Exposure measurement of aflatoxins and aflatoxin ...

Exposure measurement of aflatoxins and aflatoxin metabolites in human body fluids. A short review Yin-Hui Leong, Aishah A. Latiff, Nurul Izzah Ahmad & Ahmad Rosma

Mycotoxin Research ISSN 0178-7888 Volume 28 Number 2 Mycotoxin Res (2012) 28:79-87 DOI 10.1007/s12550-012-0129-8

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Author's personal copy Mycotoxin Res (2012) 28:79–87 DOI 10.1007/s12550-012-0129-8

REVIEW

Exposure measurement of aflatoxins and aflatoxin metabolites in human body fluids. A short review Yin-Hui Leong & Aishah A. Latiff & Nurul Izzah Ahmad & Ahmad Rosma

Received: 24 August 2011 / Revised: 6 March 2012 / Accepted: 7 March 2012 / Published online: 3 April 2012 # Society for Mycotoxin Research and Springer 2012

Abstract Aflatoxins are highly toxic secondary fungal metabolites mainly produced by Aspergillus flavus and A. parasiticus. Human exposure to aflatoxins may result directly from ingestion of contaminated foods, or indirectly from consumption of foods from animals previously exposed to aflatoxins in feeds. This paper focuses on exposure measurement of aflatoxins and aflatoxin metabolites in various human body fluids. Research on different metabolites present in blood, urine, breast milk, and other human fluids or tissues including their detection techniques is reviewed. The association between dietary intake of aflatoxins and biomarker measurement is also highlighted. Finally, aspects related to the differences between aflatoxin determination in food versus the biomarker approach are discussed. Keywords Aflatoxins . Biomarkers . Food . Exposure

Introduction Aflatoxins are highly toxic secondary metabolites produced by fungi belonging to several Aspergillus species, mainly A. Y.-H. Leong (*) : A. A. Latiff Doping Control Centre, Universiti Sains Malaysia, Penang, Malaysia e-mail: [email protected] N. I. Ahmad Environmental Health Research Centre, Institute for Medical Research, Kuala Lumpur, Malaysia A. Rosma School of Industrial Technology, Universiti Sains Malaysia, Penang, Malaysia

flavus and A. parasiticus (Gourama and Bullerman 1995; Groopman et al. 1988; O’Riordan and Wilkinson 2008). Currently, more than 14 different types of aflatoxins have been identified; the naturally occurring aflatoxins are aflatoxin B1, B2, G1 and G2 (denoted AFB1, AFB2, AFG1 and AFG2). Aflatoxins were discovered in the 1960s when a series of outbreaks in poultry and fish occurred in different parts of the world. One of the famous outbreaks was the sudden death of more than 100,000 turkeys and other farm animals (turkey “X” disease) in the UK where the cause was attributed to A. flavus-contaminated peanut meal (Bennett and Klich 2003). Aflatoxins occur mostly in tropical regions where hot and humid climates promote fungal growth. Poor harvesting practices, improper storage, and less than optimal conditions during transport and marketing can also contribute to proliferation of fungal and increase the risk of aflatoxins production (Bhat and Vasanthi 2003). Human exposure to aflatoxins can result directly from ingestion of contaminated foods, or indirectly from consumption of foods from animals previously exposed to aflatoxins in feeds. Inhalation of contaminated airborne aerosols can represent an additional route of exposure, which, however, has not yet been exhaustively investigated. Extensive epidemiological studies on dietary exposure to aflatoxin were performed during the 1980s and early 1990s. Some of the first applications of biomarkers for human exposure to food chemicals or contaminants were related to AFB1 in the late 1980s (Gan et al. 1988; Zhu et al. 1987). Use of biomarkers in epidemiological study design normally combines the tools of standard epidemiology (such as case histories, questionnaires, and monitoring of exposure) with the sensitive laboratory analysis of molecular biology (Perera 1987). It has the advantage of being directly relevant to human risk. Moreover, it has the potential to identify the hazard, and to give early warning by signalling the early effects of

