Journal of Viral Hepatitis, 2015, 22, 926–935
doi:10.1111/jvh.12410
Aflatoxin levels in chronic hepatitis B patients with cirrhosis or hepatocellular carcinoma in Balıkesir, Turkey M. Aydın,1 S. Aydın,2 M. Bacanlı2 and N. Basßaran2
1
Department of Infectious Diseases and Clinical Microbiology,
Faculty of Medicine, Istanbul Hospital of Basßkent University, Istanbul, Turkey; and Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey 2
Received August 2014; accepted for publication February 2015
SUMMARY. Aflatoxins, the secondary metabolites produced
by species of naturally occurring Aspergilli, are commonly found in food such as cereals, dried fruits and juice, wine, beer and spices. They are hepatotoxic and are well known human carcinogens based on evidence from human studies. Aflatoxins are an environmental risk factor for the development of hepatocellular carcinoma (HCC). Chronic hepatitis B-infected patients are at increased risk of cirrhosis, hepatic failure and liver cancer. This study was designed to determine the serum aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) concentrations using high-pressure liquid chromatography (HPLC) in hepatitis B-infected patients with or
INTRODUCTION Aflatoxins (AFs), formed by several fungus species of the genus Aspergilli, are naturally occurring contaminants of agricultural crops, and owing to their adverse health effects, contamination of food by AFs is of global importance [1,2]. The four major types of AFs are aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2). AFB1 is one of the most potent carcinogens occurring naturally and classified as carcinogenic to humans by the International Agency for Research on Cancer (IARC). IARC concluded that there was sufficient evidence in experimental animals for the carcinogenicity of naturally occurring mixtures of AFB1, AFG1 and AFM1; Abbreviations: Ab, antibodies; ACN, acetonitrile; AF, aflatoxin; AFP, alpha-fetoprotein; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CHB, chronic hepatitis B; ELISA, enzyme-linked immunosorbent assay; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HPLC, high-pressure liquid chromatography; IARC, International Agency for Research on Cancer; MeOH, methanol; TFA, trifluoroacetic acid; UHT, ultra-high temperature. Correspondence: Mehtap Aydın, Department of Infectious Diseases and Clinical Microbiology, Istanbul Hospital of Basßkent University, Istanbul 34662, Turkey. E-mail: [email protected]
without cirrhosis and liver cancer, alongside healthy controls in Balıkesir, Turkey. The mean AFB1 and total AF levels in patients without liver cancer and cirrhosis were significantly higher than healthy controls. The mean AFB1 and total AF levels in patients with chronic hepatitis B and HCC were significantly higher than infected patients with or without cirrhosis. These results suggest that patients with chronic hepatitis B who are exposed to AFs are at increased risk for developing HCC, which might be prevented by reducing consumption of contaminated foods. Keywords: aflatoxins, chronic hepatitis B, cirrhosis, hepatocellular carcinoma, Turkey.
limited evidence for the carcinogenicity of AFB2; and inadequate evidence for the carcinogenicity of AFG2 [1]. The toxicological importance of AFs has been extensively studied. The primary target organ for AF toxicity and carcinogenicity in humans and animals is the liver. AFB1 is metabolized in the liver by cytochrome P450 system to the highly reactive AFB1-8,9-epoxide, which is responsible for the toxicity [3]. The metabolic effects of AFs include inhibition of DNA, RNA and protein synthesis; reduction in miscellaneous enzyme activities; depression of glucose metabolism; inhibition of lipid synthesis, and depression of clotting factor synthesis [4]. Experimental animals infected with hepatitis B virus (HBV) were reported to be more sensitive to the carcinogenic effects of AF than uninfected animals confirming the carcinogenic potential of AFs in hepatitis B-infected animals [1]. Some epidemiological studies have also shown that AF exposure particularly in combination with HBV is associated with an increased risk of hepatocellular carcinoma (HCC). The potency of AFs appears to be significantly enhanced in individuals with HBV infection [5–7]. The correlation between AFs exposure and liver cancer incidence in some areas, especially in Africa and Asia has been determined [1,8]. Due to their frequent occurrence and severe toxicity, guidelines and tolerance levels of AFs have been set in several countries including Turkey [9]. Turkey has encountered AFs contamination in © 2015 John Wiley & Sons Ltd
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AF standards (AFG1, AFB1, AFG2, and AFB2) were obtained from Sigma Chemical Co. Stock solutions and standards were prepared and analysed according to AOAC Method 971.22 [17].
of the nature of the intrahepatic vessel (score 1–3), liver parenchyma (score 1–3), liver surface (1–3) and the size of the spleen (score 1–2). A spleen size of 12 cm or more was given score of 2. A total score of seven or more was indicative of cirrhosis. HCC was confirmed using elevated serum AFP levels (>200 ng/mL) with lesions occupying spaces on ultrasound characteristic of HCC (n = 20). At the time of recruitment, each study subject was interviewed to obtain information on socio-demographic characteristics, lifestyle, height, bodyweight, lifetime history of cigarette smoking and alcohol consumption, personal and family history of various chronic diseases followed by standardized clinical examination. The cumulative exposure to cigarette was calculated as ‘pack-years’ using the following formula: (number of years smoked 9 average number of cigarette smoked per day)/20. Total alcohol intake was categorized as three levels (0, 60 g/day) [20]. According to the answers obtained from the questionnaires, dietary habits of the study group are about the same. They consume more vegetables, fruits, and meat as well as locally produced food than cereals including corn and corn products. The study was approved by an ethical committee (HEK 11/118) according to the ‘Declaration of Helsinki.’ All subjects participated in the study voluntarily and written consent (in Turkish) was obtained before blood samples were drawn. Whole blood samples (7 mL) were taken from cases at the time of admission to hospital and collected from the controls at the time of recruitment. The samples were centrifuged at 800 9 g for 15 min and the serum was separated. All samples were aliquoted and stored at –20 °C until analysis.
Subjects and sampling
Biochemical analysis
A total of 38 (female:male, 16:22) CHB patients without cirrhosis and HCC (CHB patients), 26 (female:male, 10:16) CHB patients with cirrhosis (cirrhosis patients), 20 (female:male, 7:13) CHB patients with HCC (HCC patients) and 49 (female: male, 25:24) healthy controls from Balıkesir State Hospital and Balıkesir University Hospital between March 2013 and March 2014 were recruited to this retrospective study. The exclusion criteria for the study group included decompensated liver disease, haematologic disease, immunologically mediated disease, several cardiac diseases and chronic pulmonary disease. The inclusion criteria for healthy controls were as follows: no use of medications, no pregnancy or lactation for female volunteers, normal liver and renal function tests and electrocardiograms, negative for serum alpha-fetoprotein (AFP), negative hepatitis B surface antigen (HBsAg) test and negative hepatitis C virus (HCV) infection by anti-HCV test. The persistence of HBsAg for 6 months from its first detection denotes CHB infection [18]. Cirrhosis was evaluated using a validated ultrasound scoring scale as described originally by Lin et al. [19]. The scoring system consisted
The serum was collected and examined for aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total bilirubin by spectrophotometric analysis as indicative of hepatic function using standard diagnostic kits (Roche Diagnostics, Mannheim, Germany) and a Roche modular P800 clinical chemistry analyzer.
different foods [10,11], and healthy Turkish adults living in some regions of Turkey have been reported to be continuously exposed to AFs, particularly in the summer [12]. Chronic hepatitis B (CHB) is an infection of the liver caused by the oncogenic DNA virus HBV [13]. The prevalence of HBV infection in Turkey varies between 2% and 8% [14]. HCC is an important health problem in Turkey, and HBV infection is an important predisposing factor [15]. Dietary exposure to AFB1 and chronic infection of HBV are major risk factors in HCC, raising the possibility of a synergistic interaction between them [16]. The aim of this study was to determine the serum AFs levels in CHB patients with or without cirrhosis and HCC and to evaluate the relationships between AFs levels and these diseases.
