Aflatoxin B1 formamidopyrimidine adducts in human hepatocarcinogenesis: A preliminary report

GASTROENTEROLOGY 1989;97:1281-7

Aflatoxin B, Formamidopyrimidine Adducts in Human Hepatocarcinogenesis: A Preliminary Report HYO-SUK Department California

LEE, ILDIKO

SAROSI,

of Laboratory Medicine,

University

The presence of aflatoxin-B,-formamidopyrimidine, a persistent allatoxin-deoxyribonucleic acid (DNA) adduct, was investigated in vivo by immunohistochemical analysis in 14 paired hepatocellular carcinoma and nontumorous human liver tissue sections using a monoclonal anti-allatoxin-B,-formamidopy rimidine antibody. Nuclear and cytoplasmic accumulations of adducts were found in 4 of 14 nontumorous specimens but in none of 14 tumorous tissues and in none of three normal control livers. In vitro adduct formation and cellular DNA was investigated with a modified DNA immunoblot assay. These studies revealed (a) no significant difference in the amount of adduct formed by DNA samples with or without integrated hepatitis B virus DNA, (b) no difference in the amount of adduct formed with DNA from either tumorous or nontumorous tissues from a given individual, and (c) remarkable and reproducible differences in the capacity of DNA from different individuals to form in vitro adducts. Our DNA immtmoblot assay will facilitate further studies on the molecular role of aflatoxin-B,-formamidopyrimidine adducts in human hepatocarcinogenesis.

A

lthough a very strong correlation between persistent hepatitis B virus (HBV) infection and the development of hepatocellular carcinoma (HCC) has been established (l-3), there are several observations suggesting that chronic HBV infection is not the sole etiologic factor in the development of the HCC: (a) HCC tissues from several hepatitis B surface antigen (HBsAg)-positive patients do not contain any integrated or free viral deoxyribonucleic acid (DNA) (4); (b) only a small fraction of HBV carriers ever develop HCC (5); (c)epidemiologic studies from South Africa have shown that in a population with a high and uniform prevalence of chronic HBV infection the incidence of HCC varies with geographic location

and GIRISH N. VYAS of California San Francisco,

San Francisco,

(6); and (d) none of the classical mechanisms of viral oncogenesis have been demonstrated to date for HBV and HCC (7). Besides chronic HBV infection, other factors including individual susceptibility and environmental carcinogen exposure may be important determinants in the development of human HCC. One of these environmental carcinogens is aflatoxin B, (AFB,) (8). High dietary intake of AFB, has been consistently associated with high incidences of HCC in studies in Kenya, Swaziland, Thailand, and Mozambique (9,lO). A similar association of AFB, intake and HCC has been suggested for Asian countries, but no conclusive data are yet available (11,12). These observations, together with the results of extensive experimental studies on the hepatocarcinogenity of AFB, in animal models (13,14), suggest a possible association of AFB, with the development of human HCC (15,16). Although aflatoxins have been suggested to suppress cell-mediated immunity and thereby contribute to the development both of a chronic carrier state for HBsAg and of HCC (l7), the more likely molecular mechanisms underlying the carcinogenic effect of AFB, are structural modifications of the host DNA by covalent adduct formation and the consequent alteration of functional properties of the host DNA (13,18,19). Several aflatoxin metabolites are formed in the liver, and some of these metabolites are able to form adducts with host DNA. One of these adducts, Al%,-formamidopyrimidine (AFB,-FAPyr), is thought to be involved in human hepatocarcinogenesis (15). The aims of the present study were (a) to investigate the occurrence of AFB,-FAPyr adducts in hu-

Abbreviations used in this paper: AFB,, aflatoxin B,; AFB,FAPyr, aflatoxin B,-formamidopyrimidine; HCC, hepatocellular carcinoma; PBS, phosphate-bu5xed saline. 0 1989 by the American Gastroenterological Association 0018.5085/89/$3.50

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GASTROENTEROLOGY Vol. 97, No. 5

man HCC or the adjacent nontumorous liver tissue by immunohistochemical analysis; (b) to determine the variation in the ability of liver DNA from individual patients to form in vitro AFB,-FAPyr adducts; and (c) to determine any correlation with the presence or absence of integrated HBV DNA in the human DNA samples. In conjunction with these studies a DNA immunoblot assay is described.