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exposure and increased susceptibility, thus allowing opportunities to arrest disease through timely intervention (Perera 1996). Furthermore, biomarkers play an important role as a bridge between hazard characterisation and exposure assessment in cases where other components present in the food matrix affect the bioavailability and hence the systemic dose of the substance under consideration. In most cases, a biomarker is either the food contaminant itself or a metabolite thereof. The selected body fluid is frequently either urine or blood, but other target matrices such as saliva, breast milk, faeces, and adipose tissue also exist. However, biomarker methodology does not have universal applicability as a means of assessing human exposure to food contaminants. One of the most important determining factors is inter-individual variability in the pharmacokinetic and metabolic behaviour of the food contaminant (Dybing et al. 2002). This is particularly the case when the biomarker normally has a positive value even in the absence of exposure. In addition, whether the selected biomarker is able to reflect, with high specificity, the exposure to the presence of that just particular food contaminant is another factor that limits its application. Despite these limitations, a biomarker is likely to be applicable to many fields of interest within the human population, for example occupational exposure, disease prevention and therapy when the biomarker method is successfully developed and demonstrated to be a reliable and accurate measurement of dietary exposure. For aflatoxin analysis, the development of molecular and biochemical epidemiology has enabled progress beyond the determination of levels in foods and feeds. Several molecular biomarkers have been developed and applied, including the AFB1 metabolites and AFB1 macromolecular adducts. These involve AFM1 and AFB1-N7-guanine in urine as well as AFB1-albumin adducts in serum (Wang et al. 2001). The latter adduct is considered to better reflect the longer term intake of AFB1 based on the longer half-life of albumin in humans compared to urinary metabolites (Groopman et al. 1994). More stable and less fluctuation for aflatoxin–albumin adduct than urinary excretion of aflatoxin metabolites over a same period of time is observed (Groopman et al. 1992a; Wild et al. 1992). Hence, it is considered to have greater value as a biomarker and thus has been widely applied in epidemiological studies and has provided a considerable database for human aflatoxin exposure in different countries (Wild et al. 1990a, 1993).

Metabolism of aflatoxin Data from human epidemiological studies have shown that exposure to AFB1 is one of the major risk factors in the multifactorial etiology of human hepatocellular carcinoma (Qian et al. 1994). Therefore, AFB1 and/or its metabolites are normally

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taken into the account for the exposure measurement due to the fact that AFB1 is the most potent human carcinogen and the most frequent aflatoxin present at high levels in food. Several comprehensive reviews on AFB1 metabolism in animals and humans have been published (Eaton and Gallagher 1994; Guengerich et al. 1998; Wild and Turner 2002). AFB1 is metabolised by liver cytochrome P450 enzymes after ingestion to form highly reactive 8, 9-epoxide which subsequently reacts with DNA to generate guanine adducts (Bennett and Klich 2003; Groopman et al. 1993; Walton et al. 2001), or with serum albumin to generate lysine adducts (Sabbioni et al. 1990). AFB1 can be oxidised into some other derivatives, such as hydroxylated metabolites (AFM1, AFQ1), demethylated metabolite (AFP1), and the reduced metabolite (aflatoxicol). In a recent study by Partanen et al. (2010), aflatoxicol was detected as the only metabolite present in placental perfusion models, thus demonstrating that AFB1 is transferred through the placenta. The differences in susceptibility to aflatoxins across species and between persons depend largely on the fraction of the dose that is directed into the various possible pathways, with harmful “biological” exposure being the result of activation to the epoxide and the reaction of the epoxide with proteins and DNA. There is also evidence that the fractions that follow the different possible pathways are influenced by dosage, perhaps because of the saturation of the most chemically competitive processes (Eaton et al. 1993). Susceptibility to aflatoxins is greatest in the young, and there are very significant differences between species, persons of the same species (according to their differing abilities to detoxify aflatoxin by biochemical processes), and the sexes (according to the concentrations of testosterone). The toxicity of aflatoxins also varies according to many nutritional factors (Ayub and Sachan 1997; Pier et al. 1985), and recovery from protein malnutrition is delayed by exposure to aflatoxin (Adhikari et al. 1994; Rogers 1993).

