry, but the correlation coefficient between the two techniques was 0.98 (McCoy et al., 2008). In the work reported in this study we have employed a quantitative isotope dilution mass spectrometry method that permits the measurement of aflatoxin lysine obtained from serum albumin with the use of an isotopically labeled internal standard. This method is highly sensitive and specific, permitting the quantitation of this biomarker in less than 100 μL of
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Fig. 4. Aflatoxin exposure levels vary widely across regions and time periods. The data for 1995 and 2012 in China are from our recent publication (Chen et al., 2013).
plasma obtained in individuals residing in high exposure regions of the world. Despite the emphasis of many investigations on liver cancer consequent to aflatoxin exposure there have been a number of studies, primarily in West Africa, that have raised the potential etiologic contribution of this toxin to diminished child growth and development (Gong et al., 2002, 2003, 2004). Stunting is calculated to affect nearly 200,000,000 children worldwide primarily in sub-Saharan Africa and South Asia (Black et al., 2008; Smith et al., 2012). Stunting has profound associations with a variety of growth and development deficiencies in these children and to date the overall etiology of this problem remains obscure. Since aflatoxin B1 has major impacts on growth and development in a number of animal species that has been well characterized in the veterinary literature, it is reasonable hypothesis that similar growth and developmental effects can occur in people. The first step in the exploration of this hypothesis is the characterization of specific exposures in high-risk communities. The biologically effective dose biomarker, aflatoxin albumin adducts, provides an objective metric for characterizing these exposures for subsequent follow-up and potential intervention. In an attempt to place the extent of exposures in the populations reported here in Nepal and Bangladesh we compared these current data to those observed in other populations, experiencing acute toxic (Kenya) or chronic (China) exposures (Chen et al., 2013; McCoy et al., 2008), evaluated previously in our laboratory. The results depicted in Fig. 4 provide a composite perspective for the aflatoxin exposures detected in Nepal and Bangladesh in context with other documented exposures. There was an acute aflatoxicosis outbreak in Kenya in 2004 and again in 2005 in which nearly 150 people died after consuming aflatoxin-contaminated maize. The levels depicted, measured in our laboratory, were obtained from 107 people living in those affected Kenyan villages (McCoy et al., 2008; Strosnider et al., 2006). To provide perspective, the median adduct level was 10-times higher than the median for the 141 positive samples of 150 pregnant women in Nepal whose blood was collected in 1999. However, it is important to point out that many of the outlying samples in the Nepal data set overlap values of aflatoxin exposure in individuals from the Kenya toxicity event. The comparative values in Bangladesh (from Fig. 3) are shown next. Finally, three sets of data from our studies in eastern China, a region of very high liver cancer incidence, are shown in the far right of Fig. 4. Samples in China that were collected in 1983, 1995 and 2012 (100 samples for each time point) are depicted (Chen et al., 2013). While the levels found in 1983 are comparable to those measured more recently in Nepal and Bangladesh, the decline in China is seen by 1995 reflecting a transition in this population from a maize