Extraction Acid

Materials and Methods Examination Vivo Formed

of Human Adducts

Liver

plex (Vector) for 30 min at room temperature; and (e) Vector Red alkaline phosphatase substrate solution (Vector) diluted 1:50 in 0.1 M Tris buffer (pH 8.2), containing 2.5 mM levamisole for 20 min at room temperature. Between each step the slides were rinsed three times in TBS buffer (pH 7.4). Finally the slides were counterstained with hematoxylin, dehydrated in graded ethanol solutions, and mounted.

Tissues

for In

Fourteen paired HCC and adjacent nontumorous tissues were obtained surgically from patients with HCC and examined by the immunohistochemical analysis described later for the detection of in vivo formed AFB,FAPyr adducts. Three normal liver tissues obtained by autopsy from patients who died of nonhepatic diseases were simultaneously used as nontumorous negative controls.

Ability of Human Liver Deoxyribonucleic Acid to Form In Vitro Adducts Liver DNA was extracted from a different series of surgically removed, freshly frozen tissues: three paired nontumorous and tumorous (HCC) tissues (2N/2T, 6N/6T, and 7N/7T) and six tumorous tissues (llT, 12T, 13T, 14T, 15T, and 16T). In vitro AFB,-FAPyr adduct formation was performed on these DNA samples, and the adducts were detected by the immunoblot analysis described later. The control DNA samples used were as follows: (a) human placental DNA (Sigma Chemical Co., St. Louis, MO.) as a human DNA of nonhepatic origin and containing no HBV DNA: (b) DNA extracted from cultured PLUPRF-5 cells as a human DNA of hepatic origin containing integrated HBV DNA (20); (c) DNA extracted from cultured HepG2 cells as a human DNA of hepatic origin containing no HBV DNA (20); and (d) DNA extracted from HepG2-2-15 cells (HepG2 cells transfected with cloned HBV DNA) as a human DNA of hepatic origin containing both integrated and replicative HBV DNA (21).

of Genomic

Total genomic DNA was extracted from human tissues and cell culture lines by proteinase K (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) digestion, phenol-chloroform extraction, and subsequent ribonuclease (Boehringer Mannheim Biochemicals) treatment (24). The purity of the DNA samples was verified spectrophotometrically by determining the ratio of absorbance at 2601280 nm (24).

In Vitro Aflatoxin Adduct Formation

Analysis

Sections ok 7 pm were cut from the paraffinembedded tissues and processed according to a modified procedure of Hsu et al. (22). The deparaffinized and rehydrated sections were sequentially treated with the following solutions: (a) 2% normal horse serum in TBS (pH 7.4) buffer (50 mM Tris, 150 mM NaCl) for 30 min at room temperature; (b) mouse monoclonal antibody against the AFB,-DNA adduct (23) diluted 1:lOOin TBS buffer (pH 7.4) for 12 h at 4°C; (c) biotinylated horse-antimouse immunoglobulin G (Vector, Burlingame, Calif.) diluted 1:200 in TBS buffer (pH 7.4) for 30 min at room temperature; (d) alkaline phosphatase-labeled avidin-biotin com-

B,-Formamidopyrimidine

We have used a modification of a procedure first described by Martin and Garner (25). An aliquot of 10 pg of each DNA was dissolved in 2 ml of 20 mM sodium phosphate buffer (pH 7.4) and added to 2 ml of dichloromethane (Aldrich, Milwaukee, Wis.) containing 50 pg of AFB, (Hoechst Calbiochem-Behring, La Jolla, Calif.). To this two-phase system we added 10 ~1 of 10 mg/ml freshly prepared chloroperbenzoic acid (Aldrich) solution in dichloromethane. The tightly stoppered reaction tubes were kept in the dark and vigorously shaken at room temperature for 24 h. At the end of the incubation the tubes were cooled on ice and the two phases were separated by a 5-min configuration at 2000 rpm. We saved the aqueous phase, extracted it three times with chloroform, and recovered the nucleic acids containing the adducts by ethanol precipitation. After lyophylization the pellet was redissolved in 1X TE (10 mM Tris, pH 7.4, 1 mM ethylenediaminetetraacetic acid). The concentration of DNA in each solution was determined by spectrophotometry.

