Mutagenesis, Vol. 14, No. 6, 595-604,
November 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Genotoxic effects of benzyl isothiocyanate, a natural chemopreventive agent
Institute for Cancer Research, Borschkegasse 8a, A-1090 Vienna, Austria and 1 Institute of Hygiene and Toxicology, Federal Research Center for Nutrition, Karlsruhe, Germany
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Benzyl isothiocyanate (BITC) is contained in cruciferous plants which are part of the human diet. Numerous reports indicate that BITC prevents chemically induced cancer in laboratory animals and it has been postulated that BITC might also be chemoprotective in humans. On the other hand, evidence is accumulating that this compound is a potent genotoxin in mammalian cells by itself. To further elucidate the potential hazards of BITC, we investigated its genotoxic effects in different in vitro genotoxicity tests and in animal models. In in vitro experiments [differential DNA repair assay with Escherichia coli, micronucleus assay with human HepG2 cells and single cell gel electrophoresis (SCGE) assay with hepatocytes and gastrointestinal tract cells] pronounced dose-dependent genotoxic effects were found at low dose levels (
5 µg/ml). In contrast, substantially weaker effects were obtained in in vivo experiments with laboratory rodents: in the differential DNA repair assay with E.coli cells, only moderate genotoxic effects were seen in indicator cells recovered from various organs of mice after treatment with high doses (between 90 and 270 mg/kg), while in SCGE assay with rats a change in the DNA migration pattern was seen at a dose level of 220 mg/kg body wt. These findings indicate that BITC is detoxified under in vivo test conditions. This assumption was supported by the results of in vitro experiments which showed that the genotoxic effects of BITC are markedly reduced by bovine serum albumin and human body fluids such as saliva and gastric juice. Additional experiments carried out on the mechanistic aspects of the genotoxicity of BITC showed that this compound causes formation of thiobarbituric acid-reactive substances in HepG2 cells and that its DNA damaging properties are diminished by 
-tocopherol, vitamin C, sodium benzoate and ß-carotene, indicating the possible involvement of free radicals in the genotoxicity of BITC. The doses of BITC required to cause measurable DNA damage in laboratory rodents exceeded by far the dietary exposure levels of humans, but are similar to those which were required to inhibit chemically induced cancer in earlier animal experiments. |
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Isothiocyanates (ITCs) are breakdown products of glucosinolates which are secondary plant constituents of cruciferous vegetables. Numerous earlier studies on ITCs indicate that these compounds protect laboratory rodents against chemically induced cancer (Wattenberg, 1977
, 1981, 1983; Chung et al., 1985; Morse et al., 1989; Chung, 1992). The anticarcinogenic effects of ITCs were attributed to the inhibition of metabolic activation reactions (Chung et al., 1985; Chung, 1992) and/or induction of detoxifying enzymes (Sparnins et al., 1982). Recent epidemiological studies have also indicated that increased consumption of vegetables is associated with a reduction in the incidence of different human cancers, in particular tumors of the gastrointestinal tract (Negri et al., 1991; Steinmetz and Potter, 1991a,b; Block et al., 1992). One of the most intensively studied ITCs with regard to cancer chemoprevention is benzyl isothiocyanate (BITC), a product of enzymatic hydrolysis of glucotropaeolin. BITC is contained in high amounts in garden cress (Lepidium sativum), papaya (Carica papaya) and common Brassica vegetables (Tang, 1971; VanEtten et al., 1976; Fenwick et al., 1983; Pintao et al., 1995) and some studies show the potential of this compound as a chemopreventive agent in man (Wattenberg, 1977, 1981, 1983; Chung et al., 1985; Chung, 1992; Zhang and Talalay, 1994).
