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Mutagenesis, Vol. 17, No. 3, 257-260, May 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press

Fumonisin B1 is genotoxic in human derived hepatoma (HepG2) cells

Veronika Ehrlich,3, Firouz Darroudi1,3, Maria Uhl, Hans Steinkellner, Markus Zsivkovits and Siegfried Knasmueller,2

Institute of Cancer Research, University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria and 1 Department of Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Centre, 2333 AL Leiden, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and discussion
 References
 
Fumonisin B1 (FB1), a widespread Fusarium toxin which is frequently found in corn, causes liver tumors in laboratory rodents and is a suspected human carcinogen. The compound was tested in micronucleus (MN) and single cell gel electrophoresis (SCGE) assays in human derived hepatoma (HepG2) cells and caused a pronounced dose-dependent genotoxic effect at exposure concentrations 25 µg/ml. In contrast, no induction of his+ revertants was found in Salmonella microsome assays with strains TA98, TA100, TA102, TA1535 and TA1537 upon addition of HepG2-derived enzyme (S9) mix in liquid incubation assays with identical exposure concentrations. Taken together, our results indicate that FB1 is clastogenic in human derived cells. This observation supports the assumption that this compound may act as a genotoxic carcinogen in humans.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and discussion
 References
 
Fumonisin B1 (FB1) is produced by different mold species, of which Fusarium moniliforme and Fusarium proliferatum are the most important ones, as they frequently infect corn crops around the world (for a review see Bezuidenhout et al., 1998Go). In contrast to aflatoxins, fumonisins have frequently been found in corn and corn products of industrialized western countries (IARC, 1993Go). Epidemiological evidence suggests that FB1 causes acute toxic effects, such as leukoencephalomalacia in horses and pulmonary oedema in pigs (IARC, 1993Go). Consumption of contaminated corn has also been linked to the risk of esophageal cancer in humans in the Transkei region of South Africa, China and other countries (Yang, 1980Go; Marasas et al., 1981Go, 1988Go; Sydenham et al., 1990Go). Evidence for the carcinogenic properties of this compound is further supported by results of experiments with laboratory rodents (Jaskiewicz et al., 1987Go; Gelderblom et al., 1991Go, 1996Go). Gelderblom et al. (1996) investigated the mechanisms of FB1-induced neoplasia in rodents: they found initiating effects only at high, almost toxic doses, whereas promoting effects were observed at much lower concentrations. The results of currently available in vitro genotoxicity assays do not fully support the assumption that FB1 is a genotoxin: negative results were found in DNA repair assays with primary rat hepatocytes (Gelderblom et al., 1992Go; Norred et al., 1992Go) and also in Salmonella/microsome assays with rat derived S9 mix (Gelderblom et al., 1991Go; Park et al., 1992Go; Knasmüller et al., 1997Go; Aranda et al., 2000Go). Evidence for a direct genotoxic effect was only reported in a Mutatox assay with Vibrio fischeri (Sun and Stahr, 1993Go), and in a recent investigation with mice induction of micronuclei (MN) in polychromatic erythrocytes in the bone marrow was found (Aranda et al., 2000Go).

The aim of the present study was an investigation of the genotoxic effects of FB1 in a human derived hepatoma cell line (HepG2). This line has retained phase I and phase II enzyme activities and has been successfully used for the detection of genotoxic carcinogens, including compounds which gave false negative results in conventional in vitro assays (for a survey see Knasmüller et al., 1998Go; Uhl et al., 2000Go). FB1 was tested in these cells in MN assays which reflect clastogenic as well as aneugenic effects (Marzin, 1997Go). Furthermore, single cell gel electrophoresis assays (SCGE assays) were also carried out: These tests are based on the migration of DNA in an electric field and detect single- and double-strand breaks as well as alkali-labile sites (Tice et al., 2000Go). A protocol for a SCGE assay with HepG2 cells has been developed and validated recently in our laboratory (Uhl et al., 1999Go, 2000Go). Additionally, we also carried out Salmonella/microsome assays with strains TA98, TA100, TA102, TA1535 and TA1537 in order to elucidate whether an enzyme extract (S9 mix) from HepG2 cells is able to convert the mycotoxin to a bacterial mutagen. It has been shown earlier that HepG2 S9 is able to activate representatives of different classes of promutagens (Darroudi and Natarajan, 1993Go; Duverger-van Bogaert et al., 1993; Knasmüller et al., 1998Go). In all experiments aflatoxin B1 (AFB1) was included for reasons of comparison and as a positive control.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and discussion
 References
 
