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3 Centro de Investigaciones en Nutricion y Salud, Guatemala City, Guatemala 01015; 4 Instituto de Nutricion de Centro America y Panama, Guatemala City, Guatamala 09001; 5 Institute of Agricultural Science and Technology, Villa Nueva, Guatemala; 6 Center for Human Genetics, Duke University Medical Center, Durham, NC 27710; 7 School of Biology and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332-0230; 8 Microbial Genomics Research Unit, National Center for Agricultural Utilization Research, USDA-Agricultural Research Service, Peoria, IL 61604; and 9 Toxicology and Mycotoxin Research Unit, R. B. Russell Agricultural Research Center, USDA-Agricultural Research Service, Athens, GA 30604
* To whom correspondence should be addressed. E-mail: ron.riley{at}ars.usda.gov.
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ABSTRACT |
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 TOP ABSTRACT IntroductionMaterials and MethodsResults and DiscussionLITERATURE CITED |
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Introduction |
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TOPABSTRACTIntroductionMaterials and MethodsResults and DiscussionLITERATURE CITED |
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Fumonisin B1 is a known liver and kidney carcinogen in rodents (4). A provisional maximum tolerable daily intake (PMTDI) of 2 µg·kg body weight–1·d–1 for fumonisins B1, B2, and B3, alone or in combination, has been proposed (3). The PMTDI was calculated based on a safety factor of 100 and a no-observable-adverse-effect level of 0.2 mg·kg body weight–1·d–1 for the critical target (rat kidney). There is a growing body of evidence that suggests that fumonisin B1 is possibly carcinogenic to humans (4).
Humans at greatest risk from fumonisins are those for whom maize is a dietary staple. Mayan communities and other indigenous groups in Guatemala are known to consume large amounts of maize processed into nixtamalized (alkali-treated) products, which reduces the total fumonisins by 
50% (6). Nixtamalized maize products provide most of the daily energy intake for a large proportion of the population in the Central Highlands of Guatemala (7). To assess the magnitude of exposure, a 24-h recall survey was conducted in the Central Highlands in 1999–2000 (n = 92) by the Instituto de Nutricion de Centro America y Panama (INCAP) for the Consejo Nacional de Ciencia y Tecnología (Guatemala City, Guatemala). This study found that 100% of the population surveyed consumed maize as tortillas and that the mean consumption was 14 tortillas per day. In addition, 50% of the individuals surveyed consumed other maize products besides tortillas. The mean consumption of maize derived from products other than tortillas was 145 g/d. Bressani (8) reported that the daily per-capita maize consumption in Guatemala was 318 g and Fuentes-Lopez et al. (9) reported that urban maize consumption was 102 g/d and rural maize consumption was 454 g/d. Because of the large amount of maize products consumed by rural communities in Guatemala, even relatively low levels of fumonisins in maize could pose a significant health risk. Guatemala is divided into 22 geographic areas known as Departments (Departamentos). Interestingly, in the highland Departments of Guatemala where maize consumption is high, maize production is not sufficient and therefore much of the maize consumed in the highlands must be imported from lowland Departments and elsewhere (9).
NTD occur at a much higher frequency in countries where maize consumption is high and fumonisin contamination is likely (5). The association of fumonisin exposure with increased risk of NTD is plausible given that fumonisins are proven inhibitors of folate transport (10) and can induce NTD in rodents (11,12). Therefore, it is not surprising that fumonisin exposure has been implicated in increased NTD risk in humans in the United States (13). Thus, exposure to fumonisins is an important health issue for populations consuming large amounts of maize.
The goals of this study were to: 1) determine the levels of fumonisins and incidence of fumonisin-producing Fusarium species in maize grown in the highlands and lowlands of Guatemala; and 2) use the information on fumonisin levels in maize sold for human consumption and the effects of traditional processing methods (nixtamalization) on fumonisins to develop a preliminary exposure estimate.