Association between dietary intake of aflatoxin and biomarker measurement According to Zhu et al. (1987), statistically significant correlations between the dietary intake of AFB1 and urinary excretion of the metabolite AFM1 have been reported. In this study, 252 urine samples from residents in Fushui county of the Guangxi province, People's Republic of China, have been analysed for AFM 1 . Between 1.23 and 2.18 % of ingested AFB1 was found to be excreted as AFM1 in human urine. In a later study, Groopman et al. (1992b) analysed the same urine samples and found that levels of AFB1-N7-guanine adduct were also correlated with dietary AFB1 intake. The AFB1-N7-guanine amount

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that present in urine in a 3-day period was reported to range from 9–19 years), 0.04 (>19–65 years), and 0.03 ng/kg bw/day (>65 years), respectively. Thai children (3–9 years) had the highest AFM1 intake, 0.26 ng/kg bw/day, which was comparable to those in the Far East (0.20 ng/kg bw/day) (JECFA 2001), France (3–14 years, 0.22 ng/kg bw/day) (Leblanc et al. 2005), Brazil (adults, 0.188 ng/kg bw/day) (Shundo et al. 2009), and Spain (4–9 years, 0.21 ng/kg bw/day) (CanoSancho et al. 2010).

Table 1 Estimated exposure to total aflatoxins for adults (assuming a body weight of 60 kg) at the proposed maximum levels of 10 μg/kg for the three tree nuts (almonds, hazelnuts and pistachios) and other tree nuts in European Countries (EFSA 2009) Foodstuff

Three tree nutsa Other tree nutsb Other foodc

Total aflatoxins exposure (ng/kg bw/day) Cluster diet B

Cluster diet E

Cluster diet F

0.054 0.030 1.896

0.030 0.014 1.076

0.013 0.009 0.677

a

Almonds, hazelnuts and pistachios which have been assessed by EFSA (EFSA 2007) b

Tree nuts other than almonds, hazelnuts and pistachios

c

Other food groups as presented in the assessment (EFSA 2007)

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Determination of aflatoxins in food versus biomarkers approach Comparing the determination of aflatoxins in food, the methodology for measuring aflatoxins and their metabolites in human body fluids appears to be less established and involves the use of specific antibodies. These antibodies as well as the standards used are normally not commercially available and their production requires special resources including facilities for animal work and technical skills. Methods that have been employed include chromatographic techniques such as thin layer chromatography (TLC) and high performance liquid chromatography (HPLC), immunological assays using specific antibodies or antisera like enzyme-linked immunosorbent assay (ELISA), radio-immunoassay (RIA) and immunochemical visualisation in tissues as well as instrumentation-based methods such as synchronous fluorescence spectroscopy (Kensler et al. 1998). Recently, immunoaffinity column (IAC) clean-up followed by high performance liquid chromatography with fluorescence detection (HPLC-FL) is the most frequently applied method due to the advantages of both antibody selectivity and chromatographic separations. Some recent studies using this method have included analysis of AFM1 in breast milk samples from Turkey (Gürbay et al. 2010), analysis of AFM 1 in urine samples of a Brazilian population (Romero et al. 2010) and analysis of AFB1-lysine adduct in pregnant women in Kumasi, Ghana (Shuaib et al. 2010). RIA is comparably less common compared to other techniques because it requires radiolabelled substrates, access to scintillation counters, and skilled personnel. Table 2 highlights some of the recent analysis with the detection methods of aflatoxins and aflatoxin metabolites in human body fluids.

Human urine The various aflatoxins and their levels in human urine have been reviewed (Autrup and Autrup 1992; Yourtee and KirkYourtee 1989). Aflatoxins (B1, B2, G1, G2, M1, Q1, P1, B2a), aflatoxin-N7-guanine and aflatoxicol have been measured in human urine. Aflatoxins are difuranocoumarins and are chemically grouped into two main subgroups according to their chemical structure: the difurocoumarocyclopentenone series (AFB1, AFB2, AFB2a, AFM1, AFM2, AFM2a and aflatoxicol) and the difurocoumarolactone series (AFG1, AFG2, AFG2a, AFGM1, AFGM2, AFGM2a and AFB3) (International Crops Research Institute for the Semi-Arid Tropics, ICRISAT 2012). Among the various metabolites, the nucleic acid adduct aflatoxin-N7-guanine has been focused on due to it reflecting DNA damage in the presumed target cell

