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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / s c i t o t e n v

PAH exposure in a Ghanaian population at high risk for aflatoxicosis

Natalie M. Johnson a , Evans Afriyie-Gyawu a,1 , Henry Huebner a , Alicia Marroquin-Cardona a , Abraham Robinson a , Lili Tang b , Li Xu b , Nii-Ayi Ankrah c , David Ofori-Adjei c , Pauline E. Jolly d , Jonathan H. Williams e , Jia-Sheng Wang b,2 , Timothy D. Phillips a,
a

Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, Texas, USA Institute of Environmental/Human Health, Texas Tech University, Lubbock, Texas, USA c Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Accra, Ghana d Department of Epidemiology, School of Public Health, University of Alabama at Birmingham, Birmingham, AL, USA e Peanut Collaborative Research Support Program, The University of Georgia, Griffin, Georgia, USA
b

AR TIC LE D ATA
Article history: Received 2 October 2008 Received in revised form 21 November 2008 Accepted 25 November 2008 Available online 14 January 2009 Keywords: Biomarker Polycyclic aromatic hydrocarbons 1-hydroxypyrene Aflatoxin Food safety Environment

ABSTR ACT
It was postulated that a population in sub-Saharan Africa, known to be at high risk for aflatoxicosis due to frequent ingestion of aflatoxin (AF)-contaminated foods could also be exposed to polycyclic aromatic hydrocarbons (PAHs) from a variety of environmental sources. Previously, participants in this population were shown to be highly exposed to AFs, and this exposure was significantly reduced by intervention with NovaSil clay (NS). Objectives of this study were 1) to assess PAH exposure in participants from the AF study using urinary biomarker 1-hydroxypyrene (1-OHP); 2) examine the effect of NS clay and placebo (cellulose) treatment on 1-OHP levels; and 3) determine potential association(s) between AF and PAH exposures. A clinical trial was conducted in 177 Ghanaians who received either NS capsules as high dose or low dose, or placebo (cellulose) for a period of 3 months. At the start and end of the study, urine samples were analyzed for 1-OHP. Of the 279 total samples, 98.9% had detectable levels of 1OHP. Median 1-OHP excretion in nonsmokers was 0.64 mol/mol creatinine at baseline and 0.69 mol/mol creatinine after 3 months. Samples collected at both time points did not show significant differences between placebo and NS-treated groups. There was no linear correlation between 1-OHP and AF-albumin adduct levels. Results show that this population is highly exposed to PAHs (and AFs), that NS and cellulose treatment had no statistically significant effect on 1-OHP levels, and that this urinary biomarker was not linearly related with AF exposure. 2008 Elsevier B.V. All rights reserved.

1.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are unavoidable environmental pollutants produced from the incomplete com-

bustion of organic material and are detected in contaminated air, tobacco smoke, and charcoal-grilled and smoked foods (Nikolaou et al., 1984). A variety of PAHs, which normally exist in complex mixtures, have been recognized as carcinogenic (IARC,

Corresponding author. Department of Veterinary Integrative Biosciences, College of Veterinary Medicine, Texas A&M University, 4458 TAMU, College Station, TX 77843, USA. Tel.: +1 979 845 6414; fax: +1 979 862 4929. E-mail addresses: evansafriyiegyawu@georgiasouthern.edu (E. Afriyie-Gyawu), jswang@uga.edu (J.-S. Wang), tphillips@cvm.tamu.edu (T.D. Phillips). 1 Present address: Jian-Ping Hsu College of Public Health, Georgia Southern University, Statesboro, GA 30460, USA. 0048-9697/$ see front matter 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2008.11.060