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Materials and Methods |
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TOPABSTRACTIntroductionMaterials and MethodsResults and DiscussionLITERATURE CITED |
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From January to February 2005, 236 maize samples (1000 g) from the 2004 harvest (November to December) were purchased or provided by commercial vendors from 20 Departments across Guatemala. A total of 192 of the samples were white maize (blanco), 35 yellow (amarillo), 7 black (negro), and 2 were unidentified as to color. An approximation of the average altitude of the Departments was made based on the Official Visitor and Business Guide to Guatemala (14) and visual approximation using a topographical map of Guatemala (15). Departments with altitudes that were predominantly >1000 m (Central Highlands, Western Highlands, Northern Highlands, and metropolitan Guatemala City) were Alta and Baja Verapaz (n = 15), Chimaltenago (n = 9), Guatemala (n = 16), Huehuetenango (n = 5), Quetzaltenango (n = 5), Sacatepequez (n = 8), San Marcos (n = 21), Quiche (n = 8), Solola (n = 6), and Totonicapan (n = 6). Departments that were predominantly <1000 m (El Peten, Izabal, Western Plains, and Pacific Coast) were Chiquimula (n = 5), El Progreso (n = 10), Escuintla (n = 16), Izabal (n = 14), Jalapa (n = 9), Jutiapa (n = 15), Peten (n = 21), Retalhuleu (n = 8), Santa Rosa (n = 13), and Zacapa (n = 16). The vendors were asked to provide, as precisely as possible, the location where the maize was grown (source). The altitude of the source was determined using a topographical map of Guatemala (15) and the Index Mundi Web-based resource (16). After collection, all maize samples were delivered to the INCAP where they were stored dry at –20°C until processing and extraction of fumonisins. The time interval between collection of the samples and storage at –20°C ranged from 24 to 72 h and the samples were maintained dry while in transit.
Fusarium infection and species identification.
A total of 18 maize samples (intact kernels) from highland (n = 9) and lowland (n = 9) locations were selected from the 2001 harvest to test for Fusarium contamination. The maize seeds were surface sterilized by shaking in 100% commercial bleach for 3 min followed by rinsing in sterile water for 1 min. For each sample, a total of 20 sterilized kernels (4 replicates of 5 kernels per replicate) were plated on 2-benzoxazolinone agar medium (17) in 100-mm diameter petri plates. After incubation for 1 wk at 25°C in the dark, each plate was assessed for the number of kernels infected with Fusarium. For each infected kernel, a single transfer of the contaminating fungus was made to fresh 2-benzoxazolinone medium and incubated as above. Single-spore isolations were made from each of these subcultures to obtain a single, pure representative isolate from each infected kernel. A conidial suspension of each fungal strain was frozen in 15% glycerol at –80°C for long-term storage. Isolates were routinely cultured on potato dextrose agar (Difco) at 25°C in the dark. Species identification of isolates was determined by a combination of morphology, cultural characteristics, and sequencing of a portion of the translation elongation factor 1 
gene (18).
Assessment of fumonisin production.
Selected isolates were inoculated onto potato dextrose agar from glycerol stocks and grown for 1 wk. Conidial suspensions were obtained by flooding each agar plate with 10 mL of sterile water. Twice-autoclaved cracked maize [5 g hydrated with 45% water (v:w) in a 20-mL glass vial] was inoculated with 250 µL of conidial suspension (2.5 x 107 conidia). Two vials of maize were inoculated for each strain. Known fumonisin-producing strains (MRC826 and A00999) served as positive controls. After 14-d incubation at 25°C in the dark, 10 mL of acetonitrile:water (1:1) was added to each vial, which was shaken and allowed to stand for 2–3 h. Samples were frozen at –20°C until ready for further analysis.
Extraction and analysis for fumonisins. The method for collection, extraction, and solid-phase clean up is described in detail elsewhere (19). Briefly, dry-shelled maize samples (500–1000 g) were mixed and then 25 g was ground using a Romer Mill (Romer grinding/Subsampling Model 2A, Romer Lab Analytical Instruments Division). A total of 5 g of the ground maize was extracted with 25 mL acetonitrile:water (1:1) and fumonisins were isolated on C18 solid phase cartridges. Samples of the C18 solid phase cartridge eluates were analyzed at INCAP in Guatemala for fumonisin B1 by HPLC in 2000 and 2001 as described in Riley et al. (20) except that orthophthalaldehyde derivatized samples were manually loaded and injected on the column. In 2002, 2003, and 2005, analyses were accomplished using liquid chromatography-tandem-ion trap (LC) MS (19). The detection limits for HPLC and LC/MS methods were 0.3 µg/g and 0.01 µg/g, respectively. The LC/MS method also detected fumonisin B2 and fumonisin B3. Except where indicated, samples analyzed from 2002, 2003, and 2005 that had detectable fumonisin B1 but were <0.3 µg/g were assigned a value of 0 µg/g so that the results of the HPLC and LC/MS analyses for all years could be combined.
Exposure estimate. Maize consumption estimates were obtained from a 24-h recall study of women conducted in 1999–2000 in the town of Santa Maria de Jesus in the Department of Sacatepequez (n = 92) and from published data (9,10). Total fumonisins in nixtamalized maize foods, including hydrolyzed fumonisins B1, B2, and B3, were estimated based on a mass-balance study of the effects of traditional nixtamalization on total fumonisins in tortillas (6). For estimating exposure, we assumed nonhydrolyzed and hydrolyzed fumonisins of the B series to have equal toxicological potential in humans.