We recently started to investigate the antigenotoxic effects of BITC towards mutagenic heterocyclic aromatic amines in short-term genotoxicity assays (Knasmüller et al., 1996) and in the course of our studies we found, quite unexpectedly, that this compound causes DNA damage by itself. In light of the critical importance of DNA damage in the induction of cancer by genotoxic carcinogens and since a closely related compound, allyl isothiocyanate, was found to be genotoxic (Yamaguchi, 1980) and carinogenic (IARC, 1985), we aimed, in the present study, to investigate the genotoxic potential of BITC in different in vitro and in vivo short-term genotoxicity tests with a variety of end-points. In the first part of the study, we investigated the in vitro effects of BITC in a gene mutation assay with Salmonella typhimurium, a micronucleus (MN) assay with human-derived hepatoma (HepG2) cells, a differential DNA repair assay with Escherichia coli and a single cell gel electrophoresis (SCGE) assay with rat and human gastric mucosa cells as well as primary rat hepatocytes and colonocytes. In subsequent experiments, attempts were made to compare the effects seen under in vitro conditions with results from in vivo experiments, where relatively high doses of BITC were fed to mice and rats, in differential DNA repair and SCGE assays, respectively. Furthermore, since the genotoxic effect induced by BITC under in vivo condition was much weaker than that observed in in vitro tests, the potential antigenotoxic role of saliva, gastric juice and proteins was studied. Finally, in experiments where an attempt was made to shed light on the molecular mechanisms which are responsible for the genotoxicity of BITC, we investigated the protective effects of free radical scavengers towards the genotoxic effect of BITC and induction of thiobarbituric acid-reactive substances (TBARS) by this compound.
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Chemicals
BITC, vitamin E, ß-carotene, vitamin C, mannitol, sodium benzoate, ethidium bromide, NADP, glucose 6-phosposphate, benzo[a]pyrene [B(a)P], streptozotocin (SZ), ethylnitrosurea (ENU) and N-nitrosodimethylamine (NDMA), 4-nitro-o-phenylenediamine (NPD), tris-hydroxymethylaminomethane (Trizma base), 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), laurylsarcosine sodium salt, Triton X-100, dithiothreitol and proteinase type XII were from Sigma (St Louis, MO). Neutral red, trypan blue, 2,6-di-tert-butyl-4-methylphenol, trichloroacetic acid and malonaldehyde bis(diethylacetal) and inorganic salts for bacterial assays were purchased from Merck (Darmstadt, Germany). Collagenase P from Clostridium histolyticum was obtained from Boehringer Mannheim (Mannheim, Germany). Dulbecco's modified Eagle's medium and Eagle's minimal essential medium were obtained from Gibco Laboratories (Paisley, UK). Normal and low melting point agarose were from Biozym diagnostic (Hameln, Germany). Thiobarbituric acid (TBA) was from BDH Chemicals (Poole, UK). Bacterial media were from Difco (Detroit, MI), except for Nutrient Broth No. 2 which came from Oxoid (Basingstoke, UK). Liver enzyme homogenate (S9 mix) was prepared from male Aroclor 1254-induced F-344 rats and was purchased from Orgon Technics (Durham, NC).
Indicator cells
Salmonella typhimurium strains TA98 and TA100 were obtained from B.Ames (University of California, Berkley, CA). Escherichia coli strains were from G.R.Mohn (RIVM, Bilthoven, The Netherlands). The human HepG2 cells were a gift from F.Darroudi (University of Leiden, Leiden, The Netherlands).
Laboratory animals
Male Swiss albino mice (~25 g) were purchased from the Himberg breeding facility (Himberg, Austria). Male Fischer 344 rats (250 g) were bred at the animal facility of the Federal Research Centre for Nutrition (Karlsruhe, Germany). The animals were kept in a room that was maintained at a constant temperature and humidity (24 ± 1°C, 50 ± 5%) with a 12 h light/dark cycle. The animals were allowed to acclimatize to the new environment for 2 weeks before the beginning of the experiment.
Bacterial gene mutation assay
Salmonella His+ reversion assays were carried out with strains TA98 and TA100 as plate incorporation assays (Maron and Ames, 1984) or according to the preincubation protocol of Yamaguchi (1980). Per experimental point, three plates were evaluated and each experiment was repeated at least twice. The S9 homogenates were composed according to the standard recipe of Maron and Ames (1984).
