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Novel adenine adducts, N7-guanine-AFB1 adducts, and p53 mutations in patients with schistosomiasis and aflatoxin exposure
Cancer Detection and Prevention 30 (2006) 491–498 www.elsevier.com/locate/cdp
Novel adenine adducts, N7-guanine-AFB1 adducts, and p53 mutations in patients with schistosomiasis and aflatoxin exposure Samy L. Habib PhDa,*, Boctor Said PhDa, Ahmed T. Awad MDb, Mostafa H. Mostafa PhDc, Ronald C. Shank PhDa a
Department of Community and Environmental Medicine, College of Medicine, University of California at Irvine, Irvine, CA 92697, USA b Department of Surgery, College of Medicine, Alexandria University, Egypt c Institute of Graduate Studies and Research, University of Alexandria, Alexandria, Egypt Accepted 10 October 2006
Abstract Introduction: The most frequent mutation in human hepatocellular carcinoma (HCC) in populations exposed to a high dietary intake of aflatoxin B1 (AFB1) is a mutation in codon 249 of the p53 gene. Schistosomiasis is known to cause p53 mutation. We hypothesized that the combination of schistosomiasis and aflatoxin B1 increases the incidence of p53 gene mutation. Methods: Liver tissue from 21 patients with schistosomiasis and 5 patients without schistosomiasis were analyzed for occurrence of mutations of the p53 gene and levels of N7-guanineAFB1 adducts. Results: The presence of mutations in codon 249 of p53 gene was higher in patients infected with Schistosoma haematobium (S. haematobium) than in those infected with Schistosoma mansoni (S. mansoni) or a combination of both strains ( p < 0.01), compared to control subjects. No mutations were detected in p53 gene in liver DNA from schistosomiasis-free patients. Significant amounts of N7-guanineAFB1 adducts and novel adenine-adducts ( p < 0.01) were detected in patients with schistosomiasis, mostly in patients infected with S. haematobium or a combination of both strains, compared to control subjects. Conclusion: These data suggest that schistosomiasis and exposure to aflatoxin B1 act synergistically to increase the incidence of p53 gene mutation. The increase in p53 mutations may enhance progression of HCC at an early age in patients with schistosomiasis. # 2006 International Society for Preventive Oncology. Published by Elsevier Ltd. All rights reserved. Keywords: Novel adenine-adducts; N7-guanine-AFB1adducts; P53 mutation; Schistosomiasis; Synergism; Aflatoxin B1; S. mansoni; S. hematobium; Cytochrome P-450; PCR
1. Introduction Exposure to dietary aflatoxin B1 (AFB1), hepatitis virus or schistosomal infections is associated with an increased risk of hepatocellular carcinoma (HCC) in humans. Synergistic effects of multiple risk factors in the development of HCC by chronic liver disease are major contributors to carcinogenesis [1–3]. Hepatocellular carcinoma is the fifth most common cancer in the world with 80% of cases occurring in developing * Corresponding author at: The University of Texas Health Science Center, Department of Medicine, Division of Nephrology-MSC 7882, 7703 Floyd Curl Dr., San Antonio, TX 78229, USA. Tel.: +1 210 567 4699; fax: +1 210 567 4712. E-mail address: [email protected] (S.L. Habib).
countries. Liver fibrosis is the main cause of death in the 20 million individuals suffering from chronic schistosomiasis [4–6]. In Egypt, hepatitis B and C viruses, (HBV and HCV) and schistosomiasis constitute the most common causes of chronic liver disease [7,8]. Schistosomiasis increases the severity of HBV infection and the risk of HCC over that associated with the HBV infection alone [9]. A recent survey of aflatoxin contamination in Egyptian foods indicated that there is a high prevalence of AFB1 in herbs, medicinal plants, cereals, spices, nuts, seeds and vegetables which may increase the incidence of liver cancer [10]. Schistosoma mansoni (S. mansoni) causes liver fibrosis, intestinal carcinoma and splenic lymphoma while Schistosoma haematobium (S. haematobium) causes urinary schistosomiasis leading to bladder cancer [11,12]. An
0361-090X/$30.00 # 2006 International Society for Preventive Oncology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cdp.2006.10.006
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association between S. japonicum and HCC was reported in a Japanese necropsy study [13,14]. In addition, a relationship between S. mansoni infection and HCC was found in patients with schistosomiasis in Saudi Arabia [15]. Clinical and pathological studies of S. haematobium or S. mansoni have been performed on animal models to evaluate their carcinogenic effects. Infection with S. mansoni in mice was found to increase the risk of hepatic carcinoma associated with administration of the liver carcinogen, 2-amino-5azotoluene [16]. Similarly, in the case of S. haematobium infection, epithelial hyperplasia and metaplasia were reported in the bladders of parasite-infected mice exposed to aromatic amines such as acetylaminofluorene [17] or 2-naphthylamine [18]. Several epidemiological studies have established a strong association between dietary aflatoxin B1 exposure and the development of primary hepatocellular carcinoma [19–22]. AFB1 is activated by hepatic cytochrome P-450dependent monooxygenases leading to the formation of several forms of AFB1 metabolites [23]. AFB1-8-9epoxide is presumed to be the ultimate mutagen and carcinogen [24]. Upon metabolic activation, the reactive AFB1-8-9-epoxide forms DNA adducts primarily at the N7 position of guanine [25]. Aflatoxin B1 causes a significant inhibition in RNA [26,27] and protein synthesis and inhibits the activity of drug metabolizing enzymes [28,29]. Our previous studies, indicate an association between S. mansoni infections and inhibition of metabolizing enzyme activity in the liver of experimental animals [30] and humans [31]. The p53 gene is a major suppressor gene and it is well suited for analysis of the mutational spectrum in human cancer. Certain p53 mutants lose their suppressor activity and gain oncogenic activity, which is one explanation for the commonality of p53 mutations in human cancer [32]. Patients with HCC living in countries with high aflatoxin exposure have a characteristic mutational spectrum in the p53 gene, specifically, G to T transversion at the third base of codon 249 [33]. The mutation of p53 gene in HCC from African and Asian patients has been associated with high exposure to aflatoxin B1 and high prevalence of HBV infection [34,35]. The p53 gene regulates multiple components of the DNA damage control response and promotes cellular senescence. Consequently, the biological properties of mutated p53 depend mainly on the site and the nature of point mutations. These alterations in p53 gene will consequently modulate the expression of genes that regulate DNA repair and synthesis, cell cycle, cell differentiation and, cell death [36]. There is a strong correlation between infection with schistosoma or exposure to aflatoxin and the site of p53 mutation [37,38]. In the present study, we investigated the effect of the combination of schistosoma and aflatoxin B1 on the mutation of p53 gene. The level of N7-guanine-AFB1 adducts in the liver of patients exposed to aflatoxin contaminated foods was also investigated.