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1987). The association between high PAH exposure and the risk of lung cancer has been established through various molecular epidemiological studies (Tang et al., 1995; Mastrangelo et al., 1996). It has been documented that PAHs cause cellular and humoral immunotoxicity in vitro and in vivo (White, 1986); additionally, this class of compounds has been shown to cause complex effects on the immune system in humans (Davila et al., 1996; Burchiel and Luster, 2001). The urinary metabolite 1hydroxypyrene (1-OHP) has been extensively characterized and validated as a biological indicator of PAH exposure in many human populations and is widely accepted due to the presence of pyrene in most PAH mixtures (Levin, 1995; Bouchard and Viau, 1999). While researchers consider urinary metabolites of additional PAHs to be valid, 1-OHP remains the most comprehensively studied biomarker in the urine. Increased levels of 1OHP excreted in human urine have shown significant correlation with subjects exposed to mixtures of PAHs. The excretion of 1-OHP represents recent PAH exposure with ranges of half-life values reported to be: 24 to 48 h, 6 to 35 h, and 16 to 20 h (Jongeneelen et al., 1990; Buchet et al., 1992). Hence, 1-OHP is used as a short-term biomarker of PAH exposure. In the Ashanti Region of Ghana, West Africa, human exposure to PAHs may readily occur from a variety of environmental sources as well as preparation of foods by smoking (and subsequent ingestion). Traditional methods of cooking in the region include the use of fire pits and indoor woodburning stoves; often the dwellings are not well-ventilated. Furthermore, the direct drying and/or smoking of maize, a staple crop in the region, is frequently employed before storage as a means to repel insects. In this same region of Ghana, we have recently shown that people were highly exposed to aflatoxins (AFs) based on biomarkers in urine and blood (Jolly et al., 2006). AFs are naturally occurring metabolites from Aspergillus fungi that are frequently found as contaminants of commodities such as cereal grains, peanuts and tree nuts (CAST, 2003). It has been well-documented that exposure to the most potent congener (AFB1) can cause hepatotoxicity, suppression of the immune system, and anti-nutritional effects in animals and humans (Gong et al., 2002; Turner et al., 2003; Jiang et al., 2007). Chronic exposure to AFs is implicated as a major risk factor in the etiology of hepatocellular carcinoma (HCC), particularly in tropical areas of Southeast Asia, South America, and the sub-Saharan regions of Africa (Wogan, 1992; CAST, 2003). Additionally, it is estimated that 80% of all HCC cases occur in developing countries (Wild and Hall, 2000). In West Africa, the risk of developing HCC is significantly increased by infection with hepatitis B virus (HBV). Epidemiological studies and animal models have demonstrated synergistic interactions between AF exposure and HBV infection in the development of HCC (Turner et al., 2002). In a recent community-based cohort study conducted in Taiwan, an increased risk of HCC was associated with PAH exposure, while an even greater risk was found among participants who were also exposed to high levels of AFs (Wu et al., 2007). Thus, strategies that reduce multiple exposures to AFs and PAHs are highly desirable for populations at risk for both agents. One PAH, benzo[a]pyrene (BaP), is similar to AFB1 in that: 1) both AFB1 and BaP are carcinogens, 2) their mutagenic action requires metabolic activation to epoxide analogs (BaP 7,8-diol-9,10-epoxide

and AFB1 exo-8,9-epoxide), and 3) their ability to form DNA adducts primarily at the N-2 and N-7 position of the guanine residue, respectively, can lead to tumorigenesis. Among various strategies used to reduce exposures, binding of toxin by NovaSil (NS), a montmorillonite clay, in the gastrointestinal tract (enterosorption) appears to be a safe and viable approach for AFs (Phillips et al., 2007). This conclusion is based on previous studies using multiple animal models and clinical intervention trials in humans in the U.S. and Ghana (Wang et al., 2005; Afriyie-Gyawu et al., 2008). Although, no clinical interventions have been reported for PAHs using enterosorbent strategies, Viau et al. (2004) noted a trend in the reduction of urinary excretion of 1-OHP in rats fed pyrene and diets containing fiber. Additionally, bulk cellulose in the diet was shown to affect the recovery of 1-OHP from the urine suggesting that it may bind PAHs. Therefore, the main objectives of this study were to assess the overall exposure to PAHs in our population in Ghana by measuring 1-OHP and to determine if there was a relationship between AF and PAH exposures. Another objective was to determine the effect of NS versus placebo (cellulose) treatment on 1-OHP levels.