MATERIALS AND METHODS Chemicals and reagents HPLC grade methanol (MeOH), acetonitrile (ACN) were purchased from Riedel (Poole, Dorset, UK). All other chemicals and solvents were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Standards
© 2015 John Wiley & Sons Ltd
Determination of viral serology Serum samples were analysed for HBsAg antibodies (ab) according to the standard laboratory operating procedures at the Balıkesir State Hospital. Qualitative detection of HBsAg, a marker of chronic HBV carriage, in human serum was performed using commercially available enzyme-linked immunosorbent assay (ELISA; Alpco Diagnostics, Salem, NH, USA) [21].
Extraction of serum samples After digestion of serum protein [22], the extraction method modified from Nelson et al. [23] was performed.
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One millilitre of serum sample was diluted with 2 mL of nhexane and mixed for 1 min. After the centrifugation at 5000 9 g for 5 min, upper n-hexane phase containing serum lipids was removed. This process was repeated twice. One millilitre of chloroform was added to the serum and after shaking vigorously for 0.5 and 4.5 min, successively, the solution was centrifuged again at 5000 9 g for 10 min. The bottom chloroform layer was collected. This process was repeated for three more times. The chloroform phases were collected in a tube and evaporated under a nitrogen stream to complete dryness.
Determination of AFG1, AFB1, AFG2 and AFB2 levels by HPLC method The dry residue was subjected to the trifluoroacetyl (TFA) derivatization to improve fluorescence properties according to the procedure of AOAC method 971.22 [17]. Determination of AFB1, AFB2, AFG1 and AFG2 levels in the derivatized standards and samples were carried out by HPLC equipped with an auto sampler (Hewlett Packard [HP] Agilent 1100 Series, Vienna, Austria) using a fluorescence detector (excitation at 360 nm, emission at 430 nm). A Spherisorb S5ODS2 column (3.8 mm i.d. and length 25 cm, 5 lm particle size, Waters, Milford, MA, USA) was used. The mobile phase was deionized water:ACN:MeOH (62:16:22, v:v:v), and the flow rate was 1 mL/min. The injection volume was 100 lL. AF standards used were ranged between 5 and 1000 pg/mL. The retention times for the AFs were 6.2 min for AFG1, 8.2 min for AFB1, 11.8 min for AFG2 and 17.0 min for AFB2. Recovery studies were performed on blank samples of serum spiked with levels of 0.1, 0.25 and 0.5 ng/mL for each AF standard and repeated for three times. The average recoveries were 69.7% for AFG1, 79.6% for AFB1, 101.3% for AFG2 and 107.4% for AFB2. The concentrations of AFs in the samples were calculated using the calibration curves of peak area prepared for each AF standard separately. The detection limits were determined as 5 pg/mL for AFG1 and AFB1; 2.5 pg/mL for AFG2 and AFB2. The quantification limits were determined as 35.2 pg/mL for AFG1, 21.2 pg/ mL for AFB1, 60.5 pg/mL for AFG2 and 34.85 pg/mL for AFB2.
Statistical analysis All data were analysed using SPSS software version 15.0 (SPSS Inc., Chicago, IL, USA). The distribution of the data was checked for normality using the Shapiro–Wilk test. The homogeneity of the variance was verified by the Levene test. To test for significant differences between variables, the one-way analysis of variance and the Kruskal– Wallis one-way analysis of variance were used for normally and non-normally distributed continuous data, respectively. Chi-square or Fisher’s exact test was used for
categorical data. Kolmogorov–Smirnov test was used to test continuous data for normality. The Pearson correlation method for normally distributed variables and Spearman rank correlation for non-normally distributed variables were used to determine the linear relationship between the variables. A two-tailed P value of 0.05). Total pack-years of smoking of patients with cirrhosis (14.31 5.65) and patients with HCC (21.36 5.05) were significantly higher than patients with CHB (7.91 1.32) and healthy controls (11.07 6.10) (P < 0.05). There were no significant differences between patients with CHB and healthy controls for total pack-years of smoking. There were no differences between the groups for lower ( 60 g/day) intakes of alcohol. There were no differences between the groups for the history of cancer from first-degree relatives (parents, offspring and siblings). The positive history of cancer from first-degree relatives of patients with CHB, patients with cirrhosis, HCC patients with the highest value and healthy controls were 10.5%, 11.5%, 25.0%, and 8.2%, respectively. Among 16 cases with first-degree family history of cancer in the study group, there were 10 liver cancers (62.50%), three rectum cancers (18.75%), two lung cancers (12.50%) and one gastric cancer (6.25%). There were no correlations and relationships between the dependent variables (Total AF levels) and independent variables (age, r = 0.096; body mass index (BMI, kg/m2), r = 0.158; total pack-years of smoking, r = 0.136; alcohol consumption, r = 0.186; and first-degree family history of cancer, r = 0.198).
Evaluation of biochemical parameters The biochemical parameters of study group are presented in Table 2. Patients with cirrhosis or HCC had significantly elevated AST levels vs patients with CHB and healthy controls (P < 0.05). There were no significant differences © 2015 John Wiley & Sons Ltd
© 2015 John Wiley & Sons Ltd CHB patients (n = 38) 60.20 9.05 21.08 2.54a
52.85 6.12 24.74 1.06a 7 (35.0)a 13 (65.0)a 11 (55.0)a 9 (45.0)a 21.36 5.05b 15 (75.0)a 4 (20.0)a 1 (5.0)a 5 (25.0)a 15 (75.0)a
10 (38.5)a 16 (61.5)a 16 (61.5)a 10 (38.5)a 14.31 5.65b 20 (76.9)a 4 (15.4)a 2 (7.7)a 3 (11.5)a 23 (88.5)a
b
HCC patients (n = 20)
b
Cirrhosis patients (n = 26)
4 (8.2)a 45 (91.8)a
39 (79.6)a 8 (16.3)a 2 (4.1)a
21 (42.9)a 28 (57.1)a 11.07 6.10a
25 (51.0)a 24 (49.0)a
44.49 10.12a 23.52 1.69a
Healthy controls (n = 49)
SD, standard deviation; HCC, Hepatocellular carcinoma; CHB, chronic hepatitis B. Superscripts of different letters differ significantly (P < 0.05) from each other in the same line.
a
Age (years) (mean SD) 43.50 7.30 Body mass index (kg/m2) (mean SD) 22.96 0.67a Gender [n(%)] Female 16 (42.1)a Male 22 (57.9)a Current cigarette smoker [n (%)] Yes 20 (52.6)a No 18 (47.4)a Total pack-years of smoking (mean SD) 7.91 1.32a Alcohol intake [n (%)] 0 28 (73.7)a 60 g/day 2 (5.3)a History of cancer from first-degree relatives [n (%)] Yes 4 (10.5)a No 34 (89.5)a
Factor
Table 1 Demographic characteristics of the study group
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79.15 41.72 40.65 21.45a 0.79 0.32a AST (IU/L) ALT (IU/L) Total bilirubin (mg/dL)
AST, aspartate aminotransferase; ALT, alanine aminotransferase; HCC, Hepatocellular carcinoma; CHB, chronic hepatitis B. Superscripts of different letters differ significantly (P < 0.05) from each other in the same line. The results were given as mean SD (standard deviation).