2. Materials and methods 2.1. Liver specimens Liver specimens from patients with schistosomiasis were obtained from the Alexandria General Hospital (Alexandria, Egypt). A total of 21 liver biopsies were obtained from patients with schistosomal hepatic fibrosis during operations for decongestion and/or splenectomy. Five biopsies from splenectomy patients with normal liver constituted the control group. The informed consent of each patient was obtained. All samples were histologically examined confirming the clinical diagnosis of schistosomiasis. Liver tissues were frozen at 80 8C within 20 min of surgical removal. 2.2. DNA isolation DNA was isolated from frozen liver specimens following the protocol described by Strauss [39]. Briefly, frozen liver samples were homogenized in 10 mM Tris–HCl, pH 8.0. Protein and RNA were digested by proteinase K and RNase 1A then deproteinized by successive phenol/chloroform/ isoamyl alcohol extractions. DNA was extracted and recovered by ethanol precipitation. DNA was collected by centrifugation, washed twice with 70% ethanol, and resuspended in 100 ml of 10 mM Tris–HCl, pH 7.4. DNA was separated on a 0.8% agarose gel electrophoresis and quantitated by spectrophotometry. 2.3. In vitro treatment of Calf Thymus -DNA with AFB1-8,9-epoxide The Calf Thymus (CT)-DNA-AFB1-8, 9-epoxide adducts were synthesized as described by Iyer et al. [40]. Briefly, CTDNA (0.5 mg) was reacted (in triplicate) with different concentrations of AFB1-8,9-epoxide (0.1–50 mg) in 10 mM sodium phosphate buffer, pH 7.2 (total volume 200 ml) for 30 min at 10 8C. DNA was recovered by organic solvent extraction and ethanol precipitation. DNA was collected by centrifugation, washed twice with 70% ethanol, and resuspended in 100 ml of 10 mM Tris–HCl, pH 7.4. DNA was recovered by centrifugation and then resuspended in water and heated at 37 8C for 5–10 min to ensure complete dissolution. N7-guanine-AFB1 adducts were prepared from the DNA as described by Essigmann et al. [41]. Samples were subjected to mild acid hydrolysis in 0.1 N HCl for 45 min at 70 8C, to liberate N7-guanine-AFB1 adducts. Hydrolysates were cooled, filtered through a 0.45 mm filter and then analyzed by HPLC. Different concentrations of N7-guanineAFB1 were used to generate the calibration curve by HPLC. 2.4. N7-guanine-AFB1 adducts and novel adenine-adducts assay DNA extracted from control and SHF specimens were subjected to mild acid hydrolysis in 0.1N HCl for 45 min at
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Table 1 Summarized data of human liver samples obtained from patients with schistosoma and schistosoma-free patients Subject
Patients Men
Woman
Men
Woman
Number Mean age Age range S. mansoni-positive (%) S. hematobium-positive (%) S. mansoni + S hematobium-positive (%)
16 38.5 26–46 25 31 44
5 35.5 20–41 60 20 20
3 32 28–34 0 0 0
2 35 32–38 0 0 0
70 8C, to liberate N7-guanine-AFB1 adducts. The level of guanyl-N7-AFB1 adducts in liver DNA was examined by HPLC using strong cation exchange columns (Supelco LC18, 5 mm, 250 mm 4.6 mm, i.d. column; Supelco, Bellefonte, PA). Purines were eluted by using a gradient elution: 5% methanol in 10 mM potassium phosphate buffer, pH 7.0 for 5 min, followed by linear gradient of 5–80% (v/v) methanol in phosphate buffer over 30 min and 80% methanol for 10 min. The flow rate was 1.0 ml/min. Eluted compounds were detected by HPLC using a UV detector set at 360 nm. The amount of guanyl-N7-AFB1 measured in each sample was quantitated using an N7-guanine-AFB1 adducts calibration curve. The ultra-violet absorbing novel adenine-adducts were eluted from the HPLC column at retention time of 14.3 min and the N7-guanine-AFB1 adducts were detected at 25.8 min.
Control
2.7. Synthesis of AFB1-8,9 epoxide AFB1 was purchased from Sigma–Aldrich Chemicals Inc. (St. Louis, MO). Dimethyl-dioxirane was reacted with AFB1 to give the AFB1-exo-8,9-epoxide [42] at room temperature. The epoxide was dissolved at a concentration of 30 mM in methylene chloride. 2.8. Statistics Statistical differences were determined using ANOVA followed by Student Dunnett’s (exp. versus control) test using one trial analysis. p-values less than 0.01 were considered statistically significant.
3. Results 2.5. PCR reaction 3.1. Summary of patient data Polymerase chain reaction (PCR) amplification of the region containing codon 249 was performed using the primers 50 -GTTGGCTCGACTGTACCAC-30 and 50 CTGGAGTCTTCCAGTGTGAT-30 . The PCR reaction mixture contained 2 units of Taq polymerase (Qiagen, CA), 50 pM of each primer, 200 mM of each dNTP and 5 ml of extracted DNA. The PCR procedure was 35 cycles of denaturing for 30 s at 94 8C, annealing for 30 s at 60 8C and extension for 1 min at 75 8C. At the end of the PCR reaction, 2 ml of formamide loading buffer was added, and the samples were denatured by chilling on ice. The PCR product was restricted with 10 units of Hae III restriction endonuclease (Pharmacia, NJ) for 3.5 h at 37 8C in a total reaction volume of 10 ml. Amplified PCR products and lHind III DNA marker were run through 2% agarose gel electrophoresis and stained with ethidium-bromide. 2.6. Characterization of the novel adenine-adducts Significant amounts of novel adenine-adducts were collected with HPLC, lyophilized and suspended in 50% methanol. Mass spectrometer (MS) analysis using electrospray time-of-flight mass spectrometry (ESI-TOF-MS) (Model DE-STR, Applied Biosystem) was used to analyze the novel adducts. The novel adducts with mass of 415.2110 were further analyzed by MS–MS.