Differential DNA repair assay with E.coli strains
Induction of repairable DNA damage is measured in this test procedure by comparing the viability of two indicator strains which differ vastly in their DNA repair capacity (Mohn, 1983). The two strains can be distinguished by their color and morphology. Strain 343/753 (uvrB/recA) is Lac+ and forms red colonies on neutral red agar while strain 343/765 (uvr+/rec+) is Lac– and forms white colonies. The strains can be used for in vitro assays (liquid incubation experiments) as well as for animal-mediated assays in which the indicator cells are recovered from different organs of animals treated with test chemicals. Whereas the in vitro DNA repair test has been validated with a number of model compounds and has a similar sensitivity in the bacterial gene mutation assay (Hellmer and Bocsfoldi, 1992a), the predictive value of the in vivo assay is comparable with that of the bone marrow micronucleus assay with rodents (Hellmer and Bocsfoldi, 1992b).
Both the in vitro and animal-mediated assays were carried out as described in detail by Knasmüller et al. (1996, 1998). For the in vitro assay, briefly, cultures of the two indicator strains, E.coli 343/753 (uvrB/recA) and 343/765 (uvr+/rec+), were grown overnight and mixed to give a final titer of 1–2x108 cells/ml of each strain. Incubation mixtures contained 0.1 ml of the bacterial mix, 0.1 ml of test compound dissolved in DMSO and 0.8 ml of S9 mix or the same amount of phosphate-buffered saline (PBS). The incubation tubes were rotated for 2 h at 37°C in a rotary shaker. Thereafter, the mixtures were diluted 10–4 in ice-cold PBS and 0.1 ml aliquots spread over neutral red agar plates. Finally, the plates were incubated for 24 h at 37°C and kept at room temperature for another 12 h and the number of surviving repair-proficient and repair-deficient colonies was determined by manual counting. The differential survival rate is the ratio of the viability of the two strains (Knasmüller et al., 1992a,b); 100% survival indicates absence of genotoxicity while survival rates <100% indicate genotoxicity resulting from induction of repairable DNA damage. Three incubation tubes were used per experimental point and five plates were plated for each incubation tube.
In animal-mediated assays, the bacterial mix used for in vitro assays was concentrated 20-fold and 0.2 ml aliquots (4–8x109 viable cells of each strain) were injected into the lateral tail vein of male Swiss albino mice (body weight 25 g) that had been deprived of food for 24 h but had received water ad libitum. Immediately thereafter, BITC (dissolved in corn oil) was administered to the animals by gavage (30, 90 or 270 mg/kg). Two hours later, the animals were killed by cervical dislocation and liver, lung, kidneys, stomach, small intestine and colon removed, washed with PBS, transferred into tubes with PBS (2 ml) and homogenized (Yistral YG 1024). Subsequently, the organ suspensions were diluted with PBS, 0.1–0.3 ml aliquots plated on neutral red agar plates, the plates incubated and the individual survival rates of the two strains was determined by manual colony counting. Three animals were treated per experimental point and three plates were enumerated per organ.
Sampling of body fluids
Saliva was obtained from a 30-year-old male whereas gastric juice was collected from a 35-year-old male volunteer who had fasted overnight. The test body fluids were added directly to the incubation mixtures (sterilization to prevent bacterial infection was not necessary for the differential DNA repair assay with E.coli, as the plate medium contains large quantities of streptomycin).
MN assay with HepG2 cells
Prior to the MN assay, the cytotoxic effect of BITC was determined with a spectrophotometric viability assay which is based on the reduction of tetrazolium salt to dark colored formazan (Mosmann, 1983). The MN assay was performed as described by Natarajan and Darroudi (1991). Briefly, cells were grown to confluence, exposed for 1 h to various amounts of BITC, washed with PBS and grown in medium which contained cytochalasin B (3 µg/ml) for 20 h. Then the cells were fixed, trypsinized and treated with hypotonic KCl solution (0.15 M). Finally, air-dried preparations were made and stained with Giemsa (Gurr 66). Three slides were evaluated per experimental point with a total of 2100 binucleated cells (BNC) (700 BNC/slide).