2.
2.1.

Materials and methods

Materials

Authentic 1-OHP reference standard was obtained from the Midwest Research Institute, Chemical Carcinogen Reference Standard Repository (Kansas City, MO). Sep-Pak C18 cartridges were purchased from Waters (Milford, MA). HPLC-grade solvents and -glucuronidase from Helix pomatia (type HP-2S) were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO). All of the experiments were done using filtered and deionized water (18.2 M.cm) (Millipore, Milford, MA). The NS clay was supplied by Engelhard Chemical Corporation (Iselin, NJ) and was further evaluated for environmental contaminants and heavy metals to ensure their levels met US and international standards (Afriyie-Gyawu et al., 2008). Capsules were produced, under sterile conditions at a pharmacy in a GLP setting to contain the following dose forms: 1) 500 mg NS, 2) 250 mg NS + 250 mg placebo, or 3) 250 mg placebo, based on previous dosimetry protocols (Wang et al., 2005). The placebo consisted of microcrystalline cellulose, and was initially chosen for the AF study due to its lack of binding capacity for AFs, as shown by isothermal adsorption (data not shown). All other chemicals and reagents were purchased commercially at the highest degree of purity available.

2.2.

Study site and population

Volunteers were recruited from six communities located within the Ejura-Sekyedumase district in the Ashanti Region of Ghana with the help of the District Health Director and other health personnel. Preliminary data confirming AF exposure and demographic information was previously collected in 4 of the communities. The urinary metabolite AFM1 was detected in 91.2% of 91 samples, whereas 100% of 140 serum samples had detectable levels of AFB1-albumin adducts (AFB-AA) (Jolly et al., 2006). Biomarker data regarding exposure

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to PAHs was unknown for this area, and to our knowledge, this is the first study to measure 1-OHP in a human population in Ghana. A total of 507 residents volunteered to participate in the NS intervention trial. Following enrollment, participants signed informed consent documents and completed a health history survey and food frequency questionnaire. Blood and urine samples were collected in order to select qualified participants. Based on predetermined inclusion criteria, 180 individuals (male and female) of the 507 volunteers were selected as study subjects and met the following conditions: healthy status based on physical examination results, age 18 58 years, intake of corn and/or groundnut-based foods at least 4 times a week, blood AFB-AA levels N0.5 pmol AFB1/mg albumin, no history of chronic disease(s), no use of prescribed medications for chronic or acute illness, non-pregnant and/or non-breastfeeding females, normal ranges of hematological parameters, liver and renal function indicators (blood and urine parameters), and signed consent form. Prior to start of the study and administration of the treatment, three participants had to leave the trial resulting in a total of 177 study participants.

2.3.

Study design and protocol

The study protocol was approved by the Institutional Review Boards at Texas A&M University and Noguchi Memorial Institute for Medical Research in Ghana for Ethical Clearance. All participants were provided written informed consent, as well as an oral explanation prior to the start of the study. The overall study design adhered to guidelines set for a randomized, double-blind, placebo controlled Phase IIa clinical trial, described in detail by (Afriyie-Gyawu et al., 2008). Healthy individuals (177) who met the pre-defined inclusion criteria were randomly divided into one of the three groups: 3.0 g NS/ day (high dose), 1.5 g NS/day (low dose) or placebo control (cellulose) for a period of 3 mo. Participants were further classified as smokers or non-smokers; only 9 of the 177 study subjects were known tobacco-smokers. Trained study monitors collected blood and urine samples from each participant at specified time points. Aliquots of the first urines collected were stored separately (15 ml and 4.5 ml) in polypropylene tubes and shipped to Texas A&M University where they were kept frozen at 80 C for AFM1 and 1-OHP analysis. Serum, plasma and blood cells were immediately separated and transported frozen to Texas Tech University where they were stored at 80 C until analyzed for AFB-AA.