Deoxyribonucleic lmmunohistochemical

Deoxyribonucleic

Acid

lmmunoblot

We have developed this technique for the detection of the AFB,-FAPyr adducts by combining modified procedures for spotting DNA on a cationic nylon membrane (26,27) and immunologic detection (28,291 using a highly specific monoclonal antibody (23) against the AFB,-FAPyr adducts. The cationic membrane (Nytran, Schleicher and Schuell, Inc., Keene, N.H.) was soaked in 6 x SSC (1 X SSC = 0.05 M Na, citrate, 0.15 M NaCl) and layered onto the 96-well blotting apparatus (Biorad, Richmond, Calif.). Each DNA sample was spotted into the wells in serial dilutions (in a total volume of 200 ~1 each) containing 500, 250,125, and 63 ng or 200,100,50,25,12, and 6 ng DNA.

AFLATOXIN B,-DNA ADDUCTS IN HUMAN LIVER CANCER 1283

November 1989

Unlike the conventional procedure described originally by Nehls et al. (SO), the denaturation of the DNA samples was unnecessary after blotting. After the completion of the dot blot, the filter was carefully removed from the filter support under continuous vacuum, dried between two sheets of Whatman filter paper, and baked at 60°C for 1 h. The baked filter was soaked in phosphate-buffered saline (PBS) [O.OlM Na,HPO, (pH 7.4), 0.15 M NaCl], transferred into a plastic bag, heat-sealed, and incubated in PBS containing 20% fetal calf serum for 12 h at 37°C to block nonspecific binding of the monoclonal antibody. After draining the above solution from the bag, the membrane was incubated in a 1:500 diluted solution of the monoclonal antibody (3 pg/9 cm’) in PBS, containing 0.1% Tween 20 (Sigma Chemical Co.), 8% fetal calf serum, and 2% normal horse serum for 6 h at 4°C with constant shaking on a rocker platform. After extensive washing (3 x for 10 min at room temperature) in PBS/O.l% Tween 20, the membrane was transferred into a new plastic bag, heat-sealed, and incubated for 1 h at room temperature with constant shaking in a 1:200 dilution of the biotinylated horseantimouse secondary antibody (Vector) in PBS/O.l% Tween 20 containing 4% fetal calf serum and 2% normal horse serum. Then the membrane was washed 3x for 10 min each at room temperature in PBS/O.l% Tween 20, placed into new plastic bags, and incubated for 30 min at room temperature in alkaline phosphatase-labeled avidinbiotin complex reagent (Vector) diluted 1:lOO in PBS containing 4% fetal calf serum. Finally it was washed in three changes of each of the following solutions for 10 min each: PBS/O.l% Tween 20, PBS/O.5 M NaCl, and TBS (pH 7.4). The enzymatic activity was visualized by reaction with Fast Blue stain (Vector) for 3 min at room temperature, and color development was stopped by immersing the membranes into distilled water. The membranes were air dried.

Results Jmmunohistochemical B,-Formamidopyrimidine Samples

Analysis of Aflatoxin in Human Liver

Immunohistochemical analysis demonstrated the presence of AFB,-FAPyr adducts in human liver tissue sections. To our knowledge this is the first such demonstration reported. Of the 14 paired tumorous and nontumorous tissue samples, four nontumorous tissue sections contained the adducts (Table 1). We could not detect any AFB,-FAPyr adducts in the tumorous tissues. The cells showing strong staining for AFB,-FAPyr were located mostly at the periphery of cirrhotic nodules (Figure 1A). Under higher magnification, diffuse stain for AFB,-FAPyr was found in the cytoplasm as well as in the nucleus (Figure 1B). Occasionally, strong staining was observed along the nuclear membranes. No adducts were detected in three normal control autopsy livers.

Table

1. Detection of Aflatoxin B,Formamidopyrimidine by lmmunocytochemical Analysis Using Monoclonal Antibody to Ajlatoxin B,-Formamidopyrimidine in 14 Paired Hepatoceliular Carcinoma and Nontumorous Tissues Tissue (n)

HCC (14) Nontumorous (14) Normal control liver (3)

Serum HBsAg (n) + + -

AFB,-FAPyr

(10) (4) (101 (41

- (3)

0 0 3 1"

0

AFB,-FAPyr, aflatoxin B,-formamidopyrimidine; HBsAg, hepatitis B surface antigen; HCC, hepatocellular carcinoma. a Negative for all markers of hepatitis B virus (hepatitis B virus deoxyribonucleic acid, HBsAg, antibodies to hepatitis B core and surface antigens) in the HCC tissue.