SCGE assay
For the in vitro assay, primary rat hepatocytes were isolated by the two-step collagenase perfusion technique as previously described (Berry and Friend, 1969; Bradley et al., 1982). Isolation of primary cells of the gastric and colon mucosa was carried out according to the procedures of Burlinson (1989) and Brendler et al. (1992), respectively. In order to study the effect of gastric juice on the genotoxicity of BITC, the endogenous preformed mucus was removed by washing the gastric mucosa cells with PBS while allowing it to accumulate in parallel cell suspensions. Human biopsy samples obtained during gastroscopy were incubated with digestion mix (2:1 protease:collagenase). The study was approved by the ethical commission of the university of Heidelberg. The resulting suspensions were centrifuged, resuspended in medium and checked for viability by the trypan blue method (Lindl and Bauer, 1994). Treatment of cells was carried out by exposing 0.5x106 human or 2x106 rat cells/ml to various concentrations of BITC in DMSO for 1 h at 37°C in a shaking water bath. Subsequently, survival rates were determined; incubation mixtures with viability <80% were not analyzed.
To study the effects of BITC in vivo, two series of experiments were carried out. In the first, rats received a single oral dose of 220 mg/kg BITC in corn oil and the extent of DNA damage was determined after different exposure times (1–4 h) in gastric mucosa cells. In the second experiment, the effects of two dose levels (110 and 220 mg/kg) were investigated in gastric and colon mucosa cells. In the latter case, exposure time was 2 h. The animals were killed by cervical dislocation and the gastric and colon mucosa cells isolated as described for the in vitro assays.
Microgel electrophoresis was performed according to Singh et al. (1991). For preparation of the slides, 100 µl of 0.5% normal melting point agar was transferred to precleaned slides, covered with a coverslip and the agarose allowed to solidify on a cooled metal plate. After solidification of the agar, the slides were covered with another 75 µl of 0.7% low melting point agarose and then submersed into lysis solution (100 mM Na2EDTA, 1% Triton X-100, 2.5 M NaCl, 1% N-lauroylsarcosine sodium salt, 10% DMSO, 10 mM Tris, pH 10, for at least 60 min). All slides were placed in an electrophoresis chamber containing alkaline buffer (1 mM Na2EDTA, 300 mM NaOH, pH 13) for DNA unwinding. After 20 min, the current was switched on and electrophoresis was carried out at 25 V, 300 mA for 20 min. The slides were removed from the alkaline buffer and washed three times (5 min each) with neutralization buffer (0.4 M Tris, pH 7.5). Slides were stained with ethidium bromide (20 µg/ml, 100 µl/slide). All steps beginning with the isolation of cells were conducted under red light.
In the in vitro test, three incubation mixtures were prepared for each concentration of the test compound and from each individual incubation mix 101 cells were evaluated (Pool-Zobel et al., 1994) by image analyses (Perceptive Instruments, Halstead, UK). In each in vivo experiment, one animal was used per experimental point and three slides (101 cells/slide) were evaluated.
Induction of TBARS in HepG2 cells
The assay was carried out according to Yagi (1982). Briefly, 2x106 cells in 0.5 ml of 1.2% KCl were exposed for 1 h at 37°C to BITC (10–40 mg/ml) and subsequently mixed with a reaction mixture containing 500 µl of 0.5% TBA in distilled water and 500 µl of 0.001% 2,6-di-tert-butyl-4-methylphenol. This mixture was incubated at 100°C for 30 min and the reaction stopped by cooling on ice for 5 min. Afterwards, 1 ml of acetic acid and 2 ml of chloroform were added and the tubes vortexed and centrifuged at 2000 r.p.m. for 10 min to separate the aqueous and solvent phases. Finally, the upper solvent layer was removed and the fluorescence measured at an excitation of 530 nm in a luminescence spectrometer (DU640; Beckmann, USA). Two independent experiments with three different concentrations were carried out and for each concentration three parallel assays were performed.
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Induction of His+ revertants in Salmonella typhimurium strains TA98 and TA100
The results obtained in the plate incorporation assay with S.typhimurium strains TA98 and TA100 are depicted in Figure 1a and b
. It can be seen that BITC caused a moderate but dose-dependent effect. The maximum effect was seen in both strains at a concentration of 100 µg/plate. Exposure to higher doses caused a decline in the mutant frequencies in both test strains as a result of toxic effects and this was manifested as a dissolved background lawn. In the presence of a metabolic system, the effect of BITC was eliminated in both indicator strains. Liquid preincubation tests performed according to the protocol of Yamaguchi (1980) with both indicator strains gave consistently negative results (data not shown).