Chemicals
FB1, AFB1, cyclophosphamide, ethidium bromide, trypan blue and cytochalasin B were purchased from Sigma (St Louis, MO). FB1 and AFB1 were dissolved in sterile dimethylsulfoxide (DMSO). 2-Amino-3-methylimidiazo[4,5-f]quinoline (IQ) was obtained from Toronto Research Chemicals (Toronto, Canada). Dulbecco's minimal essential medium (DMEM), trypsin and antibiotics came from PAA (Linz, Austria). Low melting point (LMP) agarose, normal melting point (NMP) agarose and fetal calf serum (FCS) were obtained from Gibco (Paisely, UK). Inorganic salts for SCGE assays and DMSO came from Merck (Darmstadt, Germany). Agar and Nutrient Broth no. 2, which was used for cultivation of the Salmonella strains, were obtained from Oxoid (Basingstoke, UK).

HepG2 cells and bacterial strains
Human HepG2 cells were kindly provided by G.Dalner (University of Stockholm, Sweden). The cells were stored in deep frozen portions at –20°C and cultivated in DMEM supplemented with 15% FCS and 1% penicillin/streptomycin in 250 ml culture flasks (Greiner, Kremsmuenster, Austria). The Salmonella tester strains were a gift from B.Ames (Berkely, CA) and were kept on master plates at 4°C in the dark. The plates were replaced every 6 weeks. HepG2 S9 mix was prepared according to the protocol of Duverger-van Bogaert et al. (1993) with slight modifications: The cells were grown in large culture flasks (182 cm2) from Greiner to a final titer of 3x107 cells/flask. Then they were trypsinized and centrifuged and the pellet was resuspended in 1.0 ml of buffer per 5x106 cells. The suspensions were sonicated (Branson Sonifier 250) and subsequently the homogenates were centrifuged (20 min, 9000 g, 4°C), the supernatants collected and stored deep frozen in cryo tubes in 2.0 ml portions. The protein concentration of the enzyme homogenate was 22 mg/ml (Bradford et al., 1976).

Bacterial assays
Overnight cultures of the indicator strains TA98, TA100, TA102, TA1535 and TA1537 were grown in Nutrient Broth no. 2 for 8–12 h. Subsequently the cultures were centrifuged (10 min, 9000 g) and resuspended at 10% of the initial volume in phosphate-buffered saline, pH 7.2. The incubation mixtures consisted of 10 µl of the concentrated bacterial suspensions, 200 µl of the HepG2 S9 mix and 10 µl of FB1 solution. The mixtures were incubated for 30 min in a rotary shaker at 37°C in the dark and subsequently plated with 2.0 ml overlay agar on histidine-free selective agar plates. The plates were incubated for 2 days at 37°C in the dark, then the numbers of colonies were counted manually. Three plates were cultured in parallel per experimental point.

Micronucleus assays
The MN assays were carried out according to the protocol of Darroudi and Natarajan (1991). Briefly, the cells were cultivated for 2 days in culture flasks (25 cm2 filter top flasks; Greiner) in a CO2 atmosphere at 37.5°C and 96% relative humidity. Subsequently the medium was changed and the cells exposed to different concentrations of the test compounds or to the solvent for 24 h. All compounds were dissolved in DMSO; the final DMSO concentration in the medium did not exceed 1.0%. In order to evaluate the frequency of MN in binucleated human HepG2 cells, cytochalasin B (final concentration 3.0 µg/ml) was added to the growth medium after treatment and washing. HepG2 cells were fixed after 28 h. For fixation the cells were trypsinized and treated with cold hypertonic KCl solution (5.6 g/l) and subsequently air dried preparations were made. For the detection of MN in binucleated cells the slides were stained with 2.0% aqueous Giemsa solution (Gurr R66). For each experimental point three cultures were treated in parallel and 1000 cells were evaluated from each culture for MN induction. To study the effects of FB1 on cell division the number of binucleated cells (%) relative to the number of mono-, tri- and tetranucleated cells was determined.

In addition, a preliminary experiment was carried out in which the induction of MN by FB1 was studied after 1 and 24 h exposure. In this experiment only one concentration was used.