51.53 29.61a 25.57 16.95c 0.72 0.27a 100.30 46.12 49.35 21.24a 0.74 0.30a 132.62 64.06 68.31 39.05b 1.63 0.51b
b
HCC patients (n = 20)
b a
CHB patients (n = 38)
Cirrhosis patients (n = 26)
Healthy controls (n = 49)
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Table 2 Biochemical parameters of the study group
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between patients with CHB and healthy controls for AST levels. Alanine aminotransferase levels of patients with CHB were higher than healthy controls (P < 0.05). ALT levels in patients with cirrhosis were statistically higher than patients with CHB and patients with HCC. There were no significant differences between patients with CHB and patients with HCC for ALT levels. Total bilirubin levels in patients with cirrhosis were statistically higher than patients with CHB, patients with HCC and healthy controls (P < 0.05). There were no significant differences between patients with CHB, patients with HCC and healthy controls for total bilirubin levels.
Evaluation of serum aflatoxin levels There were no significant differences between the groups for the percentage of AF-positive samples (Table 3). The percentages of AF B1-positive samples in healthy controls, patients with CHB, patients with cirrhosis and patients with HCC were 26.5%, 21.1%, 26.9% and 35.0%, respectively. For the detectable serum AF B1 levels, the odds ratio for patients with CHB, patients with cirrhosis and patients with HCC were 0.74 (0.27–2.02), 1.02 (0.35– 2.99) and 1.49 (0.49–4.55), respectively. Aflatoxin levels in the AF-positive samples of study group are given in Table 4. The mean AFB1 level of patients with CHB was significantly higher than healthy controls (P < 0.05). Among the patients, the mean AFB1 level of patients with HCC was significantly higher than patients with CHB and patients with cirrhosis (P < 0.05). Among the patients, the mean AFB2 levels of patients with CHB and patients with HCC were significantly higher than patients with cirrhosis (P < 0.05). The mean AFG1, AFB2 and AFG2 levels of each patient group were significantly higher than healthy controls (P < 0.05). The highest AFB1, AFG1, AFB2 and AFG2 levels were found in patients with HCC. Total AF levels in patients with CHB, patients with cirrhosis and patients with HCC were 119.4%, 82.2% and 169.4% higher than healthy controls, respectively (Fig. 1).
DISCUSSION Aflatoxins have carcinogenic, teratogenic, and mutagenic effects. It is well documented that contamination of foods with AFs may cause liver cancer in humans [7,8]. AFs may also affect the epidemiology of many diseases and have health risks in the countries where the toxin is uncontrolled [3]. The contamination of food with AFs is a major concern. Major risks are inevitable climatic conditions such as unseasonable rains or high humidity [22,24]. Where trade does occur, the least contaminated foods and feeds are exported, which may lead to enhanced exposure of © 2015 John Wiley & Sons Ltd
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Table 3 Determination of aflatoxin-positive samples and the risk in relation to AF exposure in the cases and controls
Healthy controls; n = 49 (%) CHB patients; n = 38 (%) P OR (95% CI) Cirrhosis patients; n = 26 (%) P OR (95% CI) HCC patients; n = 20 (%) P OR (95% CI)
Af B1
Af G1
Af B2
Af G2
13/49 (26.5) 8/38 (21.1) 0.554 0.74 (0.27–2.02) 7/26 (26.9) 0.971 1.02 (0.35–2.99) 7/20 (35.0) 0.483 1.49 (0.49–4.55)
10/49 (20.4) 5/38 (13.2) 0.378 0.59 (0.18–1.90) 6/26 (23.1) 0.789 1.17 (0.37–3.68) 7/20 (35.0) 0.483 1.49 (0.49–4.55)
13/49 (26.5) 8/38 (21.1) 0.554 0.74 (0.27–2.02) 7/26 (26.9) 0.971 1.02 (0.35–2.99) 7/20 (35.0) 0.483 1.49 (0.49–4.55)
10/49 (20.4) 5/38 (13.2) 0.378 0.59 (0.18–1.90) 6/26 (23.1) 0.789 1.17 (0.37–3.68) 7/20 (35.0) 0.483 1.49 (0.49–4.55)
HCC, Hepatocellular carcinoma; CHB, chronic hepatitis B; OR, odds ratio; CI, confidence interval. Superscripts of different letters differ significantly (P < 0.05) from each other in the same column. Table 4 Aflatoxin levels in the aflatoxin-positive samples of the study group
AF B1 (pg/mL) [n (%)] AF G1 (pg/mL) [n (%)] AF B2 (pg/mL) [n (%)] AFG2 (pg/mL) [n (%)]
CHB patients
Cirrhosis patients
HCC patients
Healthy controls
53.96 11.72a 8/38 (21.1) 22.80 8.51a 5/38 (13.2) 23.72 7.47a,b 8/38 (21.1) 9.33 1.69a 5/38 (13.2)
41.09 6.77a 7/26 (26.9) 23.00 14.31a 6/26 (23.1) 16.84 6.17a 7/26 (26.9) 10.25 2.67a 6/26 (23.1)
63.11 13.02b 7/20 (35.0) 28.79 9.01a 7/20 (35.0) 28.17 4.00b 7/20 (35.0) 14.77 4.82a 7/20 (35.0)
18.36 10.64c 13/49 (26.5) 11.51 3.94b 10/49 (20.4) 9.98 4.76c 13/49 (26.5) 5.71 2.25b 10/49 (20.4)
HCC, Hepatocellular carcinoma; CHB, chronic hepatitis B. Superscripts of different letters differ significantly (P < 0.05) from each other in the same line. The results were given as mean SD (standard deviation).
Fig. 1 Total aflatoxin levels in the aflatoxin-positive samples of patients with CHB, patients with cirrhosis, patients with HCC and healthy controls. Bars that do not share same letters (superscripts) are significantly different from each other (P < 0.05). The results were given as Mean SD (standard deviation). HCC, Hepatocellular carcinoma; CHB, chronic hepatitis B.