A total of 21 liver biopsies from patients with schistosomiasis were examined. Of the 21 patients, 16 were men and 5 were women. The age range of the men and women patients was 26–46 and 20–41 years, respectively, with a mean age of 37 years (Table 1). The percentage of infection with S. mansoni, S. haematobium and S. mansoni + S. hematobium was 25, 31.3 and 43.8 for men and 60, 20 and 20% for women, respectively. A total of five liver specimens from schistosoma-free patients showed no evidence of schistosomiasis. 3.2. Levels of AFB1-DNA and novel adenine adducts in liver SHF The N7-guanine-AFB1 adducts were detected in patients with schistosomiasis with concentrations ranging from 0.1 to 44.3 pg/mg DNA. Predominantly, the AFB1 adducts were found in patients with S. hematobium. N7-guanineAFB1 adducts were not detected in any of the liver samples from schistosomiasis free patients (Table 2). Other DNAadducts were also found in the neutral thermal hydrolysate (Fig. 1). These novel adenine-adducts were found in patients with schistosomiasis ranging from 1 to 6574-fold (basic value of 12,141 as 1 arbitrary units measured the area under the chromatographic peak). Of the 21 patients
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Table 2 N7-guanine-AFB1 adducts, novel adenine-adducts and P53 mutation in liver DNA of patients with schistosoma and/or aflatoxin B1 Patient (#)
Type of infestation
N7 guanine-AFB1 adducts (pg adducts/mg DNA)
Area of novel adenine-adducts (fold)
p53 mutation, codon 249
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
S. mansoni, S. haembatobium None S. haembatobium None S. mansoni None S. mansoni S. mansoni None None S. mansoni, S. haembatobium S. haembatobium S. mansoni, S. haembatobium S. mansoni, S. haembatobium S. mansoni S. mansoni S. haembatobium S. mansoni, S. haembatobium S. haembatobium S. haembatobium S. mansoni, S. haembatobium S. mansoni, S. haembatobium S. mansoni, S. haembatobium S. mansoni S. mansoni S. haembatobium
44.30 – 20.80 – – – – – – – – – 26.90 – – 0.123 0.095 0.300 0.075 – – – – – – –
– – 4688 – – – 27 – – – 64 33 – – – 1 6574 19 14 1 12 2 – – – –
+ + + + + + + + +
with schistosomiasis, the novel adenine-adducts were identified in 9 patients with S. hematobium, and/or S. mansoni + S. hematobium and only 2 patients with S. mansoni. Significant amounts of the novel adenine-adducts and N7-guanine-AFB1 adducts were detected in patients infected with S. hematobium ( p < 0.01) and with S. mansoni + S. hematobium ( p < 0.01) compared to normal subjects (Table 2).
analysis of the 415.2110 ions produced three fragments (Fig. 2B); 135.0866 m/z (consistent with an adenine), 119.0916 m/z (protonated adenine-N6 missing the amino group) and 281.1500 m/z (protonated compound that was adducted to the base). The most likely formulas postulated for the novel adenine-adducts using Mass-LYNX software (Micromass, EST) are: C15H33N3O10, C11H29N9O8, C7H25N15O6, C22H29N3O5, C17H29N5O7, C13H25N11O5, and C12H29N7O9.
3.3. Mutation in the p53 gene None of the 5 patients diagnosed as free of schistosomiasis had detectable mutations in codon 249 of the p53 gene. Nine of the 21 schistosomiasis- positive patients showed mutations in the codon 249. The 9 patients with mutations in codon 249 contained the novel adenine-adducts in their hepatic DNA and 5 of them had detectable concentrations of the N7-guanine-AFB1 adducts. Only 2 of the 11 patients with the novel adenine-adducts did not test positive for p53 mutations at the codon 249 (Table 2). The statistical analysis shows a significant difference in the number of mutations in codon 249 of the p53 gene in subjects infected with schistosoma compared to normal subjects ( p < 0.01). 3.4. MS-characterization of the novel adenine-adducts A significant signal ion at 415.2110 m/z was observed in novel adenine-adducts analyzed by MS (Fig. 2A). MS–MS
Fig. 1. HPLC chromatogram of N7-guanine-AFB1 adducts and unknown DNA- adducts. N7-guanine-AFB1 adducts in liver DNA was examined by HPLC using strong cation exchange columns. Purines were eluted using a gradient elution (details in Section 2). The ultraviolet absorbing novel DNA adducts were at retention time of 14.3 min and the N7-guanine-AFB1 adducts were detected at 25.8 min.
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Fig. 2. (A) ESI-TOF-MS spectrum of internal calibration of unknown DNA adducts. Novel DNA adducts were collected with HPLC then analyzed by electrospray time-of-flight mass spectrometry (ESI-TOF-MS). The novel adducts show a signal of 415.2110 ions. (B) MS–MS spectrum of unknown adducts showed different fragments of ion 415 m/z. Further analysis of 415.2110 ions produced three fragments; 135.0866 m/z (consistent with an adenine), 119.0916 m/ z (protonated adenine-N6 missing the amino group) and 281.1500 m/z which is possibly a novel compound adducted to adenine base.