In Vitro Binding of Aflatoxin B, to Deoxyribonucleic Acid With or Without Integrated Hepatitis B Virus The susceptibility of DNA from individual specimens, with or without integrated HBV DNA, to form in vitro adducts was analyzed by immunoblot assay (Figures 2A and 2B). At least 16-fold difference in the amount of the adducts formed in vitro with individual DNA specimens was consistently recognized in repeated assays. We have found no difference in the amounts of adducts formed with DNA extracted from HepG-2 cells, without HBV DNA, and transfected HepG2-2-15 cells, containing both integrated and replicative forms of HBV DNA (Figure 2A). We also found no difference between the amounts of adducts formed in vitro with the DNA extracted from either the tumorous or the nontumorous tissues of the same individual. However, the differences between the adduct-forming capacity of DNA samples from different individuals were both remarkable and reproducible in five different experiments using tumorous and nontumorous DNA samples from patients 2, 5, 6, and 7, and tumorous DNA samples from patients 11 and 13. In Vitro Stability of Aflatoxin B,Formamidopyrimidine Adducts and Reproducibility of the Adduct Formation To test the stability of the AFB,-FAPyr adducts formed in vitro with HP DNA, aliquots of the for 12 mo (HP-l, adducts were stored at -20% Figure 2B) and compared with adducts formed freshly with DNA from the same lot of HP DNA (HP-2). A slight decrease in the intensity of HP-l compared with HP-2 was observed. The results in-

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Figure 1. Alkaline phosphatase staining of AFB,-FAPyr in the liver of the patients with HCC. A. The dark pinkish (dark gray in this black and white photomicrograph) cells (arrow] are mostly located at the periphery of a cirrhotic nodule (X160).B. Diffuse accumulation of AFB,-FAPyr was found in the cytoplasm as well as in the nucleus (arrows). Strong reactions were observed on some nuclear membranes (arrowhead) (X 640).

dicate that the in vitro adducts are quite stable for at least 1 yr. The influence of the purity of the DNA samples on their adduct forming capacity was also determined using DNA samples collected from the same liver specimen at different stages in the phenolkhloroform extraction procedure. Irrespective of the level of DNA purification, the observations of adduct formation remained unchanged for any given sample (data not shown). The lower limit of sensitivity of the immunoblot assay for detection of the adduct was found to be between 1 and 5 pm01 of AFB,, bound to DNA, per blotted sample, and the assay was shown to be linear in the range of adduct quantities used in the blotting experiments, as determined by parallel studies per-

formed with radioactively labeled aflatoxin B, (data not shown).

Discussion Aflatoxin B, is the most toxic and carcinogenic member of a family of difuranocoumarins produced as secondary metabolites by strains of Aspergillus flavus and related fungi (13). In animals it is primarily metabolized by the microsomal mixed function oxygenase system mainly localized on the endoplasmic reticulum of liver cells. During the course of AFB, metabolism, the highly reactive electrophilic epoxide can react covalently with various nucleophilic centers in cellular macromolecules including DNA, RNA, and proteins (8). Both in vivo

November

Figure

1989

AFLATOXIN B,-DNA ADDUCTS IN HUMAN LIVER CANCER

2. In vitro binding of AFB, to individual genomic DNA with or without HBV DNA, analyzed by DNA immunoblot analysis. A. Each AFB,-DNA adduct formed in vitro was spotted into six wells with serial dilutions as indicated. The amount was immunochemically detected using monoclonal antibody to AFB,-FAPyr. PLCI PRF-5, HepG2-2-15, 13T, and l5T (marked with black dots) contained integrated HBV DNA (see text). B. The identical experiments were performed 1 yr later to test the in vitro stability and reproducibility of AFB,FAPyr. HP-1 denotes AFB,-human-placental-DNA adduct formed 1 yr before HP-2 was freshly formed for this experiment.

and in vitro, the major aflatoxin-DNA adduct formed in the presence of an appropriate metabolic activation system is 2,3,-dihydro-2-(N-7-guanyl)-3-hydroxiaflatoxin B, (AFB,-N7-Gua) (8). Deoxyribonucleic acid-aflatoxin B, adducts are unstable both in vivo (31)and in vitro (32,33) and break down to AFB,FAPyr, the persistent adduct. The biologic and carcinogenic significance of this stable adduct has been emphasized by Groopman et al. (34). Aflatoxin B, has been shown to be a potent carcinogen in many species of animals, including fish, rats, and nonhuman primates (8). Susceptibility to AFB, carcinogenity is also proven in rhesus monkeys (14). As free AFB, is detectable in human liver (35,36), it is possible that AFB, might be involved in human hepatocarcinogenesis (17,3 7),especially in relation to HBV infection (9,17).However, there is not enough data to support the hepatocarcinogenic role of AFB, in humans (38,39). In an attempt to resolve some of these controversies, we have attempted to demonstrate the presence