SCGE assays
SCGE assays were carried out as described by Uhl et al. (1999, 2000) according to the guidelines developed by Tice et al. (2000). Briefly, the cells were exposed in regular size Petri dishes (60x15 mm; Greiner) to the test compound for 24 h. To terminate the treatment the cells were trypsinized (0.1%, 5 min), lysed and transferred to agarose-coated slides. After electrophoresis the DNA was stained with ethidium bromide (10 µg/ml, 5 min) and the slides evaluated under a fluorescence microscope (Nikon model 027012) with an automatic image analysis system based on the public domain program NIH Image (Helma and Uhl, 2000Go). For each experimental point, three cultures were treated in parallel and from each culture the tail lengths of 50 cells were measured. In each experiment the viability of the cells was determined by the trypan blue method (Lindl and Bauer, 1994Go) and only cultures in which the viability of the cells after exposure was >=80% were analyzed.


   Results and discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
References
 
Figure 1Go shows that dose-dependent induction of MN was found in the human derived cells in the range 25–200 µg/ml (Figure 2AGo). At the highest dose tested the MN frequency was ~3-fold higher than in the untreated control. The mutagenic effect was paralleled by a decline in cell division, which is shown in Figure 2BGo. With AFB1, which was used as a positive control, a pronounced effect was seen at much lower concentrations; 3.0 µg/ml induced a 1.7-fold increase over the background rate.



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  		Fig. 1. . The chemical structure of fumonisin B1.

 



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  		Fig. 2. . Effects of FB1 on MN formation and cell division in HepG2 cells. (A) The number of MN induced after treatment for 24 h with different concentrations of FB1. Bars represent means ± SD of MN numbers found in three cultures. In each culture, 500 binuclear cells were evaluated for MN induction. 0, negative control; 0+S, solvent control (DMSO). AFB1 was used as a positive control; 3 µg/ml induced on average 55 ± 12 MN per 1000 binuclear cells. (B) The number of binuclear cells relative to the number of mono-, tri- and tetranuclear cells under identical experimental conditions. Each bar represents the mean ± SD of results obtained with three cultures per experimental point.

 
The results of a preliminary experiment where the cells were exposed for either 1 or for 24 h are shown in Figure 3Go. It can be seen that short exposure caused a less pronounced effect compared to that seen with the longer treatment period.



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  		Fig. 3. . Effect of exposure time on the induction of MN by FB1 in HepG2 cells. The cells were exposed to FB1 for 1 and 24 h. Only one culture was made per experimental point.

 
The effects of FB1 are not restricted to MN induction. Figure 4A and BGo shows that the mycotoxin also caused a significant induction of DNA migration (comet formation) in the human derived cells. Again, a pronounced effect was seen with concentrations >=25 µg/ml.



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  		Fig. 4. . Induction of DNA migration by FB1 in HepG2 cells. The cells were exposed to FB1 under identical conditions as in the MN assays. Subsequently the cells were analyzed for induction of comets as described in Materials and methods. (A) Means ± SD of results obtained with three cultures per exposure concentration. From each culture 50 cells were evaluated. (B) The extent of DNA migration at the individual cell level. The tail lengths of the comets after exposure to AFB1 (0.3 mg/ml), which was used as a positive control, were on average 56 ± 12 µm.

 
In a further series of experiments we investigated whether HepG2 derived S9 mix is capable of converting FB1 to metabolites which are mutagenic in the Salmonella tester strains TA98, TA100, TA102, TA1535 and TA1537. It is known from earlier experiments that FB1 gives negative results in the absence of activation mix and with rat S9 mix in these strains (Gelderblom et al., 1991Go; Park et al., 1992Go; Knasmüller et al., 1997Go; Aranda et al., 2000Go). The present experiments were carried out as liquid preincubation assays and the exposure concentrations were similar to those used in the MN and SCGE experiments with intact HepG2 cells in order to create comparable experimental conditions. Table IGo shows that negative results were obtained with FB1 under all test conditions, whereas with AFB1 (10 µg/plate) and the other positive controls a pronounced induction of his+ revertants was found with HepG2 S9 mix. Note that the concentration range in the bacterial assays was limited in comparison to recommended protocols for routine testing of compounds (Gatehouse et al., 1990Go).