4. Discussion The association of schistosomiasis-induced hepatic fibrosis in patients exposed to aflatoxin exposure has received special attention due to its high prevalence in Egypt. A major finding in our study is the significant production of N7-guanine-AFB1 adducts and novel adenineadducts associated with mutations in p53 gene in patients
with schistosomasis. Interestingly, this association was seen in patients infected with either species of schistosoma. This association could play an important role in the pathogenesis and disease progression of HCC in patients with schistosomiasis. The difference in the prevalence of infection between males and females has been reported in several studies from Egypt [43]. A recent study, indicated that the prevalence of infection with S. mansoni was 85% in males
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between 11 and 40 years old, while only 58% in females at age between 15 and 30 years old [44]. The age of patients with schistosoma in our study ranged from 20 to 46 years old with a mean age of 37 years old. Prevalence and intensity of schistosomiasis peaked in the 20–30 years old group of males and was higher in males than females in the Ismailia governorate of Egypt [45]. Our results showed that 38.1% of patients were infected with both species of schistosoma, while 33.3 and 28.3% were infected with either S. mansoni or S. hematobium, respectively (Table 1). A survey from North Africa revealed that the incidence rates of HCC were 4.1/100,000 for males and 2.1/100,000 for females [46]. The overall annual age-standardized mortality rates from HCC in Egypt were 4.1 and 2.4/100,000 for males and females, respectively [47]. The most recent report from the Alexandria cancer registry from Egypt shows a substantial increase in the frequency of HCC associated with schistosomal and hepatitis viral infections to 4.9 and 3.0% in males and females, respectively [48]. Another study showed that the formation of AFB1-N7guanine adducts was linear over the low-dose range examined, and the liver, which is the primary target organ, had the highest level of adducts in all animals species [48]. Formation of initial AFB1-N7-guanine adducts has been correlated with the incidence of HCC in trout and rats [49] and several human studies have shown a similar correlation between dietary exposure to AFB1 and excretion of AFB1N7-guanine in the urine [50,51]. High concentrations of N7guanine-AFB1 adducts ranging from 20.8 to 44.3 pg/mg DNA were identified in patients infected with S. hematobium, and S. mansoni + S. hematobium, suggesting that synergism between AFB1-DNA adducts and schistosomiasis leads to and increased incidence of liver cancer. Novel adenine-adducts were found in patients with schistosomiasis ranging from 1 to 6574 arbitrary units (a basic value of 12141 is equivalent to 1 arbitrary unit of the area measured under the chromatographic peak). Of the 21 patients with schistosomiasis, the novel adducts were identified in 7 patients with S. hematobium and/or S. mansoni + S. hematobium and 2 patients with S. mansoni (Table 2). Significant levels of novel adenine-adducts were detected in patients infected with S. hematobium compared to patients infected with S. mansoni or S. mansoni + S. hematobium, suggesting that the infection with S. hematobium may induce specific isoforms of hepatic cytochrome P450 enzymes present in S. heamatobium, which catalyze the formation of novel adenine-adducts. A previous report indicated that the extract of the adult worms of both S. hematobium or S. mansoni has shown enzymatic systems involved in the metabolism of xenobiotics. Moreover, the cytochrome P450 enzymes produced by adult worms of both schistosoma species can metabolize some cytochrome P450 substrates such as aminopyrine more efficiently than rat liver microsomes [52]. Novel adenine-adducts analyzed by MS displayed high spectra corresponding to protonated adducts with sodium
ion at 437.1927 m/z and the parent adducts without sodium ion at 415.2110 m/z (Fig. 2A). Further analysis of fragments from the novel adenine-adducts characterized by MS–MS shows 2 fragments of 281.1500 and 135.0866 m/z (Fig. 2B). The fragment ion of 281.1500 m/z is a protonated compound that was adducted to the mass of 135.0866 m/z, a known adenine mass. Ion 135.0866 m/z is likely a protonated adenine base and could be a radical cation. The fragment ion at 119.0916 m/z arising from the parent adducts observed in the collisionally activated dissociation (CAD) spectra is probably due to loss of adducts and NH3+, leaving an adenine fragment missing the N6-amino group (loss of 16 Dalton). The identity of ion at m/z 281.1500 could be a covalent novel adduct to N6 or N1 on an adenine base. Replication of DNA containing the AFB1-N7-guanine adducts induces G ! T transversion at codon 249 of p53 gene. High frequency of p53 mutations have been found to occur in HCC specimens collected from populations exposed to high levels of dietary aflatoxin in China and Southern Africa [53,54]. The sequence in human DNA for codons 248–250 is 50 -CGGAGGCCC-30 ; restriction in this region by Hae III would cut the DNA as follows: 50 -CGGAGG#CCC-30 to 2 subunits of 75 and 35 bp. A mutation at the internal guanine (underscored) in codon 249 (50 -AGG-30 ) would alter the Hae III restriction site (50 GG#CC-30 ), making the site unrecognizable to the endonuclease, resulting in lack of restriction at this site. The identification of mutations in the p53 gene of liver DNA from patients with schistosmomiasis is based on the lack of restriction by Hae III in PCR products containing this site. All the patients who had p53 mutations were infected with schistosoma, 5 with S. haematobium alone, 2 with S. mansoni alone and 2 with both species. A previous study, from Egypt found p53 mutations in bladder cancer specimens in 90% of patients infected with S. haematobium [37]. Mutations in the p53 gene could be induced by AFB1N7-guanine adducts and/or novel adenine-adducts. A mutation in the internal guanine in codon 249 of the p53 gene was detected in patients with liver cancer who consumed aflatoxin B1 contaminated food in Senegal and Southern Africa [55,56]. In addition, a mutation in codon 249 of p53 gene has been found in plasma from 52% of patients with liver tumors by electrospray ionization mass spectrometry in Gambia et al. [57]. Mutant p53 protein, mutations in the p53 gene, and specific codon 249 mutations were detected in 37, 29, and 13% of the HCC cases from patients with chronic HBV infection and AFB1 exposure, respectively [58]. In our study, we show that a combination of schistosomal infection with AFB1-N7-guanine adducts and novel adenineadducts increased the incidence of p53 mutation. The prevalence of infection with a single or combined species of schistosoma and chronic exposure to potent toxic and carcinogenic chemicals such as aflatoxin B1 in Egyptian populations should be considered as a potential major factor impacting on the increased incidence of hepatocellular
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carcinoma. However, the effect of the interaction between parasitic infections and aflatoxin exposure in the pathogenesis of HCC requires further understanding. Acknowledgements We thank Dr. Hernan Rincon-Choles for critical editing of the manuscript.