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dominated diet to one in which rice quickly became the dominate staple. Maize is always more contaminated with aflatoxin compared to rice. By 2012, virtually all of the samples collected in Qidong, China were non-detectable for aflatoxin biomarkers. This outcome documents the reduction in aflatoxin exposures that can be obtained through primary prevention: in this case, improved food quality and diversity. Finally, we have found that this relatively recent reduction in aflatoxin exposure in Qidong, China, is associated with a nearly 50% reduction in liver cancer mortality among people under the age of 40 (but not older) in this population, suggesting strongly that reduction of early life exposure can have a major protective effect on cancer outcomes. Data from several published studies can be utilized to calculate approximations of daily aflatoxin exposure in adults and children. These data sets include information gleaned from micro-dosing experiments in humans and plate food-based analyses of exposure where aflatoxin–albumin adduct analysis was also determined (Cupid et al., 2004; Wild and Gong, 2010; Wild et al., 1992). Extrapolating previously published findings with the aflatoxin B1 albumin adduct levels reported in this work indicates that daily exposure in women and their children range from 1 to 20 μg per day. Assuming average body weight of 50 and 10 kg in women and children, respectively, the adult exposures range from 10 to 200 ng per kilogram body weight per day. The children’s exposure was substantially higher due to lower body mass resulting in an exposure range of 50 to 1000 ng per kilogram body weight per day. These exposure calculations provide a framework for determining targeted strategies to reduce dietary exposure to aflatoxin in these populations. In conclusion, in this study we have found that aflatoxin exposure is ubiquitous among at least some of the rural populations of Nepal and Bangladesh, as assessed by a robust mass spectrometrybased albumin adduct biomarker. High levels of aflatoxin B 1 lysine adducts are observed consistently across the first and third trimesters of pregnancy in both of these populations. Aflatoxin B1lysine albumin adducts in human cord blood samples demonstrate that the fetus has the capacity to metabolize aflatoxin to levels comparable to that of the mother. Substantial aflatoxin B1-lysine albumin adducts measured in the 2 year-old children, equivalent to adult levels, strongly suggests life-time exposures to this potent hepatotoxic and immunotoxic food contaminant occur. These data strongly suggest a need to further explore the sources of aflatoxin contamination in the staple grains to establish a process for intervention to lower these exposures. These data strongly indicate the need to develop interventions similar to those used in other highrisk high exposure settings. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgments Supported in part by the Bill and Melinda Gates Foundation (GH 614 and GCE 1046227, GCE 1046221, proteomics), Seattle, WA, USA and the US NIH P30 CA006973. References Begum, F., Samajpati, N., 2000. Mycotoxin production on rice, pulses and oilseeds. Naturwissenschaften 87, 275–277.
Bhutta, Z.A., Das, J.K., Rizvi, A., Gaffey, M.F., Walker, N., Horton, S., et al., 2013. Evidence-based interventions for improvement of maternal and child nutrition: what can be done and at what cost? Lancet 382, 452–477. Black, R.E., Allen, L.H., Bhutta, Z.A., Caulfield, L.E., de Onis, M., Ezzati, M., et al., 2008. Maternal and child undernutrition: global and regional exposures and health consequences. Lancet 371, 243–260. Black, R.E., Victora, C.G., Walker, S.P., Bhutta, Z.A., Christian, P., de Onis, M., et al., 2013. Maternal and child undernutrition and overweight in low-income and middleincome countries. Lancet 382, 427–451. CAST, 2003. Mycotoxins: risk in plants, animals and human systems. Council for Agricultural Science and Technology, p. 217. Chen, J.G., Egner, P.A., Ng, D., Jacobson, L.P., Munoz, A., Zhu, Y.