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  		Table I. . Effect of fumonisin B1 on the induction of his+ revertants in Salmonella strains TA98, TA100, TA102, TA1535 and TA1537 after incubation with HepG2 S9 mixa
 
The most important finding of the present study is that it is the first report which gives evidence that FB1 is mutagenic in human derived liver cells. As described above, information on the DNA-damaging effects on FB1 are scarce and controversial. Our findings clearly show that the mycotoxin causes MN induction as well as DNA migration in intact cells, whereas no effects were seen in the Ames test with HepG2 derived enzyme homogenate. These characteristics allow us to draw some conclusions on the mechanisms involved. The formation of MN is either due to chromosomal breakage or spindle disruption. Since chromosome loss is not detectable in the Comet assay, which reflects only single- and double-strand breaks and alkali-labile sites (Tice et al., 2000Go), it is likely that MN formation is due to clastogenic effects. Note in this context that we reported earlier that FB1 causes structural chromosome aberrations in primary rat hepatocytes in vitro (Knasmüller et al., 1997Go).

Furthermore, the kinetics of MN induction (Figure 3Go) indicate that the compound requires metabolic activation by enzymes present in HepG2 cells. The lack of a positive result in the bacterial assays might be due to the fact that phase II enzymes are not adequately represented in the enzyme homogenate (Wiebel et al., 1980Go). However, since the activities of certain phase I enzymes are also low in HepG2 S9 mix prepared from non-induced cells (Knasmüller et al., 1998Go), it cannot be excluded that this accounts for the negative Salmonella assays. This latter assumption is also supported by the fact that AFB1 and the other positive controls (which are activated by phase I enzymes) caused only moderate effects in the HepG2 S9 mix/Salmonella assay. Although the lowest effective dose levels of AFB1 determined in previous studies with conventional Aroclor-induced rat liver S9 mix in strains TA100 and TA98 differ over a broad range, they were consistently 2–3 orders of magnitude lower than those required to cause measurable effects in the present experiments with HepG2 S9 mix (IARC, 1993Go). Consistently negative results were also obtained with FB1 in earlier bacterial experiments with rat S9 mix (Knasmüller et al., 1997Go).

To our knowledge no reports on serum levels and tissue concentrations of FB1 in exposed humans are available, but several articles on the pharmacokinetics of this compound in different animal species have been published (Prelusky et al., 1994Go, 1995Go; Shepard et al., 1995Go). Treatment of vervet monkeys (Cercopithecus aethiopus) with FB1 (6.5 mg/kg body wt) led to maximal plasma concentrations between 90 and 210 ng/ml. Extrapolation of these data to humans indicates that consumption of strongly contaminated foods (with FB1 levels between 50 and 150 mg/kg; IARC, 1993Go) may lead to maximal exposure levels between 0.6 and 2 mg/kg body wt. This would lead to serum levels <=70 ng/ml. These concentrations, which may occur in humans only under extreme exposure conditions, are still more than 2 orders of magnitude lower than the doses required to cause measurable effects in hepatoma cells. However, it cannot be excluded that primary human cells are more sensitive to FB1. In our in vitro experiments with primary rat liver cells induction of chromosome breaks was seen with FB1 doses as low as 1.0 µg/ml (Knasmüller et al., 1997Go). As mentioned above, several drug-metabolizing enzymes are less active in HepG2 cells than in primary human hepatocytes (Knasmüller et al., 1998Go). Therefore, it is possible that fresh liver cells are more sensitive to FB1 than HepG2 cells. We know from earlier studies that hepatoma cells are less sensitive to DNA damage caused by certain carcinogens than intact hepatocytes, typical examples being nitrosamines and heterocyclic aromatic amines (Knasmüller et al., 1999; Uhl et al., 2000Go).

Taken together our findings support the assumption that FB1 might be a genotoxic carcinogen in humans, but in order to elucidate whether DNA-damaging effects can be expected under realistic exposure conditions in man, further experiments are required.


   Acknowledgments
 
The experimental work was sponsored by an EU grant (HEPADNA, QLRT-1999-00810).


   Notes
 
2 To whom correspondence should be addressed. Tel: +43 01 4277 65142; Fax: +43 01 4277 9651; Email: siegfried.knasmueller{at}univie.ac.at Back

3 The first two authors contributed equally to this work Back


   References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 

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Received on August 6, 2001; accepted on January 16, 2002.


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