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) and presence (•) of rat liver S9 mix. Each experimental point represents the mean ± SD of revertant numbers of three plates. In experiments with TA98, NPD (20 µg/plate) was used as a positive control for tests without activation and AFB1 (2 µg/plate) was used in assays with metabolic activation; the revertant numbers were 1438 ± 241 for NPD and 431 ± 32 for AFB1. In tests with TA100, ENU (400 µg/plate) was used as a positive control in experiments without S9 and AFB1 (2 µg/plate) was used in tests with S9 activation; the corresponding revertant numbers were 272 ± 32 and 552 ± 40, respectively.Induction of MN in human-derived HepG2 cells
The results obtained in the MN induction assay with HepG2 cells are depicted in Figure 2
. For reasons of comparison, B(a)P was tested under identical conditions. It can be seen that low BITC concentrations (1–4 µg/ml) caused a dose-dependent induction of MN. At an exposure concentration of 4 µg/ml, the MN frequency was ~3-fold higher than the background level. B(a)P induced a pronounced effect even at the lowest dose (1 µg/ml). Figure 2 also shows that BITC is a potent cytotoxic compound; viability of HepG2 cells was reduced by ~70% upon exposure to 1 µg/ml BITC for 1 h.
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). The cells were exposed to various concentrations of the test chemicals for 1 h. The MN frequency was determined in binucleated cells (BNC) as described in Materials and methods.The upper part of the panel depicts the effect of the test compounds on the viability of the cells. Each experimental point represents the mean ± SD of micronucleated cells from 2100 BNC, 700 cells/slide.Induction of repairable DNA damage in E.coli strains
Induction of genotoxic effects by BITC in differential DNA repair assays with E.coli strains (343/753 and343/765) under in vitro conditions is depicted in Figure 3
. Exposure of indicator bacteria to low concentrations of the compound led to a pronounced reduction in the viability of the repair-deficient strain compared with the repair-proficient counterpart. This effect was of the same order of magnitude as that seen with the positive control SZ, a potent alkylating agent. A pronounced decline in the relative survival rate (>50%) was measured at a concentration of 1.5 µg BITC/ml. As in experiments with Salmonella, addition of liver homogenate resulted in a reduction in the genotoxic effect of BITC.
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Exposure of E.coli cells to BITC in different organs of mice resulted in clear-cut and dose-dependent differential DNA damage in the repair-deficient strain (Figure 4
). Moreover, regardless of the type of tissue from which the bacteria were recovered, the degree of DNA damage was almost similar. The concentrations required to cause pronounced effects were between 90 and 270 mg/kg body wt.
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SCGE assay
In the in vitro experiments, BITC caused a dose-dependent increase in DNA migration in human and rat gastric mucosa cells as well as primary rat hepatocytes. With rat gastric mucosa cells, a 6-fold increase in the tail moment over the background level was observed at a concentration 5 µg/ml (Figure 5a
). Due to the restricted availability of human cells, only one experiment was carried out and at an exposure concentration of 5 µg/ml the percentage of cells with comet-like structure increased from 8 to 17% (data not shown). The effect observed with primary rat hepatocytes was also clear cut and dose dependent (Figure 5b), however, hepatocytes were less sensitive than gastric mucosa cells to the genotoxicity of BITC.
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The results of various series of in vivo SCGE assays with rats are shown in Figure 6a–c
. These data are preliminary as only one animal was used per experimental point. Nevertheless, the results indicate that a high dose of BITC (220 mg/kg body wt) is required to cause a change in the pattern of DNA migration in gastric and colonic mucosa cells and that prolongation of exposure time for >2 h leads to a decrease in DNA damage.
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Effects of protein and body fluids on BITC-induced DNA damage
The impetus for this work came from results of earlier experiments where the genotoxic effects of BITC were dramatically reduced by S9 (with and without cofactors) and under in vivo test conditions. To determine whether direct non-specific binding to proteins is responsible for this reduction in the genotoxicity of BITC, we investigated the effect of bovine serum albumin (BSA), human saliva and human gastric juice using the in vitro differential DNA repair assay with E.coli cells. The effects of BITC were indeed reduced as a function of the concentration of BSA present in the incubation mix and totally abolished at a concentration of 9 mg/ml (Figure 7
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Though not as potent as BSA, human saliva and gastric juice also reduced repairable DNA damage by 40 and 45%, respectively (Figure 7
). A similar effect was seen with gastric juice in in vitro SCGE experiments with rat gastric mucosa cells (Figure 5a).