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for aflatoxin carcinogenesis (Groopman et al. 1993). A good correlation between the total dietary of aflatoxin intake and total AFM1 excretion in human urine during a 3-day study was reported by Zhu et al. (1987), whereby a regression equation of 0.143 plus 0.0135 multiplied by the amount of AFB1 consumed was observed. Several studies on human exposure to aflatoxins using urinary AFM1 as biomarkers have been reported. In a study of Cheng et al. (1997), 64–66 % of people from China and Taiwan had detectable AFM1, with the concentration ranged from 3.2 to 108 ng for 12 h and from 2.7 to 17 ng for 12 h among Chinese and Taiwanese, respectively. In Sierra Leone, Africa, the highest concentration of 374 ng/ml was reported by Jonsyn-Ellis (2000). In the Czech Republic, data obtained by Malir et al. (2006) revealed occurrence of AFM1 in 57.6 % of analysed urine samples, with the concentration ranging from 0.019 to 19.219 ng/g of creatinine. Seventy-eight percent (n054) of the urine samples contained >0.6 pg/ml of AFM1 in the Brazilian population (Romero et al. 2010). Detection of the presence of aflatoxins, particularly AFM1, in human urine involves TLC, ELISA and HPLC with fluorescence detection. In the largest cross-sectional survey of AFM1, excretion into over 1,200 urine samples were collected and extracted with chloroform and quantified by TLC and HPLC (Nyathi et al. 1987). Wild et al. (1986, 1992) have extracted aflatoxins from urine using immunopurification, and quantitated using polyclonal antibody in a highly sensitive ELISA. Determination of total and individual aflatoxin B1, B2, G1, G2, M1, M2, B2a, G2a, B3, GM1, and P as well as aflatoxicol in urine of Egyptian infants was performed using two-dimensional TLC (Hatem et al. 2005). The use of immunoaffinity clean-up and detection by HPLC with fluorescence detector has been reported in determination of the presence of AFM1 in urine samples from the Brazilian population (Romero et al. 2010), and in the analysis of urinary aflatoxin metabolites (AFB1, AFB2, AFG1, AFG2 and AFM1) in young children from Egypt and Guinea (Polychronaki et al. 2008). Urinary aflatoxin-N7-guanine levels have been correlated with dietary intake of aflatoxins in Gambian (Groopman et al. 1992a) and Chinese adults (Groopman et al. 1992b). However, the levels of these urinary metabolites or adducts only reflect intake on the previous day due to the short half-life of such metabolites in urine (Groopman et al. 1994).

Human blood Detection of free aflatoxins was initially focused on (Hendrickse et al. 1982; Tsuboi et al. 1984), but in the subsequent identification of the metabolic fate of aflatoxins, aflatoxin-albumin adduct is preferred because it can integrate exposure over a period of months based on the

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Table 2 Some of the recent analysis of aflatoxins and aflatoxin metabolites in human body fluids Sample/ Country

Urine Brazil Urine Guinea (children 1– 2.5 years old)

Urine Egypt (infants)

Aflatoxins and metabolites

AFM1 AFB1 AFB2 AFG1 AFG2 AFM1 AFB1, B2, G1, G2, M1, M2, B2a, G2a, B3, GM1, P and Aflatoxicol AFB1-lysine adduct

Serum Ghana (pregnant women) Serum Ghana AFB1-albumin Serum Egypt

Serum Gambia

Serum Gambia

AFB1-albumin Total HCC cases Control AFB1-albumin Mothers Umbilical cord Infants (16 weeks) AFB1 AFB2

Concentrationa Mean

Range

5.96 pg/ml 189 pg/ml 1.4 76.6 2.2 5.5 Totals: 0.1114e 0.1496d ng/ml

1.8–39.9 pg/ml – pg/ml 0.8–2.2 72.1–81.1 0.9–8.0 5.0–6.2 0.05–0.28 ng/ml 0.07–0.20