were evaporated to dryness under N2 and reconstituted in MeOH. The analyses were conducted using an HPLC system with fluorescence detection (Waters, Milford, MA). Excitation and emission parameters were set at 240 and 388 nm, respectively. Aliquots of the extracts were injected and analyzed using a 125 4.6 mm Spherisorb ODS2 HPLC column (Waters) with a particle size of 3 m. The mobile phase was comprised of 75% MeOH in water, and chromatographic separation was achieved by isocratic elution at a flow rate of 1.1 ml/min for 15 min. The 1-OHP peak was detected at a retention time of approximately 6.2 min, and the limit of detection was approximately 0.25 nmol/L of urine. Creatinine (Cr) concentrations in individual urine samples were measured at St. Joseph's Regional Health Center Laboratory using an auto-analyzer. Urinary concentrations of 1-OHP were expressed as mol/mol Cr in order to correct for variations in urine dilution. Blank urines spiked with 1-OHP standard in the range of 0.012 ng were prepared, incubated and extracted as described in the procedure minus the enzyme addition to generate a sixpoint calibration curve that was linear over the tested range (r2 = .9904) for the instrument. The repeatability precision was 7.0% and recoveries averaged 99%. The machine was calibrated daily by injecting multiple aliquots of 1-OHP reference standard, which was prepared bi-weekly and stored in the dark at 4 C.

2.5.

Statistical analysis

Data generated from HPLC analysis were transferred into an Excel database for management. Mean, median, standard deviation (SD) and range were calculated for concentrations of 1-OHP. To show the consequence of NS ingestion on 1-OHP levels, statistical evaluation focused on the comparisons among different treatment groups and different time points (baseline and 3 mo). Since the parameters were not normally distributed, the Kruskal-Wallis test or Wilcoxon rank sum test was used to compare the differences among treatment groups and each time point. To evaluate the effect of dose and time interactions, a nonparametric mixed-effect model was applied. Correlation analysis was done to examine the relationship of AF exposure (AFB-AA level) and levels of 1OHP; p-values were generated based on Spearman correlation coefficients. A p-value of less than 0.05 (two-tailed) was considered significant. Statistical analyses were done using SAS software version 9.3 (SAS Institute Inc., Cary, NC).

2.4.

1-OHP analysis in urine

3.
Urinary 1-OHP levels were measured using an HPLC-fluorescence method based on a procedure described by (Gardiner et al., 1992). Briefly, urine samples (4.0 ml) were adjusted to pH 5.0 with an equal volume of 1.0 M acetate buffer (pH 5.0). -Glucuronidase (50 l) from Helix pomatia possessing sulfatase activity was added, and samples were incubated for 4 h at 37 C while gently shaking in a water bath. The hydrolyzed samples were passed through primed Sep-Pak C18 columns on a vacuum manifold followed by sequential washing steps of 3.0 ml deionized H20 followed by 3.0 ml 50% MeOH in water. 1-OHP was eluted from the column with 100% MeOH; the eluates

Results

A sum of 162 out of 177 participants (91.5%) that began the clinical trial completed the 3-month period. A total of 279 urine samples collected at baseline and after 3 months of NS intervention were analyzed for urinary 1-OHP. Out of the total urines, 98.9% showed detectable levels of 1-OHP. Median, mean SD, and the range of 1-OHP levels at both time points are presented in Table 1; participants are further delineated as smokers or non-smokers. Since the data was not normally distributed, non-parametric analysis was applied for all statistical evaluations. There was not a significant difference

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Table 1 Levels of urinary 1-hydroxypyrene at both time points 1-OHP (mol/mol creatinine) Baseline
Non-smokers (n = 149) Smokers (n = 9) 0.64, 0.80 0.68 (b LOD to 3.37) 0.42, 0.42 0.25 (0.10 to 0.78)

3-month
0.69, 1.08 1.09 (b LOD to 5.11) 0.61, 1.00 0.81 (0.32 to 2.37)