Statistical analysis. Statistical analysis was performed using SigmaStat software (Jandel Scientific). Where many groups were compared, 1-way ANOVA was used, followed by post hoc multiple comparisons using the Holm-Sidak method or Dunn method when treatment group size was unequal. A chi-square test was performed to determine the significance of incidence data. Where only 2 groups were compared, the Student's t test was used for parametric data and the Mann-Whitney rank sum test was used for nonparametric data. All data were expressed as means ± SEM and differences among means were considered significant at P < 0.05. The detection limit for the 2000 and 2001 samples analyzed by HPLC was 0.3 µg/g. The detection limit for analyses conducted by LC/MS in 2002 and 2003 was 0.01 µg/g; however, all values <0.3 µg/g were treated as "not detected" and assigned values of 0 µg/g for calculating the incidences and means.
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Results and Discussion |
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TOPABSTRACTIntroductionMaterials and MethodsResults and DiscussionLITERATURE CITED |
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Because the size of the maize crop in the highlands is insufficient to supply local needs between harvests (9, and anecdotal testimony of Central Highland residents), we hypothesized that the quality of the stored maize would decrease as highland-grown supplies dwindled and that the reduced quality of the remaining stored maize would be reflected by increased levels of fumonisin in the stored maize. To test this hypothesis, samples of the 2002 highland crop were collected from storage immediately before harvest of the 2003 crop from the same growers that supplied maize at harvest in 2002; 27% of the samples contained >0.3 µg/g fumonisin B1 compared with only 2% in the samples collected at harvest in 2002 (Fig. 2; Table 1). Nonetheless, the incidence of fumonisin B1-positive samples in the highland maize that had been stored (27%) was still less (P < 0.05) than the 78% incidence (51/65) in 2002 lowland maize collected at harvest. There are several possible explanations for the difference in fumonisin levels at harvest compared with the maize from storage 12 mo later in the highlands. It is possible that over the course of the year, the higher quality maize was preferentially consumed or that the stored maize had been supplemented with lowland-grown maize purchased in the local highland markets. Alternatively, it is possible that the increased fumonisin level was a result of production in storage. However, the latter scenario seems unlikely given the cool temperate conditions typical in the highlands.
Based on the analysis of samples collected from 2000 to 2003, it was evident that maize grown in the lowland environment was significantly more contaminated with fumonisins than maize grown in the highlands. In 1999, 6 maize samples were collected from households in Santa Maria de Jesus (>2000 m) and although several Fusaria were isolated in high frequency from all 6 samples, there was no F. verticillioides found based on morphology and culture characteristics. These preliminary results suggested that F. verticillioides was uncommon in highland maize. The lowlands in Guatemala generally are much warmer and humid than the highlands, which are sometimes referred to as the "land of eternal spring" (14). A warm and humid climate is much more conducive to F. verticillioides growth and fumonisin production (21). Thus, we hypothesized that the maize from the lowlands might have a higher incidence of fumonisin-producing Fusaria. This hypothesis was tested using kernels selected from samples collected in 2001. All (100%) of the Fusarium-infected kernels (Table 2) analyzed from 9 random lowland samples (20 kernels per sample) were infected with F. verticillioides (60/60) and no other Fusarium species, whereas in samples from the highlands (n = 9), only 5% (2/43) of the Fusarium-positive kernels yielded F. verticillioides. Moreover, all of the F. verticillioides isolates were able to produce fumonisin in in vitro culture on maize and predominantly as fumonisin B1. The fumonisin B1 production by the Guatemalan F. verticillioides isolates tested was 652 ± 80 µg/g (n = 20), which was not different from (P = 0.68) the fumonisin B1 production by the known fumonisin-producing strains MRC826 and A00999 (748 ± 163 µg/g; n = 4). None of the other 4 Fusarium species isolated from highland maize produced fumonisins in in vitro culture on maize (data not shown), which is consistent with previous mycotoxin analyses of these species (23,24). In the highlands, the predominant Fusarium species were F. subglutinans and Fusarium sp. NRRL 25622 (Table 2). NRRL 25622 is in the American clade of the Gibberella fujikoroi species complex and is most closely related to F. subglutinans, which does not produce trichothecenes. Indeed, no member of the G. fujikoroi species complex is known to produce trichothecenes. F. boothii can produce deoxynivalenol and zearalenone and F. meridoniale produces nivalenol, 4-acetyl nivalenol, and zearalenone (23); however, deoxynivalenol and zearalenone are readily broken down by the alkali treatment (nixtamalization) used by the local Mayan populations (25).