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Novel adenine adducts, N7-guanine-AFB1 adducts, and p53 mutations in patients with schistosomiasis and aflatoxin exposure Samy L. Habib PhDa,*, Boctor Said PhDa, Ahmed T. Awad MDb, Mostafa H. Mostafa PhDc, Ronald C. Shank PhDa a
Department of Community and Environmental Medicine, College of Medicine, University of California at Irvine, Irvine, CA 92697, USA b Department of Surgery, College of Medicine, Alexandria University, Egypt c Institute of Graduate Studies and Research, University of Alexandria, Alexandria, Egypt Accepted 10 October 2006
Abstract Introduction: The most frequent mutation in human hepatocellular carcinoma (HCC) in populations exposed to a high dietary intake of aflatoxin B1 (AFB1) is a mutation in codon 249 of the p53 gene. Schistosomiasis is known to cause p53 mutation. We hypothesized that the combination of schistosomiasis and aflatoxin B1 increases the incidence of p53 gene mutation. Methods: Liver tissue from 21 patients with schistosomiasis and 5 patients without schistosomiasis were analyzed for occurrence of mutations of the p53 gene and levels of N7-guanineAFB1 adducts. Results: The presence of mutations in codon 249 of p53 gene was higher in patients infected with Schistosoma haematobium (S. haematobium) than in those infected with Schistosoma mansoni (S. mansoni) or a combination of both strains ( p < 0.01), compared to control subjects. No mutations were detected in p53 gene in liver DNA from schistosomiasis-free patients. Significant amounts of N7-guanineAFB1 adducts and novel adenine-adducts ( p < 0.01) were detected in patients with schistosomiasis, mostly in patients infected with S. haematobium or a combination of both strains, compared to control subjects. Conclusion: These data suggest that schistosomiasis and exposure to aflatoxin B1 act synergistically to increase the incidence of p53 gene mutation. The increase in p53 mutations may enhance progression of HCC at an early age in patients with schistosomiasis. # 2006 International Society for Preventive Oncology. Published by Elsevier Ltd. All rights reserved. Keywords: Novel adenine-adducts; N7-guanine-AFB1adducts; P53 mutation; Schistosomiasis; Synergism; Aflatoxin B1; S. mansoni; S. hematobium; Cytochrome P-450; PCR
1. Introduction Exposure to dietary aflatoxin B1 (AFB1), hepatitis virus or schistosomal infections is associated with an increased risk of hepatocellular carcinoma (HCC) in humans. Synergistic effects of multiple risk factors in the development of HCC by chronic liver disease are major contributors to carcinogenesis [1–3]. Hepatocellular carcinoma is the fifth most common cancer in the world with 80% of cases occurring in developing * Corresponding author at: The University of Texas Health Science Center, Department of Medicine, Division of Nephrology-MSC 7882, 7703 Floyd Curl Dr., San Antonio, TX 78229, USA. Tel.: +1 210 567 4699; fax: +1 210 567 4712. E-mail address: [email protected] (S.L. Habib).
countries. Liver fibrosis is the main cause of death in the 20 million individuals suffering from chronic schistosomiasis [4–6]. In Egypt, hepatitis B and C viruses, (HBV and HCV) and schistosomiasis constitute the most common causes of chronic liver disease [7,8]. Schistosomiasis increases the severity of HBV infection and the risk of HCC over that associated with the HBV infection alone [9]. A recent survey of aflatoxin contamination in Egyptian foods indicated that there is a high prevalence of AFB1 in herbs, medicinal plants, cereals, spices, nuts, seeds and vegetables which may increase the incidence of liver cancer [10]. Schistosoma mansoni (S. mansoni) causes liver fibrosis, intestinal carcinoma and splenic lymphoma while Schistosoma haematobium (S. haematobium) causes urinary schistosomiasis leading to bladder cancer [11,12]. An
0361-090X/$30.00 # 2006 International Society for Preventive Oncology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cdp.2006.10.006
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association between S. japonicum and HCC was reported in a Japanese necropsy study [13,14]. In addition, a relationship between S. mansoni infection and HCC was found in patients with schistosomiasis in Saudi Arabia [15]. Clinical and pathological studies of S. haematobium or S. mansoni have been performed on animal models to evaluate their carcinogenic effects. Infection with S. mansoni in mice was found to increase the risk of hepatic carcinoma associated with administration of the liver carcinogen, 2-amino-5azotoluene [16]. Similarly, in the case of S. haematobium infection, epithelial hyperplasia and metaplasia were reported in the bladders of parasite-infected mice exposed to aromatic amines such as acetylaminofluorene [17] or 2-naphthylamine [18]. Several epidemiological studies have established a strong association between dietary aflatoxin B1 exposure and the development of primary hepatocellular carcinoma [19–22]. AFB1 is activated by hepatic cytochrome P-450dependent monooxygenases leading to the formation of several forms of AFB1 metabolites [23]. AFB1-8-9epoxide is presumed to be the ultimate mutagen and carcinogen [24]. Upon metabolic activation, the reactive AFB1-8-9-epoxide forms DNA adducts primarily at the N7 position of guanine [25]. Aflatoxin B1 causes a significant inhibition in RNA [26,27] and protein synthesis and inhibits the activity of drug metabolizing enzymes [28,29]. Our previous studies, indicate an association between S. mansoni infections and inhibition of metabolizing enzyme activity in the liver of experimental animals [30] and humans [31]. The p53 gene is a major suppressor gene and it is well suited for analysis of the mutational spectrum in human cancer. Certain p53 mutants lose their suppressor activity and gain oncogenic activity, which is one explanation for the commonality of p53 mutations in human cancer [32]. Patients with HCC living in countries with high aflatoxin exposure have a characteristic mutational spectrum in the p53 gene, specifically, G to T transversion at the third base of codon 249 [33]. The mutation of p53 gene in HCC from African and Asian patients has been associated with high exposure to aflatoxin B1 and high prevalence of HBV infection [34,35]. The p53 gene regulates multiple components of the DNA damage control response and promotes cellular senescence. Consequently, the biological properties of mutated p53 depend mainly on the site and the nature of point mutations. These alterations in p53 gene will consequently modulate the expression of genes that regulate DNA repair and synthesis, cell cycle, cell differentiation and, cell death [36]. There is a strong correlation between infection with schistosoma or exposure to aflatoxin and the site of p53 mutation [37,38]. In the present study, we investigated the effect of the combination of schistosoma and aflatoxin B1 on the mutation of p53 gene. The level of N7-guanine-AFB1 adducts in the liver of patients exposed to aflatoxin contaminated foods was also investigated.