the producers, because the more highly contaminated products are retained at home for consumption by a population that is already at the greatest risk of AF exposure © 2015 John Wiley & Sons Ltd
[25]. Although AFM1, the main monohydroxylated derivate of AFB1, is less carcinogenic and mutagenic than AFB1, it exhibits a high level of genotoxic activity and certainly represents a health risk because of its possible accumulation and linkage to DNA [26]. The levels of AFM1 in 30 (73.2%, n = 30/41) samples of ultra-high temperature (UHT) milk randomly collected from different supermarkets in Burdur, near to Balıkesir during 2011 were found to be 6.42 to 71.33 ng/L (mean level, 17.76 ng/L), and only three (7.3%, n = 3/41) samples of UHT milk were contaminated at levels above the Turkish legal limit (50 ng/L) [27]. There are some studies indicating that the Turkish population is continuously exposed to AFs. Giray et al. [11] reported that the total AFs levels were ranged between 10.4 and 643.5 ng/kg in the 41 wheat samples grown and consumed in some regions of Turkey. Fifty-nine percentage of the samples were positive for total AFs. The percentage of positive samples for AFB1, AFB2, AFG1 and AFG2 were 42%, 12%, 37% and 12%, respectively, although the detected levels were under the permitted levels for AFs in cereals [11]. Total AF levels in corn samples
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from various street bazaars and market outlets in different regions of Turkey were found to be ranged between 1.75 and 120.3 lg/kg, and 4% of the samples contained AFs above the acceptable limit of 10 lg/kg in Turkey [28]. Kabak [29] analysed the 85 walnut samples collected between October 2012 and April 2013 in different provinces of Turkey for the presence of AFs. The daily intakes of AFB1 and total AFs from walnuts for Turkish consumers were estimated to be 0.020 and 0.026 ng/kg bw per day, respectively. Hacıbekiro glu and Kolak [30] reported that 76% of 62 food samples from Istanbul, Turkey, exceeded legal limits of total AF, and the highest levels were found in chestnut (232.9 lg/kg), nutmeg (206.1 lg/kg) and sumac (182.5 lg/kg). The concentrations of the total AFs, AFB1, AFB2 and AFG1 in the three different corn flour samples purchased from different markets of Balıkesir, Turkey, were found to be 1.08 0.02, 0.59 0.01, 0.16 0.01 and 0.34 0.013 lg/kg, respectively [31]. A number of approaches have been used to determine AF exposure in human populations, including analysis of AF metabolites and AF-DNA or AF-protein adducts [32– 34]. However, these studies are more complicated and expensive. Generally, AFB1 in blood is not recognized as a biomarker compared to these adducts and there is limited data validating this as a biomarker. The detection of AF B2, AF G1 and AFG2 subtypes, determined at low levels in food, has not been confirmed as a sensitive method in blood for exposure. There are a few studies in literature demonstrating the AF levels directly in biological samples [12,23,35]. Corcuera et al. [36] reported that the level of AFB1 in plasma from rats given a single dose of 0.25 mg/ kg of AFB1 by oral gavage was 24.8 ng/mL and 9.5 ng/ mL at 10 min and 30 min, respectively. Studies on human serum samples mostly have limited sample sizes vs the current study. In a study performed by Hassan et al. [37], they measured the AFB1 levels in the serum and milk of mothers (n = 50) and serum of infants (n = 50) in Egypt. Twenty-four of fifty mothers and their infants had been contaminated with AF with the following mean contamination levels of 8.9 4.2 ng/mL (mothers’ serum), 1.9 0.6 ng/mL (mothers’ milk) and 1.8 0.9 ng/mL (infants’ serum). In a study conducted in UK using ELISA, AF levels of blood donors (n = 27) were found not to be higher than 20 pg/mL and it was reported that present UK guideline tolerances for AF in imported food were effective in limiting human exposure to toxic AFs in the UK diet [38]. Hepatocellular carcinoma is one of the most common malignancies worldwide. There is a marked geographical variation in the incidence of HCC due to the various aetiological factors including viral (HBV and HCV), chemical and several other factors such as AFs, alcohol, cigarette smoke and genetic tendencies [39]. AFB1 has been suggested to play a causative role in the aetiology of HCC. The correlations between human HCC and food
contamination with AF have been reported in various studies [6,7,35,40]. A number of mechanisms have been suggested, the most widely accepted of which is that HBV infection sensitizes hepatocytes to the carcinogenic effects of AFB1 [41]. Prospective studies in Shanghai, China, showed that urinary excretion of AF metabolites increased the risk of HCC up to fourfold, and HBV infection increased the risk sevenfold. However, individuals who excreted AFB1 metabolites and were carriers of HBV had as much as a 60-fold increase in risk of HCC [42]. In comparison with the study of the interaction between AFs and HBV, there has been little focus on the potential of HCV to cause HCC. AFs also have a synergistic effect on HCV-induced liver cancer. A study in Taiwan suggested that AF-albumin adducts were associated with more advanced liver disease in individuals infected with HCV [43]. However, the accurate molecular mechanism of liver carcinogenesis is not yet clearly understood. The incidence of AFB1-DNA adducts in patients with HBsAg and NBNC (non-HBsAg and non-HCV ab patients with hepatitis B core ab) was found to be significantly higher than in the patients with HCV ab. The hepatocarcinogenic effect of AFB1 might be associated with HCC, particularly in patients with HBV and in patients without HBsAg and HCV ab, despite low AFB1 exposure [44]. Most patients with HCC and HBV infection had prevalent GC ? TA transversion mutation at the third position of codon 249 of the p53 gene. The HBV X protein of HBV also promotes cell cycle progression, increases the expression of telomerase reverse transcriptase, inactivates negative growth regulators and binds to and inhibits the expression of p53 (anti-apoptotic activity) and other tumour suppressor genes and senescence-related factors [16]. International expert committees concluded that even very low levels of exposure to AFs, that is 1 ng/kg bw per day still contribute to the risk of liver cancer [45]. Tchana et al. [7] showed that AFB1 was present in the 63.9% of the serum samples obtained from patients with liver diseases (n = 36) in Cameroon using HPLC. To our knowledge, there is not enough study in the literature evaluating the AF levels in patients with chronic hepatitis in Turkey. Our study is the first to assess the blood AF levels in patients with chronic hepatitis related to cirrhosis and HCC in Balıkesir, Turkey. Although the present study contributes to the literatures by presenting the AF levels of the patients and healthy controls living in Balıkesir, it has some limitations: In our study, the number of the subjects could have been increased. The analyses of AFs contamination of foods consumed in Balıkesir could have given more strength to the current study, where this would reflect the relation between the consumption of AFs contaminated food and blood AFs levels. In the present study, we determined AF levels in serum samples after the digestion of serum protein and this provided instantaneous © 2015 John Wiley & Sons Ltd
Aflatoxin in chronic hepatitis B patient monitoring of AF exposure. The highest AFs (AFB1, AFG1, AFB2, AFG2) concentration and the highest risk in relation to AFs exposure was found in patients with HCC. AFB1 levels in patients with HCC were found to be significantly higher than patients with CHB, cirrhosis and also healthy controls (P < 0.05). In Turkey, the cancer incidence has been increased over the years. The liver cancer is the eleventh common type of cancer in males, affecting 2.1 (per hundred thousand) males, while in females it is the 15th common type of cancer, affecting 1.3 (per hundred thousand) females in Turkey [46]. In Izmir, the major commercial and industrial centre of the Aegean region and near to Balıkesir in western Turkey, age standardized incidence rates (world standard) of liver cancer for males and females were reported to be 1.5 and 0.8, respectively. The rough rate of liver carcinoma determined by a direct method using the world standard population for males and females was reported to be 1.3 (per hundred thousand) and 0.7 (per hundred thousand), respectively [47]. The incidence of liver cancer in Izmir was rather low; however, HBV and HCV and AFs are major risk factors for HCC [1] and the international incidence pattern of liver cancer correlates with the prevalence of HBV infection. AFs exposure is also low in Izmir, although no information on the exposure levels is available and the exposure of AF in the population living in the region may be one of the factors affecting the induction of liver cancer. In a cross-sectional observational and a single-centre study (between January 2006 and June 2007),
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it was reported that in patients with viral hepatitis from Ankara University, Gastroenterology Clinic in Turkey, AF exposure was significantly higher than in the healthy subjects and it was suggested to play an important role in the development of HCC [48]. Mokhles et al. [40] reported that there was a higher statistical significance of AF prevalence and concentration in serum of cirrhotic than patients with HCC and control. In conclusion, mycotoxins have a negative effect both on public health and on international trade of foodstuffs. It is not clear how these toxins are metabolized in human; therefore, how well they predict exposure remains unknown. The population living in Turkey is continuously exposed to AFs, and the importance of overall daily dietary intake of AFs in patients with CHB should not be underestimated because of possible risk in the development of HCC. It is important to determine AFs exposure for the patients with hepatitis. Further studies on the viral hepatitis or other diseases and AFs exposure should be performed to clarify the toxicity mechanisms of AFs.
CONFLICT OF INTEREST The authors declare that there are no conflict of interests.
FUNDING This work was supported by Hacettepe University Research Fund (Contract grant number: 012D12301001).