-R., et al., 2013. Reduced aflatoxin exposure presages decline in liver cancer mortality in an endemic region of China. Cancer Prev. Res. 6, 1038–1045. Christian, P., Schulze, K., Stoltzfus, R.J., West, K.P., Jr., 1998. Hyporetinolemia, illness symptoms, and acute phase protein response in pregnant women with and without night blindness. Am. J. Clin. Nutr. 67, 1237–1243. Christian, P., Khatry, S.K., Katz, J., Pradhan, E.K., LeClerq, S.C., Shrestha, S.R., et al., 2003a. Effects of alternative maternal micronutrient supplements on low birth weight in rural Nepal: double blind randomised community trial. BMJ 326, 571. Christian, P., West, K.P., Jr., Khatry, S.K., Leclerq, S.C., Pradhan, E.K., Katz, J., et al., 2003b. Effects of maternal micronutrient supplementation on fetal loss and infant mortality: a cluster-randomized trial in Nepal. Am. J. Clin. Nutr. 78, 1194– 1202. Cole, R.N., Ruczinski, I., Schulze, K., Christian, P., Herbrich, S., Wu, L., et al., 2013. The plasma proteome identifies expected and novel proteins correlated with micronutrient status in undernourished Nepalese children. J. Nutr. 143, 1540– 1548. doi: 1510.3945/jn.1113.175018. Epub 172013 Aug 175021. Cupid, B.C., Lightfoot, T.J., Russell, D., Gant, S.J., Turner, P.C., Dingley, K.H., et al., 2004. The formation of AFB 1-macromolecular adducts in rats and humans at dietary levels of exposure. Food Chem. Toxicol. 42, 559–569. Dawlatana, M., Coker, R.D., Nagler, M.J., Wild, C.P., Hassan, M.S., Blunden, G., 2002. The occurrence of mycotoxins in key commodities in Bangladesh: surveillance results from 1993 to 1995. J. Nat. Toxins 11, 379–386. Gong, Y., Hounsa, A., Egal, S., Turner, P.C., Sutcliffe, A.E., Hall, A.J., et al., 2004. Postweaning exposure to aflatoxin results in impaired child growth: a longitudinal study in Benin, West Africa. Environ. Health Perspect. 112, 1334–1338. Gong, Y.Y., Cardwell, K., Hounsa, A., Egal, S., Turner, P.C., Hall, A.J., et al., 2002. Dietary aflatoxin exposure and impaired growth in young children from Benin and Togo: cross sectional study. BMJ 325, 20–21. Gong, Y.Y., Egal, S., Hounsa, A., Turner, P.C., Hall, A.J., Cardwell, K.F., et al., 2003. Determinants of aflatoxin exposure in young children from Benin and Togo, West Africa: the critical role of weaning. Int. J. Epidemiol. 32, 556–562. Guengerich, F.P., 2008. Cytochrome p450 and chemical toxicology. Chem. Res. Toxicol. 21, 70–83. Katz, J., Lee, A.C., Kozuki, N., Lawn, J.E., Cousens, S., Blencowe, H., et al., 2013. Mortality risk in preterm and small-for-gestational-age infants in low-income and middle-income countries: a pooled country analysis. Lancet 382, 417–425. Kensler, T.W., Roebuck, B.D., Wogan, G.N., Groopman, J.D., 2011. Aflatoxin: a 50-year odyssey of mechanistic and translational toxicology. Toxicol. Sci. 120 (Suppl. 1), S28–S48. Khlangwiset, P., Shephard, G.S., Wu, F., 2011. Aflatoxins and growth impairment: a review. Crit. Rev. Toxicol. 41, 740–755. Koirala, P., Kumar, S., Yadav, B.K., Premarajan, K.C., 2005. Occurrence of aflatoxin in some of the food and feed in Nepal. Indian J. Med. Sci. 59, 331–336. Krishnamachari, K.A., Bhat, V.R., Nagarajan, V., Tilak, T.B., Tulpule, P.G., 1977. The problem of aflatoxic human disease in parts of India-epidemiological and ecological aspects. Ann. Nutr. Aliment. 