10.9 pg/ albumin

0.44–268.73 pg/mg albumin 0.94 pmol/ albumin 0.1–4.44 pmol/mg albumin 5.0 pg/mgb 2.6b 9.0b 40.4 pg/mg albuminb 10.1b 8.7b 2.5 ng/ml 0.2 3.6 0.1 0.7 2.1 0.1 Total: 0.7058d ng/ml 0.2521e

Serum China

AFG1 AFG2 AFM1 AFM2 Aflatoxicol AFB1, B2, G1, G2, M1, M2, B2a, G2a, B3, GM1, P and Aflatoxicol AFB1-albumin

Breast milk Turkey Breast milk Iran Breast milk Brazil

AFM1 AFM1 AFM1

0.9972 pmol/mg albumin NSf 8.2 ng/kg –

Urine China

AFQ1 AFM1 AFB1-N7-guanine

10.4 ng/mlg 0.04g 0.38g

Serum Egypt (Infants)

a

The unit is expressed only in the first row

b

Geometric means

c

ND non-detectable

d

Infants with kwashiorkor

e

Infants with marasmus

f

Not specified

g

Report as median

h

Report as interquartile range (IQR)

Detection method

Reference

IAC, HPLC-FL HPLC-FL

Romero et al. (2010) Polychronaki et al. (2008)

Two-dimensional TLC

Hatem et al. (2005)

HPLC-FL

Shuaib et al. (2010)

RIA

Tang et al. (2009)

ELISA

Turner et al. (2008)

ELISA

Turner et al. (2007)

HPLC

Jonsyn-Ellis (2007)

Two-dimensional TLC

Hatem et al. (2005)

RIA

Jiang et al. (2005)

IAC, HPLC-FL ELISA IAC, HPLC-FL

Gürbay et al. (2010) Sadeghi et al. (2009) Navas et al. (2005)

HPLC using UV and FL detector

Mykkänen et al. (2005)

NDc–32.8 ND–32.8 ND–25.8 4.8–260.8 pg/mg albumin 5.0–189.6 5.0–30.2 0.2–74 ng/ml 0.02–2.2 0.3–56 0.01–1.2 0.03–6.8 0.04–48 0.01–3.2 0.37–1.11 ng/ml 0.15–0.38

0.3325–2.2703 pmol/mg albumin 60.10–299.99 ng/l 0.3–26.7 ng/kg One positive sample 0.024 ng/ml 3.39–23.3 ng/mlh 0.01–0.33h 0.0–2.15h

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half-life of albumin in humans (Sabbioni et al. 1987). The aflatoxin which bound to albumin can be AFB1 and AFG1 but not AFB2 and AFG2 which can be metabolised to 8, 9-epoxide (Egal et al. 2005). However, the aflatoxin-albumin adduct levels should be regarded as a measure of the levels of AFB1 ingested, due to the fact that the presence of AFG1 in the contaminated food is rare. Albumin is the only protein in serum which binds AFB1 to form a high level of adducts in rats (Skipper et al. 1985), while haemoglobin binds AFB1 in a very low yield (Tannenbaum and Skipper 1984). Moreover, albumin is readily extracted from human blood, and it provides a relatively non-intrusive measure of the biologically effective dose of ingested AFB1 (Wild et al. 1996). This approach may be useful for rapid screening of samples for acute exposures and it also reflects chronic exposure that is not available from other markers such as the aflatoxinN7-guanine adduct in urine. The major analytical techniques currently available for measuring AFB1-albumin adduct levels in human blood are detected using ELISA, RIA, and IAC followed by HPLC with fluorescence detection and LC-MS/MS techniques (Chapot and Wild 1991; Gan et al. 1988; McCoy et al. 2005; Sabbioni et al. 1990; Shuaib et al. 2010; Walton et al. 2001; Wang et al. 1996; Wild et al. 1990b; Xu et al. 2010). AFB1-lysine is the only AFB1 adduct which has been structurally identified in enzymatically digested plasma albumin (Guengerich et al. 2002). Therefore, AFB1-lysine adduct is considered to give more accurate results than AFB1albumin adduct in which the latter targets the whole AFB1-albumin adduct and tends to report higher yield. In addition, this has led to the development of a specific monoclonal antibody (IIA4B3) for AFB 1-lysine adduct using a synthetic AFB1-lysine-cationised bovine serum albumin conjugate (Wang et al. 2001). Previous studies either used a polyclonal antiserum-based ELISA (Chapot and Wild, 1991; Wild et al. 1990b) or a monoclonal antibody 2B11-based RIA (Gan et al. 1988; Wang et al. 1996) to measure AFB1-albumin adducts. This IIA4B3 was found about 12-fold more sensitive than 2B11 for binding to AFB1-lysine when 3H-AFB1lysine was used as the tracer (Wang et al. 2001). Furthermore, a more specific, precise and sensitive detection of AFB1lysine with D4-AFB1-lysine as the internal standard using an isotope dilution liquid chromatography mass spectrometry (IDMS) has also been developed by McCoy et al. (2005). The measured adduct concentration is expressed as aflatoxin equivalents per mg albumin. Highly correlated data generated from various methods, for example, ELISA and HPLC-fluorescence (Wild et al. 1990a), RIA and HPLC-fluorescence (Wang et al. 1996) as well as ELISA and LC-MS/MS (Scholl et al. 2006) have been reported. However, aflatoxin levels measured by antibody-