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Aflatoxin in chronic hepatitis B patient 45 European Food Safety Authority. Opinion on the scientific panel on contaminants in the food chain on a request from the commission related to the potential increase of consumer health risk by a possible increase of the existing maximum levels for aflatoxins in almonds, hazelnuts and pistachios and
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doi:10.1111/jvh.12410
Aflatoxin levels in chronic hepatitis B patients with cirrhosis or hepatocellular carcinoma in Balıkesir, Turkey M. Aydın,1 S. Aydın,2 M. Bacanlı2 and N. Basßaran2
1
Department of Infectious Diseases and Clinical Microbiology,
Faculty of Medicine, Istanbul Hospital of Basßkent University, Istanbul, Turkey; and Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey 2
Received August 2014; accepted for publication February 2015
SUMMARY. Aflatoxins, the secondary metabolites produced
by species of naturally occurring Aspergilli, are commonly found in food such as cereals, dried fruits and juice, wine, beer and spices. They are hepatotoxic and are well known human carcinogens based on evidence from human studies. Aflatoxins are an environmental risk factor for the development of hepatocellular carcinoma (HCC). Chronic hepatitis B-infected patients are at increased risk of cirrhosis, hepatic failure and liver cancer. This study was designed to determine the serum aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) concentrations using high-pressure liquid chromatography (HPLC) in hepatitis B-infected patients with or
INTRODUCTION Aflatoxins (AFs), formed by several fungus species of the genus Aspergilli, are naturally occurring contaminants of agricultural crops, and owing to their adverse health effects, contamination of food by AFs is of global importance [1,2]. The four major types of AFs are aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2). AFB1 is one of the most potent carcinogens occurring naturally and classified as carcinogenic to humans by the International Agency for Research on Cancer (IARC). IARC concluded that there was sufficient evidence in experimental animals for the carcinogenicity of naturally occurring mixtures of AFB1, AFG1 and AFM1; Abbreviations: Ab, antibodies; ACN, acetonitrile; AF, aflatoxin; AFP, alpha-fetoprotein; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CHB, chronic hepatitis B; ELISA, enzyme-linked immunosorbent assay; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HPLC, high-pressure liquid chromatography; IARC, International Agency for Research on Cancer; MeOH, methanol; TFA, trifluoroacetic acid; UHT, ultra-high temperature. Correspondence: Mehtap Aydın, Department of Infectious Diseases and Clinical Microbiology, Istanbul Hospital of Basßkent University, Istanbul 34662, Turkey. E-mail: [email protected]
without cirrhosis and liver cancer, alongside healthy controls in Balıkesir, Turkey. The mean AFB1 and total AF levels in patients without liver cancer and cirrhosis were significantly higher than healthy controls. The mean AFB1 and total AF levels in patients with chronic hepatitis B and HCC were significantly higher than infected patients with or without cirrhosis. These results suggest that patients with chronic hepatitis B who are exposed to AFs are at increased risk for developing HCC, which might be prevented by reducing consumption of contaminated foods. Keywords: aflatoxins, chronic hepatitis B, cirrhosis, hepatocellular carcinoma, Turkey.
limited evidence for the carcinogenicity of AFB2; and inadequate evidence for the carcinogenicity of AFG2 [1]. The toxicological importance of AFs has been extensively studied. The primary target organ for AF toxicity and carcinogenicity in humans and animals is the liver. AFB1 is metabolized in the liver by cytochrome P450 system to the highly reactive AFB1-8,9-epoxide, which is responsible for the toxicity [3]. The metabolic effects of AFs include inhibition of DNA, RNA and protein synthesis; reduction in miscellaneous enzyme activities; depression of glucose metabolism; inhibition of lipid synthesis, and depression of clotting factor synthesis [4]. Experimental animals infected with hepatitis B virus (HBV) were reported to be more sensitive to the carcinogenic effects of AF than uninfected animals confirming the carcinogenic potential of AFs in hepatitis B-infected animals [1]. Some epidemiological studies have also shown that AF exposure particularly in combination with HBV is associated with an increased risk of hepatocellular carcinoma (HCC). The potency of AFs appears to be significantly enhanced in individuals with HBV infection [5–7]. The correlation between AFs exposure and liver cancer incidence in some areas, especially in Africa and Asia has been determined [1,8]. Due to their frequent occurrence and severe toxicity, guidelines and tolerance levels of AFs have been set in several countries including Turkey [9]. Turkey has encountered AFs contamination in © 2015 John Wiley & Sons Ltd
Aflatoxin in chronic hepatitis B patient
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AF standards (AFG1, AFB1, AFG2, and AFB2) were obtained from Sigma Chemical Co. Stock solutions and standards were prepared and analysed according to AOAC Method 971.22 [17].
of the nature of the intrahepatic vessel (score 1–3), liver parenchyma (score 1–3), liver surface (1–3) and the size of the spleen (score 1–2). A spleen size of 12 cm or more was given score of 2. A total score of seven or more was indicative of cirrhosis. HCC was confirmed using elevated serum AFP levels (>200 ng/mL) with lesions occupying spaces on ultrasound characteristic of HCC (n = 20). At the time of recruitment, each study subject was interviewed to obtain information on socio-demographic characteristics, lifestyle, height, bodyweight, lifetime history of cigarette smoking and alcohol consumption, personal and family history of various chronic diseases followed by standardized clinical examination. The cumulative exposure to cigarette was calculated as ‘pack-years’ using the following formula: (number of years smoked 9 average number of cigarette smoked per day)/20. Total alcohol intake was categorized as three levels (0, 60 g/day) [20]. According to the answers obtained from the questionnaires, dietary habits of the study group are about the same. They consume more vegetables, fruits, and meat as well as locally produced food than cereals including corn and corn products. The study was approved by an ethical committee (HEK 11/118) according to the ‘Declaration of Helsinki.’ All subjects participated in the study voluntarily and written consent (in Turkish) was obtained before blood samples were drawn. Whole blood samples (7 mL) were taken from cases at the time of admission to hospital and collected from the controls at the time of recruitment. The samples were centrifuged at 800 9 g for 15 min and the serum was separated. All samples were aliquoted and stored at –20 °C until analysis.
Subjects and sampling
Biochemical analysis
A total of 38 (female:male, 16:22) CHB patients without cirrhosis and HCC (CHB patients), 26 (female:male, 10:16) CHB patients with cirrhosis (cirrhosis patients), 20 (female:male, 7:13) CHB patients with HCC (HCC patients) and 49 (female: male, 25:24) healthy controls from Balıkesir State Hospital and Balıkesir University Hospital between March 2013 and March 2014 were recruited to this retrospective study. The exclusion criteria for the study group included decompensated liver disease, haematologic disease, immunologically mediated disease, several cardiac diseases and chronic pulmonary disease. The inclusion criteria for healthy controls were as follows: no use of medications, no pregnancy or lactation for female volunteers, normal liver and renal function tests and electrocardiograms, negative for serum alpha-fetoprotein (AFP), negative hepatitis B surface antigen (HBsAg) test and negative hepatitis C virus (HCV) infection by anti-HCV test. The persistence of HBsAg for 6 months from its first detection denotes CHB infection [18]. Cirrhosis was evaluated using a validated ultrasound scoring scale as described originally by Lin et al. [19]. The scoring system consisted
The serum was collected and examined for aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total bilirubin by spectrophotometric analysis as indicative of hepatic function using standard diagnostic kits (Roche Diagnostics, Mannheim, Germany) and a Roche modular P800 clinical chemistry analyzer.
different foods [10,11], and healthy Turkish adults living in some regions of Turkey have been reported to be continuously exposed to AFs, particularly in the summer [12]. Chronic hepatitis B (CHB) is an infection of the liver caused by the oncogenic DNA virus HBV [13]. The prevalence of HBV infection in Turkey varies between 2% and 8% [14]. HCC is an important health problem in Turkey, and HBV infection is an important predisposing factor [15]. Dietary exposure to AFB1 and chronic infection of HBV are major risk factors in HCC, raising the possibility of a synergistic interaction between them [16]. The aim of this study was to determine the serum AFs levels in CHB patients with or without cirrhosis and HCC and to evaluate the relationships between AFs levels and these diseases.