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Although it is difficult to pinpoint the origin of the maize sold in the local highland markets, the source information provided by the vendors and the levels of fumonisins in the maize suggest that maize from lowland locations is frequently sold in highland markets. Because the cool temperate highland environment does not appear to be conducive for F. verticillioides infection and fumonisin production on maize (Table 2), the frequency distribution and levels of fumonisin contamination (Fig. 3C) appear to closely reflect those of the maize grown in the lowlands (Fig. 1B) where F. verticillioides is common. This conclusion is supported by the reports of the vendors as to the source of the maize. A total of 109 samples of maize was analyzed from Departments where the average altitude was predominantly >1000 m (14). Of these, 107 were tracked to specific locations where the altitude could be estimated using a combination of a topographical map (15) and a Web-based resource (16). Over 50% of the maize samples were from lowland locations (<1000 m) where the total fumonisin concentration was 3.4 ± 0.7 µg/g (n = 56). In contrast, the concentration of the samples from higher altitude locations (>1000 m) was 0.2 ± 0.1 µg/g (n = 51) (P < 0.05). Only 7 of the 110 sourced samples from Departments where the mean altitude was <1000 m were sourced by the vendors to highland locations and the others (n = 103) were sourced to lowland locations. The source of only 19 samples could not be identified by the vendors. For these samples, the total fumonisin concentration (4.8 ± 2.1 µg/g) was not significantly different from the maize tracked to lowland locations, suggesting that this maize was most likely from the lowlands.
Taken together, the results suggest that fumonisin exposure appears to be greatest in individuals who consume large amounts of maize products and obtain maize in the market place from commercial vendors who sell maize from lowland locations (including Mexico and elsewhere). In Guatemala, as in many other countries in Central America, maize is consumed as nixtamalized maize products. Many highland Departments cannot produce sufficient maize to meet the local demand. Thus, even though environmental factors in highland areas of Guatemala are not conducive to F. verticillioides growth and fumonisin production, fumonisin exposure in the highlands has the potential to be quite high in individuals who consume large amounts of lowland maize purchased from vendors in highland markets.
In Central America and Mexico, processed maize consumption varies based on many factors including age, sex, reproductive status, and location (8). Regardless, urban populations consume much less than rural populations. Utilizing the existing consumption data (8,9; data from the recall study in the Central Highlands) and the fumonisin data (summarized in Table 3 and Fig. 3A), it is possible to estimate the potential fumonisin exposure to consumers of nixtamalized maize products in Guatemala based on the following assumptions. First, nixtamalization reduces total fumonisins and converts a large portion of the fumonisins in the final product to hydrolyzed fumonisins that may be less toxic than the parent compounds (26,27). However, because the toxicity of the hydrolyzed products is uncertain, for our exposure assessment, we assumed that hydrolyzed fumonisins and fumonisins of the B series are equally toxic to humans. In addition, we assumed that the reduction in total fumonisins as described in Palencia et al. (6) is similar throughout Guatemala for traditional processing. In the United States where water is not limiting, commercial processing appears to reduce total fumonisins more than in the traditional Mayan process (28,29). It is unknown whether commercial processing in Guatemala is of similar efficacy. However, it is possible that fumonisin exposure may be different for rural consumers of nixtamalized maize products made by traditional processing vs. urban populations consuming products made from nixtamalized maize products made by commercial methods.
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The commercial nixtamalization process can reduce fumonisin levels by as much as 80% (28,29), although the reduction was slightly less (56%) with the traditional Mayan method. One-half of the remaining fumonisins were present as the hydrolyzed forms, which may be less biologically active. If the hydrolyzed forms of fumonisins are proven to be not toxic to humans, then the exposure estimates in Table 4 would be reduced by 50%. Regardless, careful nixtamalization using sufficient alkaline steeping and water rinsing of the kernels is perhaps the best approach for minimizing fumonisin exposure. Because consumption of maize products is of such great importance in Central America, excluding lowland maize from rural markets would be unreasonable. However, care should be taken to prevent low quality lowland maize from being sold for human consumption in markets throughout Guatemala.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Author disclosures: O. A. Torres, E. Palencia, L. Lopez de Pratdesaba, R. Grajeda, M. Fuentes, M. C. Speer, A. H. Merrill, Jr., K. O'Donnell, C. W. Bacon, A. E. Glenn, and R. T. Riley, no conflicts of interest.
10 Abbreviations used: INCAP, Instituto de Nutricion de Centro America y Panama; LC, liquid chromatography; NTD, Neural tube defect; PMTDI, provisional maximum tolerable daily intake.
Manuscript received 29 June 2007. Initial review completed 1 August 2007. Revision accepted 24 September 2007.
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LITERATURE CITED |
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TOPABSTRACTIntroductionMaterials and MethodsResults and DiscussionLITERATURE CITED |
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