2. Materials and methods 2.1. Liver specimens Liver specimens from patients with schistosomiasis were obtained from the Alexandria General Hospital (Alexandria, Egypt). A total of 21 liver biopsies were obtained from patients with schistosomal hepatic fibrosis during operations for decongestion and/or splenectomy. Five biopsies from splenectomy patients with normal liver constituted the control group. The informed consent of each patient was obtained. All samples were histologically examined confirming the clinical diagnosis of schistosomiasis. Liver tissues were frozen at 80 8C within 20 min of surgical removal. 2.2. DNA isolation DNA was isolated from frozen liver specimens following the protocol described by Strauss [39]. Briefly, frozen liver samples were homogenized in 10 mM Tris–HCl, pH 8.0. Protein and RNA were digested by proteinase K and RNase 1A then deproteinized by successive phenol/chloroform/ isoamyl alcohol extractions. DNA was extracted and recovered by ethanol precipitation. DNA was collected by centrifugation, washed twice with 70% ethanol, and resuspended in 100 ml of 10 mM Tris–HCl, pH 7.4. DNA was separated on a 0.8% agarose gel electrophoresis and quantitated by spectrophotometry. 2.3. In vitro treatment of Calf Thymus -DNA with AFB1-8,9-epoxide The Calf Thymus (CT)-DNA-AFB1-8, 9-epoxide adducts were synthesized as described by Iyer et al. [40]. Briefly, CTDNA (0.5 mg) was reacted (in triplicate) with different concentrations of AFB1-8,9-epoxide (0.1–50 mg) in 10 mM sodium phosphate buffer, pH 7.2 (total volume 200 ml) for 30 min at 10 8C. DNA was recovered by organic solvent extraction and ethanol precipitation. DNA was collected by centrifugation, washed twice with 70% ethanol, and resuspended in 100 ml of 10 mM Tris–HCl, pH 7.4. DNA was recovered by centrifugation and then resuspended in water and heated at 37 8C for 5–10 min to ensure complete dissolution. N7-guanine-AFB1 adducts were prepared from the DNA as described by Essigmann et al. [41]. Samples were subjected to mild acid hydrolysis in 0.1 N HCl for 45 min at 70 8C, to liberate N7-guanine-AFB1 adducts. Hydrolysates were cooled, filtered through a 0.45 mm filter and then analyzed by HPLC. Different concentrations of N7-guanineAFB1 were used to generate the calibration curve by HPLC. 2.4. N7-guanine-AFB1 adducts and novel adenine-adducts assay DNA extracted from control and SHF specimens were subjected to mild acid hydrolysis in 0.1N HCl for 45 min at
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Table 1 Summarized data of human liver samples obtained from patients with schistosoma and schistosoma-free patients Subject
Patients Men
Woman
Men
Woman
Number Mean age Age range S. mansoni-positive (%) S. hematobium-positive (%) S. mansoni + S hematobium-positive (%)
16 38.5 26–46 25 31 44
5 35.5 20–41 60 20 20
3 32 28–34 0 0 0
2 35 32–38 0 0 0
70 8C, to liberate N7-guanine-AFB1 adducts. The level of guanyl-N7-AFB1 adducts in liver DNA was examined by HPLC using strong cation exchange columns (Supelco LC18, 5 mm, 250 mm 4.6 mm, i.d. column; Supelco, Bellefonte, PA). Purines were eluted by using a gradient elution: 5% methanol in 10 mM potassium phosphate buffer, pH 7.0 for 5 min, followed by linear gradient of 5–80% (v/v) methanol in phosphate buffer over 30 min and 80% methanol for 10 min. The flow rate was 1.0 ml/min. Eluted compounds were detected by HPLC using a UV detector set at 360 nm. The amount of guanyl-N7-AFB1 measured in each sample was quantitated using an N7-guanine-AFB1 adducts calibration curve. The ultra-violet absorbing novel adenine-adducts were eluted from the HPLC column at retention time of 14.3 min and the N7-guanine-AFB1 adducts were detected at 25.8 min.
Control
2.7. Synthesis of AFB1-8,9 epoxide AFB1 was purchased from Sigma–Aldrich Chemicals Inc. (St. Louis, MO). Dimethyl-dioxirane was reacted with AFB1 to give the AFB1-exo-8,9-epoxide [42] at room temperature. The epoxide was dissolved at a concentration of 30 mM in methylene chloride. 2.8. Statistics Statistical differences were determined using ANOVA followed by Student Dunnett’s (exp. versus control) test using one trial analysis. p-values less than 0.01 were considered statistically significant.
3. Results 2.5. PCR reaction 3.1. Summary of patient data Polymerase chain reaction (PCR) amplification of the region containing codon 249 was performed using the primers 50 -GTTGGCTCGACTGTACCAC-30 and 50 CTGGAGTCTTCCAGTGTGAT-30 . The PCR reaction mixture contained 2 units of Taq polymerase (Qiagen, CA), 50 pM of each primer, 200 mM of each dNTP and 5 ml of extracted DNA. The PCR procedure was 35 cycles of denaturing for 30 s at 94 8C, annealing for 30 s at 60 8C and extension for 1 min at 75 8C. At the end of the PCR reaction, 2 ml of formamide loading buffer was added, and the samples were denatured by chilling on ice. The PCR product was restricted with 10 units of Hae III restriction endonuclease (Pharmacia, NJ) for 3.5 h at 37 8C in a total reaction volume of 10 ml. Amplified PCR products and lHind III DNA marker were run through 2% agarose gel electrophoresis and stained with ethidium-bromide. 2.6. Characterization of the novel adenine-adducts Significant amounts of novel adenine-adducts were collected with HPLC, lyophilized and suspended in 50% methanol. Mass spectrometer (MS) analysis using electrospray time-of-flight mass spectrometry (ESI-TOF-MS) (Model DE-STR, Applied Biosystem) was used to analyze the novel adducts. The novel adducts with mass of 415.2110 were further analyzed by MS–MS.