MATERIALS AND METHODS Chemicals and reagents HPLC grade methanol (MeOH), acetonitrile (ACN) were purchased from Riedel (Poole, Dorset, UK). All other chemicals and solvents were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Standards
© 2015 John Wiley & Sons Ltd
Determination of viral serology Serum samples were analysed for HBsAg antibodies (ab) according to the standard laboratory operating procedures at the Balıkesir State Hospital. Qualitative detection of HBsAg, a marker of chronic HBV carriage, in human serum was performed using commercially available enzyme-linked immunosorbent assay (ELISA; Alpco Diagnostics, Salem, NH, USA) [21].
Extraction of serum samples After digestion of serum protein [22], the extraction method modified from Nelson et al. [23] was performed.
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One millilitre of serum sample was diluted with 2 mL of nhexane and mixed for 1 min. After the centrifugation at 5000 9 g for 5 min, upper n-hexane phase containing serum lipids was removed. This process was repeated twice. One millilitre of chloroform was added to the serum and after shaking vigorously for 0.5 and 4.5 min, successively, the solution was centrifuged again at 5000 9 g for 10 min. The bottom chloroform layer was collected. This process was repeated for three more times. The chloroform phases were collected in a tube and evaporated under a nitrogen stream to complete dryness.
Determination of AFG1, AFB1, AFG2 and AFB2 levels by HPLC method The dry residue was subjected to the trifluoroacetyl (TFA) derivatization to improve fluorescence properties according to the procedure of AOAC method 971.22 [17]. Determination of AFB1, AFB2, AFG1 and AFG2 levels in the derivatized standards and samples were carried out by HPLC equipped with an auto sampler (Hewlett Packard [HP] Agilent 1100 Series, Vienna, Austria) using a fluorescence detector (excitation at 360 nm, emission at 430 nm). A Spherisorb S5ODS2 column (3.8 mm i.d. and length 25 cm, 5 lm particle size, Waters, Milford, MA, USA) was used. The mobile phase was deionized water:ACN:MeOH (62:16:22, v:v:v), and the flow rate was 1 mL/min. The injection volume was 100 lL. AF standards used were ranged between 5 and 1000 pg/mL. The retention times for the AFs were 6.2 min for AFG1, 8.2 min for AFB1, 11.8 min for AFG2 and 17.0 min for AFB2. Recovery studies were performed on blank samples of serum spiked with levels of 0.1, 0.25 and 0.5 ng/mL for each AF standard and repeated for three times. The average recoveries were 69.7% for AFG1, 79.6% for AFB1, 101.3% for AFG2 and 107.4% for AFB2. The concentrations of AFs in the samples were calculated using the calibration curves of peak area prepared for each AF standard separately. The detection limits were determined as 5 pg/mL for AFG1 and AFB1; 2.5 pg/mL for AFG2 and AFB2. The quantification limits were determined as 35.2 pg/mL for AFG1, 21.2 pg/ mL for AFB1, 60.5 pg/mL for AFG2 and 34.85 pg/mL for AFB2.
Statistical analysis All data were analysed using SPSS software version 15.0 (SPSS Inc., Chicago, IL, USA). The distribution of the data was checked for normality using the Shapiro–Wilk test. The homogeneity of the variance was verified by the Levene test. To test for significant differences between variables, the one-way analysis of variance and the Kruskal– Wallis one-way analysis of variance were used for normally and non-normally distributed continuous data, respectively. Chi-square or Fisher’s exact test was used for
categorical data. Kolmogorov–Smirnov test was used to test continuous data for normality. The Pearson correlation method for normally distributed variables and Spearman rank correlation for non-normally distributed variables were used to determine the linear relationship between the variables. A two-tailed P value of 0.05). Total pack-years of smoking of patients with cirrhosis (14.31 5.65) and patients with HCC (21.36 5.05) were significantly higher than patients with CHB (7.91 1.32) and healthy controls (11.07 6.10) (P < 0.05). There were no significant differences between patients with CHB and healthy controls for total pack-years of smoking. There were no differences between the groups for lower ( 60 g/day) intakes of alcohol. There were no differences between the groups for the history of cancer from first-degree relatives (parents, offspring and siblings). The positive history of cancer from first-degree relatives of patients with CHB, patients with cirrhosis, HCC patients with the highest value and healthy controls were 10.5%, 11.5%, 25.0%, and 8.2%, respectively. Among 16 cases with first-degree family history of cancer in the study group, there were 10 liver cancers (62.50%), three rectum cancers (18.75%), two lung cancers (12.50%) and one gastric cancer (6.25%). There were no correlations and relationships between the dependent variables (Total AF levels) and independent variables (age, r = 0.096; body mass index (BMI, kg/m2), r = 0.158; total pack-years of smoking, r = 0.136; alcohol consumption, r = 0.186; and first-degree family history of cancer, r = 0.198).
Evaluation of biochemical parameters The biochemical parameters of study group are presented in Table 2. Patients with cirrhosis or HCC had significantly elevated AST levels vs patients with CHB and healthy controls (P < 0.05). There were no significant differences © 2015 John Wiley & Sons Ltd
© 2015 John Wiley & Sons Ltd CHB patients (n = 38) 60.20 9.05 21.08 2.54a
52.85 6.12 24.74 1.06a 7 (35.0)a 13 (65.0)a 11 (55.0)a 9 (45.0)a 21.36 5.05b 15 (75.0)a 4 (20.0)a 1 (5.0)a 5 (25.0)a 15 (75.0)a
10 (38.5)a 16 (61.5)a 16 (61.5)a 10 (38.5)a 14.31 5.65b 20 (76.9)a 4 (15.4)a 2 (7.7)a 3 (11.5)a 23 (88.5)a
b
HCC patients (n = 20)
b
Cirrhosis patients (n = 26)
4 (8.2)a 45 (91.8)a
39 (79.6)a 8 (16.3)a 2 (4.1)a
21 (42.9)a 28 (57.1)a 11.07 6.10a
25 (51.0)a 24 (49.0)a
44.49 10.12a 23.52 1.69a
Healthy controls (n = 49)
SD, standard deviation; HCC, Hepatocellular carcinoma; CHB, chronic hepatitis B. Superscripts of different letters differ significantly (P < 0.05) from each other in the same line.
a
Age (years) (mean SD) 43.50 7.30 Body mass index (kg/m2) (mean SD) 22.96 0.67a Gender [n(%)] Female 16 (42.1)a Male 22 (57.9)a Current cigarette smoker [n (%)] Yes 20 (52.6)a No 18 (47.4)a Total pack-years of smoking (mean SD) 7.91 1.32a Alcohol intake [n (%)] 0 28 (73.7)a 60 g/day 2 (5.3)a History of cancer from first-degree relatives [n (%)] Yes 4 (10.5)a No 34 (89.5)a
Factor
Table 1 Demographic characteristics of the study group
Aflatoxin in chronic hepatitis B patient 929
79.15 41.72 40.65 21.45a 0.79 0.32a AST (IU/L) ALT (IU/L) Total bilirubin (mg/dL)
AST, aspartate aminotransferase; ALT, alanine aminotransferase; HCC, Hepatocellular carcinoma; CHB, chronic hepatitis B. Superscripts of different letters differ significantly (P < 0.05) from each other in the same line. The results were given as mean SD (standard deviation).