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based methods are usually higher than those measured using the LC/MS method, the isotope internal standard of which was coupled because the ELISA may be measuring other aflatoxin adducts in addition to the target adduct. Aflatoxin-albumin has been used to measure the aflatoxin exposure in West African countries, including The Gambia, Guinea, Ghana, Benin and Togo (Egal et al. 2005; Gong et al. 2002, 2003; Jolly et al. 2006; Wild et al. 2000), in China (Qian et al. 1994; Ross et al. 1992), and in infectious disease-linked immune suppression (Jiang et al. 2005; Turner et al. 2003). Moreover, aflatoxin-albumin has been regularly used as the surrogate efficacy biomarker of AFB1 exposure for assessment of different therapeutic/intervention agents and techniques in human intervention trials (Kensler et al. 1998).

Breast milk Aflatoxin metabolites can also be found in lactating animals and, thus, in human breast milk. Several papers have described the presence of aflatoxins in human milk (Coulter et al. 1984; Maxwell et al. 1989). The method of ELISA, and complimentary with HPLC-fluorescence detector, have been reported to measure AFM1 in human breast milk obtained from women in Zimbabwe and Sudan at levels of 10–50 ppt (Wild et al. 1987). Assessment of child or infant exposure of AFM1 in breast milk from lactating mothers in The Gambia, West Africa (Zarba et al. 1992) and Iran (Sadeghi et al. 2009) have also been reported. Polychronaki et al. (2006) has found that 37 % of Egyptian women had detectable aflatoxins in breast milk in a cross-sectional study. AFM1 in the range of 60.10–299.99 ng/l in breast milk samples from Ankara, Turkey, was reported recently (Gürbay et al. 2010).

Other human fluids or tissues Although AFB1 is a potent liver toxin, it also affects other tissues such as lung, kidney and colon, in both rodents and humans (Larsson and Tjalve 1995). Aflatoxins have been detected in nasal secretions, sputum and tissue biopsies (lung, liver, brain) from patients exposed to toxinproducing moulds in their environment (Hooper et al. 2009). In addition, faeces have been proposed to be a marker for aflatoxins exposure because they are an important route of excretion of both the unabsorbed AFB1 and metabolites formed from the absorbed toxin. Excretion of AFQ1 and AFM1 in human faeces has been quantified in 83 young Chinese males selected from 300 subjects based on detectable urinary AFM1 in Guangzhou, southern China

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(Mykkänen et al. 2005). This study showed that faecal AFQ1 was 60 times higher than that of AFM1. Acknowledgement This work is supported by research grants from Food Safety and Quality Division, Ministry of Health, Malaysia. Conflict of interest The authors declare that there are no conflicts of interest.

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