A total of 21 liver biopsies from patients with schistosomiasis were examined. Of the 21 patients, 16 were men and 5 were women. The age range of the men and women patients was 26–46 and 20–41 years, respectively, with a mean age of 37 years (Table 1). The percentage of infection with S. mansoni, S. haematobium and S. mansoni + S. hematobium was 25, 31.3 and 43.8 for men and 60, 20 and 20% for women, respectively. A total of five liver specimens from schistosoma-free patients showed no evidence of schistosomiasis. 3.2. Levels of AFB1-DNA and novel adenine adducts in liver SHF The N7-guanine-AFB1 adducts were detected in patients with schistosomiasis with concentrations ranging from 0.1 to 44.3 pg/mg DNA. Predominantly, the AFB1 adducts were found in patients with S. hematobium. N7-guanineAFB1 adducts were not detected in any of the liver samples from schistosomiasis free patients (Table 2). Other DNAadducts were also found in the neutral thermal hydrolysate (Fig. 1). These novel adenine-adducts were found in patients with schistosomiasis ranging from 1 to 6574-fold (basic value of 12,141 as 1 arbitrary units measured the area under the chromatographic peak). Of the 21 patients
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Table 2 N7-guanine-AFB1 adducts, novel adenine-adducts and P53 mutation in liver DNA of patients with schistosoma and/or aflatoxin B1 Patient (#)
Type of infestation
N7 guanine-AFB1 adducts (pg adducts/mg DNA)
Area of novel adenine-adducts (fold)
p53 mutation, codon 249
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
S. mansoni, S. haembatobium None S. haembatobium None S. mansoni None S. mansoni S. mansoni None None S. mansoni, S. haembatobium S. haembatobium S. mansoni, S. haembatobium S. mansoni, S. haembatobium S. mansoni S. mansoni S. haembatobium S. mansoni, S. haembatobium S. haembatobium S. haembatobium S. mansoni, S. haembatobium S. mansoni, S. haembatobium S. mansoni, S. haembatobium S. mansoni S. mansoni S. haembatobium
44.30 – 20.80 – – – – – – – – – 26.90 – – 0.123 0.095 0.300 0.075 – – – – – – –
– – 4688 – – – 27 – – – 64 33 – – – 1 6574 19 14 1 12 2 – – – –
+ + + + + + + + +
with schistosomiasis, the novel adenine-adducts were identified in 9 patients with S. hematobium, and/or S. mansoni + S. hematobium and only 2 patients with S. mansoni. Significant amounts of the novel adenine-adducts and N7-guanine-AFB1 adducts were detected in patients infected with S. hematobium ( p < 0.01) and with S. mansoni + S. hematobium ( p < 0.01) compared to normal subjects (Table 2).
analysis of the 415.2110 ions produced three fragments (Fig. 2B); 135.0866 m/z (consistent with an adenine), 119.0916 m/z (protonated adenine-N6 missing the amino group) and 281.1500 m/z (protonated compound that was adducted to the base). The most likely formulas postulated for the novel adenine-adducts using Mass-LYNX software (Micromass, EST) are: C15H33N3O10, C11H29N9O8, C7H25N15O6, C22H29N3O5, C17H29N5O7, C13H25N11O5, and C12H29N7O9.
3.3. Mutation in the p53 gene None of the 5 patients diagnosed as free of schistosomiasis had detectable mutations in codon 249 of the p53 gene. Nine of the 21 schistosomiasis- positive patients showed mutations in the codon 249. The 9 patients with mutations in codon 249 contained the novel adenine-adducts in their hepatic DNA and 5 of them had detectable concentrations of the N7-guanine-AFB1 adducts. Only 2 of the 11 patients with the novel adenine-adducts did not test positive for p53 mutations at the codon 249 (Table 2). The statistical analysis shows a significant difference in the number of mutations in codon 249 of the p53 gene in subjects infected with schistosoma compared to normal subjects ( p < 0.01). 3.4. MS-characterization of the novel adenine-adducts A significant signal ion at 415.2110 m/z was observed in novel adenine-adducts analyzed by MS (Fig. 2A). MS–MS
Fig. 1. HPLC chromatogram of N7-guanine-AFB1 adducts and unknown DNA- adducts. N7-guanine-AFB1 adducts in liver DNA was examined by HPLC using strong cation exchange columns. Purines were eluted using a gradient elution (details in Section 2). The ultraviolet absorbing novel DNA adducts were at retention time of 14.3 min and the N7-guanine-AFB1 adducts were detected at 25.8 min.
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Fig. 2. (A) ESI-TOF-MS spectrum of internal calibration of unknown DNA adducts. Novel DNA adducts were collected with HPLC then analyzed by electrospray time-of-flight mass spectrometry (ESI-TOF-MS). The novel adducts show a signal of 415.2110 ions. (B) MS–MS spectrum of unknown adducts showed different fragments of ion 415 m/z. Further analysis of 415.2110 ions produced three fragments; 135.0866 m/z (consistent with an adenine), 119.0916 m/ z (protonated adenine-N6 missing the amino group) and 281.1500 m/z which is possibly a novel compound adducted to adenine base.