51.53 29.61a 25.57 16.95c 0.72 0.27a 100.30 46.12 49.35 21.24a 0.74 0.30a 132.62 64.06 68.31 39.05b 1.63 0.51b
b
HCC patients (n = 20)
b a
CHB patients (n = 38)
Cirrhosis patients (n = 26)
Healthy controls (n = 49)
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Table 2 Biochemical parameters of the study group
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between patients with CHB and healthy controls for AST levels. Alanine aminotransferase levels of patients with CHB were higher than healthy controls (P < 0.05). ALT levels in patients with cirrhosis were statistically higher than patients with CHB and patients with HCC. There were no significant differences between patients with CHB and patients with HCC for ALT levels. Total bilirubin levels in patients with cirrhosis were statistically higher than patients with CHB, patients with HCC and healthy controls (P < 0.05). There were no significant differences between patients with CHB, patients with HCC and healthy controls for total bilirubin levels.
Evaluation of serum aflatoxin levels There were no significant differences between the groups for the percentage of AF-positive samples (Table 3). The percentages of AF B1-positive samples in healthy controls, patients with CHB, patients with cirrhosis and patients with HCC were 26.5%, 21.1%, 26.9% and 35.0%, respectively. For the detectable serum AF B1 levels, the odds ratio for patients with CHB, patients with cirrhosis and patients with HCC were 0.74 (0.27–2.02), 1.02 (0.35– 2.99) and 1.49 (0.49–4.55), respectively. Aflatoxin levels in the AF-positive samples of study group are given in Table 4. The mean AFB1 level of patients with CHB was significantly higher than healthy controls (P < 0.05). Among the patients, the mean AFB1 level of patients with HCC was significantly higher than patients with CHB and patients with cirrhosis (P < 0.05). Among the patients, the mean AFB2 levels of patients with CHB and patients with HCC were significantly higher than patients with cirrhosis (P < 0.05). The mean AFG1, AFB2 and AFG2 levels of each patient group were significantly higher than healthy controls (P < 0.05). The highest AFB1, AFG1, AFB2 and AFG2 levels were found in patients with HCC. Total AF levels in patients with CHB, patients with cirrhosis and patients with HCC were 119.4%, 82.2% and 169.4% higher than healthy controls, respectively (Fig. 1).
DISCUSSION Aflatoxins have carcinogenic, teratogenic, and mutagenic effects. It is well documented that contamination of foods with AFs may cause liver cancer in humans [7,8]. AFs may also affect the epidemiology of many diseases and have health risks in the countries where the toxin is uncontrolled [3]. The contamination of food with AFs is a major concern. Major risks are inevitable climatic conditions such as unseasonable rains or high humidity [22,24]. Where trade does occur, the least contaminated foods and feeds are exported, which may lead to enhanced exposure of © 2015 John Wiley & Sons Ltd
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Table 3 Determination of aflatoxin-positive samples and the risk in relation to AF exposure in the cases and controls
Healthy controls; n = 49 (%) CHB patients; n = 38 (%) P OR (95% CI) Cirrhosis patients; n = 26 (%) P OR (95% CI) HCC patients; n = 20 (%) P OR (95% CI)
Af B1
Af G1
Af B2
Af G2
13/49 (26.5) 8/38 (21.1) 0.554 0.74 (0.27–2.02) 7/26 (26.9) 0.971 1.02 (0.35–2.99) 7/20 (35.0) 0.483 1.49 (0.49–4.55)
10/49 (20.4) 5/38 (13.2) 0.378 0.59 (0.18–1.90) 6/26 (23.1) 0.789 1.17 (0.37–3.68) 7/20 (35.0) 0.483 1.49 (0.49–4.55)
13/49 (26.5) 8/38 (21.1) 0.554 0.74 (0.27–2.02) 7/26 (26.9) 0.971 1.02 (0.35–2.99) 7/20 (35.0) 0.483 1.49 (0.49–4.55)
10/49 (20.4) 5/38 (13.2) 0.378 0.59 (0.18–1.90) 6/26 (23.1) 0.789 1.17 (0.37–3.68) 7/20 (35.0) 0.483 1.49 (0.49–4.55)
HCC, Hepatocellular carcinoma; CHB, chronic hepatitis B; OR, odds ratio; CI, confidence interval. Superscripts of different letters differ significantly (P < 0.05) from each other in the same column. Table 4 Aflatoxin levels in the aflatoxin-positive samples of the study group
AF B1 (pg/mL) [n (%)] AF G1 (pg/mL) [n (%)] AF B2 (pg/mL) [n (%)] AFG2 (pg/mL) [n (%)]
CHB patients
Cirrhosis patients
HCC patients
Healthy controls
53.96 11.72a 8/38 (21.1) 22.80 8.51a 5/38 (13.2) 23.72 7.47a,b 8/38 (21.1) 9.33 1.69a 5/38 (13.2)
41.09 6.77a 7/26 (26.9) 23.00 14.31a 6/26 (23.1) 16.84 6.17a 7/26 (26.9) 10.25 2.67a 6/26 (23.1)
63.11 13.02b 7/20 (35.0) 28.79 9.01a 7/20 (35.0) 28.17 4.00b 7/20 (35.0) 14.77 4.82a 7/20 (35.0)
18.36 10.64c 13/49 (26.5) 11.51 3.94b 10/49 (20.4) 9.98 4.76c 13/49 (26.5) 5.71 2.25b 10/49 (20.4)
HCC, Hepatocellular carcinoma; CHB, chronic hepatitis B. Superscripts of different letters differ significantly (P < 0.05) from each other in the same line. The results were given as mean SD (standard deviation).
Fig. 1 Total aflatoxin levels in the aflatoxin-positive samples of patients with CHB, patients with cirrhosis, patients with HCC and healthy controls. Bars that do not share same letters (superscripts) are significantly different from each other (P < 0.05). The results were given as Mean SD (standard deviation). HCC, Hepatocellular carcinoma; CHB, chronic hepatitis B.