Values are median, mean SD and (range).

that the combustion of wood and other fuels for cooking constitutes a significant source of PAHs; furthermore, the consumption of grilled meat has been linked to elevated levels of 1-OHP in the urine (van Maanen et al., 1994). The elevated levels of urinary 1-OHP measured in study participants suggest that inhabitants are exposed to large doses of PAHs likely from the indoor preparation of foods by using wood as fuel, socializing by fires, and the ingestion of smoked foods. In addition, drinking water is sometimes allowed to condition in smoked containers to develop a desirable smoke-flavor. Traditionally, plant material (e.g., palm nut fiber) may be burned

in 1-OHP levels in participants classified as smokers as compared to non-smokers in urines measured at baseline (p = 0.098) or after 3 months of intervention (p = 0.822). The distribution of urinary 1-OHP levels in participants at baseline and after 3 months of intervention is illustrated in Fig. 1. There were no significant differences in median 1-OHP levels among the three study groups at baseline (p = 0.890) or after 3 months of intervention (p = 0.384) (Fig. 1). The median 1-OHP levels were comparable between the placebo and low dose group in urine samples collected at baseline (p = 0.820) and after 3 months of intervention (p = 0.186). Additionally, no significant differences were found in median levels of 1-OHP between the placebo and high dose group at baseline (p = 0.843) or after 3 months of intervention (p = 0.325). Furthermore, no significant time effect for 1-OHP levels were found in the high dose (p = 0.373), low dose (p = 0.113) or placebo (p = 0.758) groups over the 3 months study period. Nonparametric mixed-effect model analysis did not show a significant dose-time interaction (p = 0.436) delineating that there was not a reduction in urinary 1-OHP levels due to the NS intervention. Correlation analysis was done to examine the relationship of AF exposure (AFB-AA level) and levels of 1OHP; results are illustrated in Fig. 2. No significant correlations were found between levels of AFB-AA and 1-OHP at baseline (Fig. 2A, CC = 0.080, p = 0.394) or after 3 months of intervention (Fig. 2B, CC = 0.029, p = 0.717); CC represents the Spearman correlation coefficients.

4.

Discussion

Results illustrate that the majority of the study population had a measurable exposure to PAHs. 1-OHP levels measured in this Ghanaian population were higher than those previously recorded for non-smoking individuals in numerous countries. For instance, the median 1-OHP level measured in nonsmokers at baseline in our study population (0.64 mol/mol Cr) is considerably higher than median 1-OHP concentrations measured in non-smokers from the US (0.27 mol/mol Cr) and Canada (0.07 mol/mol Cr) (Levin, 1995). Viau et al. (2000) measured 1-OHP in populations in Burundi and found elevated 1-OHP levels in individuals living in traditional rural houses comparable to levels we found in Ghana. In many developing countries, especially in rural areas, indoor wood burning fireplaces and stoves are employed as means of cooking and heating, suggesting that individuals may be prone to an increased risk of PAH exposure. It has been documented

Fig. 1 The dose effects of NovaSil intervention on urinary 1-OHP levels over the 3 months study duration. The box plots show distributions of 1-OHP levels in each group at baseline and after 3 months of intervention. The box represents values ranging from 25 to 75 percentile, the line within indicates the median value. The bars on both sides of the box represent values ranging from 5 to 25 percentile and from 75 to 95 percentile, respectively.

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Fig. 2 Correlation analyses between 1-OHP and AFB1-albumin adduct levels in study participants at baseline (A) and after 3 months with intervention (B). P-values were generated based on Spearman correlation coefficients (CC).