4. Discussion The association of schistosomiasis-induced hepatic fibrosis in patients exposed to aflatoxin exposure has received special attention due to its high prevalence in Egypt. A major finding in our study is the significant production of N7-guanine-AFB1 adducts and novel adenineadducts associated with mutations in p53 gene in patients
with schistosomasis. Interestingly, this association was seen in patients infected with either species of schistosoma. This association could play an important role in the pathogenesis and disease progression of HCC in patients with schistosomiasis. The difference in the prevalence of infection between males and females has been reported in several studies from Egypt [43]. A recent study, indicated that the prevalence of infection with S. mansoni was 85% in males
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between 11 and 40 years old, while only 58% in females at age between 15 and 30 years old [44]. The age of patients with schistosoma in our study ranged from 20 to 46 years old with a mean age of 37 years old. Prevalence and intensity of schistosomiasis peaked in the 20–30 years old group of males and was higher in males than females in the Ismailia governorate of Egypt [45]. Our results showed that 38.1% of patients were infected with both species of schistosoma, while 33.3 and 28.3% were infected with either S. mansoni or S. hematobium, respectively (Table 1). A survey from North Africa revealed that the incidence rates of HCC were 4.1/100,000 for males and 2.1/100,000 for females [46]. The overall annual age-standardized mortality rates from HCC in Egypt were 4.1 and 2.4/100,000 for males and females, respectively [47]. The most recent report from the Alexandria cancer registry from Egypt shows a substantial increase in the frequency of HCC associated with schistosomal and hepatitis viral infections to 4.9 and 3.0% in males and females, respectively [48]. Another study showed that the formation of AFB1-N7guanine adducts was linear over the low-dose range examined, and the liver, which is the primary target organ, had the highest level of adducts in all animals species [48]. Formation of initial AFB1-N7-guanine adducts has been correlated with the incidence of HCC in trout and rats [49] and several human studies have shown a similar correlation between dietary exposure to AFB1 and excretion of AFB1N7-guanine in the urine [50,51]. High concentrations of N7guanine-AFB1 adducts ranging from 20.8 to 44.3 pg/mg DNA were identified in patients infected with S. hematobium, and S. mansoni + S. hematobium, suggesting that synergism between AFB1-DNA adducts and schistosomiasis leads to and increased incidence of liver cancer. Novel adenine-adducts were found in patients with schistosomiasis ranging from 1 to 6574 arbitrary units (a basic value of 12141 is equivalent to 1 arbitrary unit of the area measured under the chromatographic peak). Of the 21 patients with schistosomiasis, the novel adducts were identified in 7 patients with S. hematobium and/or S. mansoni + S. hematobium and 2 patients with S. mansoni (Table 2). Significant levels of novel adenine-adducts were detected in patients infected with S. hematobium compared to patients infected with S. mansoni or S. mansoni + S. hematobium, suggesting that the infection with S. hematobium may induce specific isoforms of hepatic cytochrome P450 enzymes present in S. heamatobium, which catalyze the formation of novel adenine-adducts. A previous report indicated that the extract of the adult worms of both S. hematobium or S. mansoni has shown enzymatic systems involved in the metabolism of xenobiotics. Moreover, the cytochrome P450 enzymes produced by adult worms of both schistosoma species can metabolize some cytochrome P450 substrates such as aminopyrine more efficiently than rat liver microsomes [52]. Novel adenine-adducts analyzed by MS displayed high spectra corresponding to protonated adducts with sodium
ion at 437.1927 m/z and the parent adducts without sodium ion at 415.2110 m/z (Fig. 2A). Further analysis of fragments from the novel adenine-adducts characterized by MS–MS shows 2 fragments of 281.1500 and 135.0866 m/z (Fig. 2B). The fragment ion of 281.1500 m/z is a protonated compound that was adducted to the mass of 135.0866 m/z, a known adenine mass. Ion 135.0866 m/z is likely a protonated adenine base and could be a radical cation. The fragment ion at 119.0916 m/z arising from the parent adducts observed in the collisionally activated dissociation (CAD) spectra is probably due to loss of adducts and NH3+, leaving an adenine fragment missing the N6-amino group (loss of 16 Dalton). The identity of ion at m/z 281.1500 could be a covalent novel adduct to N6 or N1 on an adenine base. Replication of DNA containing the AFB1-N7-guanine adducts induces G ! T transversion at codon 249 of p53 gene. High frequency of p53 mutations have been found to occur in HCC specimens collected from populations exposed to high levels of dietary aflatoxin in China and Southern Africa [53,54]. The sequence in human DNA for codons 248–250 is 50 -CGGAGGCCC-30 ; restriction in this region by Hae III would cut the DNA as follows: 50 -CGGAGG#CCC-30 to 2 subunits of 75 and 35 bp. A mutation at the internal guanine (underscored) in codon 249 (50 -AGG-30 ) would alter the Hae III restriction site (50 GG#CC-30 ), making the site unrecognizable to the endonuclease, resulting in lack of restriction at this site. The identification of mutations in the p53 gene of liver DNA from patients with schistosmomiasis is based on the lack of restriction by Hae III in PCR products containing this site. All the patients who had p53 mutations were infected with schistosoma, 5 with S. haematobium alone, 2 with S. mansoni alone and 2 with both species. A previous study, from Egypt found p53 mutations in bladder cancer specimens in 90% of patients infected with S. haematobium [37]. Mutations in the p53 gene could be induced by AFB1N7-guanine adducts and/or novel adenine-adducts. A mutation in the internal guanine in codon 249 of the p53 gene was detected in patients with liver cancer who consumed aflatoxin B1 contaminated food in Senegal and Southern Africa [55,56]. In addition, a mutation in codon 249 of p53 gene has been found in plasma from 52% of patients with liver tumors by electrospray ionization mass spectrometry in Gambia et al. [57]. Mutant p53 protein, mutations in the p53 gene, and specific codon 249 mutations were detected in 37, 29, and 13% of the HCC cases from patients with chronic HBV infection and AFB1 exposure, respectively [58]. In our study, we show that a combination of schistosomal infection with AFB1-N7-guanine adducts and novel adenineadducts increased the incidence of p53 mutation. The prevalence of infection with a single or combined species of schistosoma and chronic exposure to potent toxic and carcinogenic chemicals such as aflatoxin B1 in Egyptian populations should be considered as a potential major factor impacting on the increased incidence of hepatocellular
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carcinoma. However, the effect of the interaction between parasitic infections and aflatoxin exposure in the pathogenesis of HCC requires further understanding. Acknowledgements We thank Dr. Hernan Rincon-Choles for critical editing of the manuscript.
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