the producers, because the more highly contaminated products are retained at home for consumption by a population that is already at the greatest risk of AF exposure © 2015 John Wiley & Sons Ltd
[25]. Although AFM1, the main monohydroxylated derivate of AFB1, is less carcinogenic and mutagenic than AFB1, it exhibits a high level of genotoxic activity and certainly represents a health risk because of its possible accumulation and linkage to DNA [26]. The levels of AFM1 in 30 (73.2%, n = 30/41) samples of ultra-high temperature (UHT) milk randomly collected from different supermarkets in Burdur, near to Balıkesir during 2011 were found to be 6.42 to 71.33 ng/L (mean level, 17.76 ng/L), and only three (7.3%, n = 3/41) samples of UHT milk were contaminated at levels above the Turkish legal limit (50 ng/L) [27]. There are some studies indicating that the Turkish population is continuously exposed to AFs. Giray et al. [11] reported that the total AFs levels were ranged between 10.4 and 643.5 ng/kg in the 41 wheat samples grown and consumed in some regions of Turkey. Fifty-nine percentage of the samples were positive for total AFs. The percentage of positive samples for AFB1, AFB2, AFG1 and AFG2 were 42%, 12%, 37% and 12%, respectively, although the detected levels were under the permitted levels for AFs in cereals [11]. Total AF levels in corn samples
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from various street bazaars and market outlets in different regions of Turkey were found to be ranged between 1.75 and 120.3 lg/kg, and 4% of the samples contained AFs above the acceptable limit of 10 lg/kg in Turkey [28]. Kabak [29] analysed the 85 walnut samples collected between October 2012 and April 2013 in different provinces of Turkey for the presence of AFs. The daily intakes of AFB1 and total AFs from walnuts for Turkish consumers were estimated to be 0.020 and 0.026 ng/kg bw per day, respectively. Hacıbekiro glu and Kolak [30] reported that 76% of 62 food samples from Istanbul, Turkey, exceeded legal limits of total AF, and the highest levels were found in chestnut (232.9 lg/kg), nutmeg (206.1 lg/kg) and sumac (182.5 lg/kg). The concentrations of the total AFs, AFB1, AFB2 and AFG1 in the three different corn flour samples purchased from different markets of Balıkesir, Turkey, were found to be 1.08 0.02, 0.59 0.01, 0.16 0.01 and 0.34 0.013 lg/kg, respectively [31]. A number of approaches have been used to determine AF exposure in human populations, including analysis of AF metabolites and AF-DNA or AF-protein adducts [32– 34]. However, these studies are more complicated and expensive. Generally, AFB1 in blood is not recognized as a biomarker compared to these adducts and there is limited data validating this as a biomarker. The detection of AF B2, AF G1 and AFG2 subtypes, determined at low levels in food, has not been confirmed as a sensitive method in blood for exposure. There are a few studies in literature demonstrating the AF levels directly in biological samples [12,23,35]. Corcuera et al. [36] reported that the level of AFB1 in plasma from rats given a single dose of 0.25 mg/ kg of AFB1 by oral gavage was 24.8 ng/mL and 9.5 ng/ mL at 10 min and 30 min, respectively. Studies on human serum samples mostly have limited sample sizes vs the current study. In a study performed by Hassan et al. [37], they measured the AFB1 levels in the serum and milk of mothers (n = 50) and serum of infants (n = 50) in Egypt. Twenty-four of fifty mothers and their infants had been contaminated with AF with the following mean contamination levels of 8.9 4.2 ng/mL (mothers’ serum), 1.9 0.6 ng/mL (mothers’ milk) and 1.8 0.9 ng/mL (infants’ serum). In a study conducted in UK using ELISA, AF levels of blood donors (n = 27) were found not to be higher than 20 pg/mL and it was reported that present UK guideline tolerances for AF in imported food were effective in limiting human exposure to toxic AFs in the UK diet [38]. Hepatocellular carcinoma is one of the most common malignancies worldwide. There is a marked geographical variation in the incidence of HCC due to the various aetiological factors including viral (HBV and HCV), chemical and several other factors such as AFs, alcohol, cigarette smoke and genetic tendencies [39]. AFB1 has been suggested to play a causative role in the aetiology of HCC. The correlations between human HCC and food
contamination with AF have been reported in various studies [6,7,35,40]. A number of mechanisms have been suggested, the most widely accepted of which is that HBV infection sensitizes hepatocytes to the carcinogenic effects of AFB1 [41]. Prospective studies in Shanghai, China, showed that urinary excretion of AF metabolites increased the risk of HCC up to fourfold, and HBV infection increased the risk sevenfold. However, individuals who excreted AFB1 metabolites and were carriers of HBV had as much as a 60-fold increase in risk of HCC [42]. In comparison with the study of the interaction between AFs and HBV, there has been little focus on the potential of HCV to cause HCC. AFs also have a synergistic effect on HCV-induced liver cancer. A study in Taiwan suggested that AF-albumin adducts were associated with more advanced liver disease in individuals infected with HCV [43]. However, the accurate molecular mechanism of liver carcinogenesis is not yet clearly understood. The incidence of AFB1-DNA adducts in patients with HBsAg and NBNC (non-HBsAg and non-HCV ab patients with hepatitis B core ab) was found to be significantly higher than in the patients with HCV ab. The hepatocarcinogenic effect of AFB1 might be associated with HCC, particularly in patients with HBV and in patients without HBsAg and HCV ab, despite low AFB1 exposure [44]. Most patients with HCC and HBV infection had prevalent GC ? TA transversion mutation at the third position of codon 249 of the p53 gene. The HBV X protein of HBV also promotes cell cycle progression, increases the expression of telomerase reverse transcriptase, inactivates negative growth regulators and binds to and inhibits the expression of p53 (anti-apoptotic activity) and other tumour suppressor genes and senescence-related factors [16]. International expert committees concluded that even very low levels of exposure to AFs, that is 1 ng/kg bw per day still contribute to the risk of liver cancer [45]. Tchana et al. [7] showed that AFB1 was present in the 63.9% of the serum samples obtained from patients with liver diseases (n = 36) in Cameroon using HPLC. To our knowledge, there is not enough study in the literature evaluating the AF levels in patients with chronic hepatitis in Turkey. Our study is the first to assess the blood AF levels in patients with chronic hepatitis related to cirrhosis and HCC in Balıkesir, Turkey. Although the present study contributes to the literatures by presenting the AF levels of the patients and healthy controls living in Balıkesir, it has some limitations: In our study, the number of the subjects could have been increased. The analyses of AFs contamination of foods consumed in Balıkesir could have given more strength to the current study, where this would reflect the relation between the consumption of AFs contaminated food and blood AFs levels. In the present study, we determined AF levels in serum samples after the digestion of serum protein and this provided instantaneous © 2015 John Wiley & Sons Ltd
Aflatoxin in chronic hepatitis B patient monitoring of AF exposure. The highest AFs (AFB1, AFG1, AFB2, AFG2) concentration and the highest risk in relation to AFs exposure was found in patients with HCC. AFB1 levels in patients with HCC were found to be significantly higher than patients with CHB, cirrhosis and also healthy controls (P < 0.05). In Turkey, the cancer incidence has been increased over the years. The liver cancer is the eleventh common type of cancer in males, affecting 2.1 (per hundred thousand) males, while in females it is the 15th common type of cancer, affecting 1.3 (per hundred thousand) females in Turkey [46]. In Izmir, the major commercial and industrial centre of the Aegean region and near to Balıkesir in western Turkey, age standardized incidence rates (world standard) of liver cancer for males and females were reported to be 1.5 and 0.8, respectively. The rough rate of liver carcinoma determined by a direct method using the world standard population for males and females was reported to be 1.3 (per hundred thousand) and 0.7 (per hundred thousand), respectively [47]. The incidence of liver cancer in Izmir was rather low; however, HBV and HCV and AFs are major risk factors for HCC [1] and the international incidence pattern of liver cancer correlates with the prevalence of HBV infection. AFs exposure is also low in Izmir, although no information on the exposure levels is available and the exposure of AF in the population living in the region may be one of the factors affecting the induction of liver cancer. In a cross-sectional observational and a single-centre study (between January 2006 and June 2007),
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it was reported that in patients with viral hepatitis from Ankara University, Gastroenterology Clinic in Turkey, AF exposure was significantly higher than in the healthy subjects and it was suggested to play an important role in the development of HCC [48]. Mokhles et al. [40] reported that there was a higher statistical significance of AF prevalence and concentration in serum of cirrhotic than patients with HCC and control. In conclusion, mycotoxins have a negative effect both on public health and on international trade of foodstuffs. It is not clear how these toxins are metabolized in human; therefore, how well they predict exposure remains unknown. The population living in Turkey is continuously exposed to AFs, and the importance of overall daily dietary intake of AFs in patients with CHB should not be underestimated because of possible risk in the development of HCC. It is important to determine AFs exposure for the patients with hepatitis. Further studies on the viral hepatitis or other diseases and AFs exposure should be performed to clarify the toxicity mechanisms of AFs.
CONFLICT OF INTEREST The authors declare that there are no conflict of interests.
FUNDING This work was supported by Hacettepe University Research Fund (Contract grant number: 012D12301001).
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