1285

of the permanent AFB,-FAPyr adduct rather than free AFB, in human liver. We have shown its presence in four nontumorous liver samples of 14 patients with HCC. The most plausible explanation for the absence of AFB,-FAPyr in the tumorous samples of the 4 patients is that the AFB,-adducts have been highly diluted by the rapid cell proliferation in the tumor. Our immunohistochemical demonstration of AFB,-FAPyr adducts in nontumorous human liver from HCC patients suggests a possible causative role for the adducts in human hepatocarcinogenesis. The microscopic findings of cytoplasmic as well as nuclear reaction are consistent with the observations made in rats injected with large amounts of AFB, (40). The prominent cytoplasmic reaction is consistent with the findings of Niranjan et al. (4l), who have described a preferential attack of the AFB, on mitochondrial DNA during hepatocarcinogenesis in rats. However, we were able to demonstrate the presence of AFBi-FAPyr adducts in only four of 14 nontumorous liver sections from HCC patients, while the adducts were absent in three normal autopsy liver control samples. If increases in the detection sensitivity of the current monoclonal antibody assay were able to demonstrate adducts in nontumorous liver tissue of additional HCC patients without demonstration of such adducts in additional normal control patients, these observations would support a role for AFB,-FAPyr adducts in the pathogenesis of HCC. Aflatoxins are among the few chemically identified widely disseminated environmental carcinogens. The character and intensity of human exposure may vary depending on factors such as age, nutritional status, concurrent exposure to other agents (e.g., HBV), as well as the level and the duration of the exposure. The differences between species in susceptibility to AFB,-FAPyr adduct formation (13,42) and the differences we have found between the individual patients suggest that the susceptibility to AFB,-FAPyr adduct formation might be genetically restricted. The presence or absence of aflatoxin adducts in liver specimens is readily detected by immunohistologic methods. There is no known method to determine the extent of aflatoxin exposure of a given individual during his or her lifetime. The commonly used methods determine the amount of free aflatoxin or aflatoxin adducts present at one point in time in a urine or a liver sample (15,43-49). However, over time, the dietary intake of aflatoxin can be extremely variable. Therefore, there is no way to determine the extent of cumulative aflatoxin exposure, and it is difficult to establish a dose-response relationship between the amount of intake and the amount of

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6.

7. 8.

9.

10. Figure 3. Diagramatic scheme illustrating the conceptual scope of investigational studies of HCC in humans. The shaded areas represent other background studies and unshaded areas identify new opportunities for research, specifically focusing on the host genetic factors.

11.

12.

adduct found in tissue samples at a given time. The examination of urine samples can give information about the level of exposure only if performed regularly over a long period of time. Studies performed in different species, including primates, suggest that aflatoxin exerts its carcinogenic effect only if administered often enough, in the appropriate dosage, and for a certain period of time (8,14,38). The accumulation of the permanent adduct may well depend on host factors as well as on the amount and the frequency of the dietary exposure (6,8,39,43,44). Thus, the variability in dietary exposure to AFB, and genetic susceptibility to adduct formation do not lend themselves to precise analysis. However, our protocols for in vitro adduct formation and immunoblot analysis provide possible means for the dissection of the host factors involved in hepatocarcinogenesis. To integrate the viral and mutagenic roles of AFB,-FAPyr in the etiology of HCC, we propose a conceptual framework for testing a hypothesis depicted in Figure 3.

13.

14.

15.

16.

17. 18.

19.

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Received September 27, 1988. Accepted May 5, 1989. Address requests for reprints to: Girish N. Vyas, Ph.D., Department of Laboratory Medicine, (M-501F), University of California San Francisco, California 94143-0100. Supported in part by grant 86A73 from the American Institute of Cancer Research and grant PO1 HL-36589 from the National Heart, Lung and Blood Institute. The authors thank Drs. Robert C. Lim, David C. Hohn, and Melappalayam S. Rajagopalan of UCSF for the surgically removed liver tissue specimens, Dr. George Acs of Mount Sinai Medical Center, New York, for the HepG2-2-15 cell lines, Dr. Philip K. Lane of UCSF for internal peer review of the manuscript, and Sadie McFarlane of UCSF for manuscript preparation.