at low temperatures in clay pots before the addition of water. Although the Ghana Food and Drugs Board has suspended commercial production of smoke-flavored water until the safety of the product is ensured, some individuals may still practice this process. Tano-Debrah et al. (2007) conducted a series of tests assessing PAH content in the smoke-flavored water and concluded that PAH contamination may be possible depending on the type and amount of organic material initially used to smoke the containers. Another possible route of PAH exposure in this Ghanaian population may be through the ingestion of dried maize. Maize dried directly on hot asphalt roads suggests an additional mode of PAH contamination. The direct drying of this staple crop is regularly employed as the first step in food preparation or as a way to decrease mold contamination in storage. Maize is sometimes smoked as a means to repel insects and therefore may be contaminated with PAHs. Additionally, AFcontaminated maize constitutes a major food safety problem in Ghana due to poor handling and storage. Exposure to both AFs and PAHs may result in an even greater risk of significant negative health impacts in this population. We investigated the correlation between 1-OHP and AFB-AA levels as a means of indirectly linking a high intake of AF-contaminated food with high PAH exposure. Correlation analysis showed that there was not a significant linear correlation between urinary 1-OHP levels and serum levels of AFB-AA (Fig. 2). This is likely due to multiple and different pathways of exposure to these compounds. Ultimately, subjects exposed to both PAHs and AFs may be at increased risk for disease associated with these exposures. Our results suggest that the study participants in Ghana may represent a population at high risk for PAH exposure (as well as AFs). Data did not show a significant difference in 1-OHP levels in participants classified as tobacco-smokers as compared to non-smokers. The influence of cigarette smoke on 1-OHP levels has yielded differing results in previous studies. When individuals were exposed to background levels of environmental PAHs, the consequence of smoking resulted in increased excretion of 1-OHP (Levin et al., 1995; Viau et al., 1995). Conversely, at higher exposure levels, smoking failed to

produce any differences in urinary 1-OHP levels as compared to not smoking (Buchet et al., 1992; Bouchard and Viau, 1999). Our results did not clearly show an effect of smoking cigarettes on 1-OHP levels possibly because the environmental exposure was predominant. Furthermore, of the small percentage classified as smokers in our study population (only 9 subjects), details such as frequency of smoking was unknown. A second objective of this portion of our clinical intervention study was to determine the consequence of NS and cellulose treatment on urinary 1-OHP. Our previous work (in vitro and in vivo) demonstrated that NS clay was a high affinity sorbent for AFs (Phillips et al., 2007). However, the potential affect of NS (and cellulose) on bioavailability of PAHs from the gastrointestinal tract had not been investigated. After administration of NS for a period of 3 months, serum levels of AFB-AA and urinary AFM1 levels were significantly reduced, confirming the aflatoxin-sorption efficacy of NS in humans (Wang et al., 2008). Results from this study illustrate that there were no statistically significant differences in 1-OHP levels between NS treatment arms and the placebo group in baseline samples or in samples collected after 3 months of intervention. While there was a trend for reduction in 1-OHP levels in participants treated with cellulose, (median reduced from 0.66 mol/mol Cr at baseline to 0.58 mol/mol Cr after 3 months), these differences were not statistically significant. This may be due to variation in the route of exposures, i.e., inhalation versus ingestion. The enterosorption strategy would not be expected to reduce exposure from air. Clearly, further research is warranted to test cellulose and similar materials for their ability to decrease exposures from food and water. In summary, this study illustrates that a population living in a rural area of Ghana is highly exposed to PAHs, as well as AFs (as previously shown); however, the exposure levels are independently distributed. Future research should focus on this population to better comprehend the sources of PAH exposure. Additionally, long-term biomarkers, like the PAH albumin adduct could be utilized in prospective studies to further investigate associations between PAH exposure and

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its public health impact. These objectives are of particular relevance in rural communities and developing countries where the incidence and negative health effects of environmental and food-borne exposures are often elevated. Further investigation is warranted to evaluate if the combined decrease of exposures to AFs and PAHs may have additional benefits to reduced incidence of liver cancer.

Acknowledgements
We gratefully acknowledge the on-site physicians, technicians and other health personnel at the Ejura District Hospital. We thank all volunteers whose participation made this study possible. This work was supported by the United States Agency for International Development (USAID LAG-G-00-96-90013-00) through Peanut CRSP of the University of Georgia.

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