31, 991–996. McCoy, L.F., Scholl, P.F., Schleicher, R.L., Groopman, J.D., Powers, C.D., Pfeiffer, C.M., 2005. Analysis of aflatoxin B1-lysine adduct in serum using isotope-dilution liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 19, 2203–2210. McCoy, L.F., Scholl, P.F., Sutcliffe, A.E., Kieszak, S.M., Powers, C.D., Rogers, H.S., et al., 2008. Human aflatoxin albumin adducts quantitatively compared by ELISA, HPLC with fluorescence detection, and HPLC with isotope dilution mass spectrometry. Cancer Epidemiol. Biomarkers Prev. 17, 1653–1657. Okumura, H., Kawamura, O., Kishimoto, S., Hasegawa, A., Shrestha, S.M., Okuda, K., et al., 1993. Aflatoxin M1 in Nepalese sera, quantified by combination of monoclonal antibody immunoaffinity chromatography and enzyme-linked immunosorbent assay. Carcinogenesis 14, 1233–1235. Sabbioni, G., Ambs, S., Wogan, G.N., Groopman, J.D., 1990. The aflatoxin B1-lysine adduct quantified by high-performance liquid chromatography from human serum albumin samples. Carcinogenesis 11, 2063–2066. Scholl, P.F., Groopman, J.D., 2008. Long-term stability of human aflatoxin B1 albumin adducts assessed by isotope dilution mass spectrometry and high-performance liquid chromatography-fluorescence. Cancer Epidemiol. Biomarkers Prev. 17, 1436–1439. Scholl, P.F., Turner, P.C., Sutcliffe, A.E., Sylla, A., Diallo, M.S., Friesen, M.D., et al., 2006. Quantitative comparison of aflatoxin B1 serum albumin adducts in humans by isotope dilution mass spectrometry and ELISA. Cancer Epidemiol. Biomarkers Prev. 15, 823–826. Scholl, P.F., Cole, R.N., Ruczinski, I., Gucek, M., Diez, R., Rennie, A., et al., 2012. Maternal serum proteome changes between the first and third trimester of pregnancy in rural southern Nepal. Placenta 33, 424–432. Smith, L.E., Stoltzfus, R.J., Prendergast, A., 2012. Food chain mycotoxin exposure, gut health, and impaired growth: a conceptual framework. Adv. Nutr. 3, 526–531.
J.D. Groopman et al./Food and Chemical Toxicology 74 (2014) 184–189
Strosnider, H., Azziz-Baumgartner, E., Banziger, M., Bhat, R.V., Breiman, R., Brune, M.N., et al., 2006. Workgroup report: public health strategies for reducing aflatoxin exposure in developing countries. Environ. Health Perspect. 114, 1898–1903. Tandon, H.D., Tandon, B.N., Ramalingaswami, V., 1978. Epidemic of toxic hepatitis in India of possible mycotoxic origin. Arch. Pathol. Lab. Med. 102, 372–376. Turner, P.C., Sylla, A., Gong, Y.Y., Diallo, M.S., Sutcliffe, A.E., Hall, A.J., et al., 2005. Reduction in exposure to carcinogenic aflatoxins by postharvest intervention measures in West Africa: a community-based intervention study. Lancet 365, 1950–1956. Turner, P.C., Collinson, A.C., Cheung, Y.B., Gong, Y., Hall, A.J., Prentice, A.M., et al., 2007. Aflatoxin exposure in utero causes growth faltering in Gambian infants. Int. J. Epidemiol. 36, 1119–1125. Wang, L.Y., Hatch, M., Chen, C.J., Levin, B., You, S.L., Lu, S.N., et al., 1996. Aflatoxin exposure and risk of hepatocellular carcinoma in Taiwan. Int. J. Cancer 67, 620–625.
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West, K.P., Shamim, A.A., Labrique, A.B., Ali, H., Shaikh, S., Mehra, S., et al., 2013. Efficacy of antenatal multiple micronutrient (MM) vs iron-folic acid (ifa) supplementation in improving gestational and postnatal viability in rural Bangladesh: the JiVitA-3 Trial. FASEB J. 27. Wild, C.P., Gong, Y.Y., 2010. Mycotoxins and human disease: a largely ignored global health issue. Carcinogenesis 31, 71–82. Wild, C.P., Hudson, G.J., Sabbioni, G., Chapot, B., Hall, A.J., Wogan, G.N., et al., 1992. Dietary intake of aflatoxins and the level of albumin-bound aflatoxin in peripheral blood in The Gambia, West Africa. Cancer Epidemiol. Biomarkers Prev. 1, 229– 234. Wogan, G.N., Kensler, T.W., Groopman, J.D., 2012. Present and future directions of translational research on aflatoxin and hepatocellular carcinoma: a review. Food Addit. Contam Part A Chem. Anal. Control Expo. Risk Assess. 29, 249–257. Yadgiri, B., Reddy, V., Tulpule, P.G., Srikantia, S.G., Gopalan, C., 1970. Aflatoxin and Indian childhood cirrhosis. Am. J. Clin. Nutr. 23, 94–98.