Effects of radical scavengers on the genotoxic effects of BITC in E.coli cells
To find out if radical scavengers reduce the genotoxic effects of BITC, a series of differential DNA repair assays with E.coli cells was carried out. -Tocopherol caused a clear dose-dependent inhibition of DNA damage (Figure 8a). Protection was also exhibited by all antioxidants tested (ß-carotene, sodium benzoate and vitamin C) but mannitol under identical experimental conditions (Figure 8b).
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Induction of lipid peroxidation by BITC
To support the assumption that free radicals are involved in the genotoxic effects of BITC, the potential of this compound to induce TBARS was studied using HepG2 cells. A dose-dependent formation of TBARS was indeed observed (Figure 9
), however, the concentrations required to cause a measurable effect were substantially (~103-fold) higher than those which were effective in the MN assays.
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Numerous reports indicate that cruciferous vegetables and their constituents, in particular ITCs, protect animals against chemically induced cancer and it has been hypothesized that the inverse association between the incidence of certain forms of cancer and an increased consumption of cruciferous vegetables might be due, at least in part, to the protective effects of organic ITCs (Steinmetz and Potter, 1991a
,b; Watzl and Leitzman, 1995). On the other hand, we recently reported that juices of cruciferous vegetables contain compounds that are direct-acting gentoxins in bacteria and mammalian cells (Kassie et al., 1995, 1996; Kassie, 1996). It is likely that these effects are due to the presence of ITCs. Musk and co-workers (Musk and Johnson, 1992, 1993; Musk et al., 1995) have repeatedly reported that ITCs induce genotoxic effects in rodent cell lines. Moreover, induction of His+ revertants in bacterial gene mutation assays was reported with a number of ITCs (Yamaguchi, 1980). Due to the causal relationship between genetic damage in somatic cells and initiation of cancer, these findings suggest that ITCs might be carcinogenic by themselves. Indeed, in the case of allyl-ITC it has been found that it causes bladder tumors in rats (IARC, 1985); to our knowledge, no data from rodent bioassays are available for other ITCs. The aim of the present investigation was to further elucidate the potential health hazards of BITC, a compound which is contained in Brassica vegetables and has been used in a large number of antimutagenicity/antigenotoxicity/anticarcinogenicity studies. In the first part of the present study we investigated the cytotoxic and genotoxic properties of BITC in bacteria as well as in human-derived HepG2 cells. Furthermore, we carried out comparative experiments in which the induction of DNA damage by BITC was measured under in vitro conditions and in laboratory rodents using identical indicator cells.
The results of the in vitro experiments indicated that BITC is an extremely potent genotoxin in E.coli and in human-derived hepatoma cells (Figures 2 and 3). The concentrations required to induce pronounced effects in both test systems were <2 µg/ml. Comparison with the effects caused by genotoxic model carcinogens in earlier differential DNA repair assays with E.coli strains and in Hep G2/MN tests revealed that BITC ranks among the most potent genotoxins ever tested in both systems (Hellmer and Bocsfoldi, 1992a; Knasmüller et al., 1998). Even more pronounced genotoxic effects were found by Musk and co-workers (Musk and Johnson, 1993; Musk et al., 1995) in experiments with Chinese hamster ovary cells where the percentage of chromosomal aberrations was doubled upon short exposure to 0.3 µg BITC/ml. The reason for the lower susceptibility of HepG2 cells is probably their metabolic capacity. In contrast to other cell lines which are currently used in genetic toxicology, HepG2 cells possess phase I and phase II enzymes (Knasmüller et al., 1998) and it is known that cytochrome P450 isozymes and glutathione S-transferases (GSTs) are involved in the metabolism of BITC (Bruggemann et al., 1986; Lee, 1996). However, compared with primary rat hepatocytes, HepG2 cells were more susceptible to the cytotoxic effects of BITC: exposure of HepG2 cells to 1 µg BITC/ml reduced cell viability to 30% while viability of primary rat hepatocytes was >80% even after being exposed to 5 µg BITC/ml. These apparent differences in susceptibility to the toxic effects of BITC might be attributed to variations in the expression of enzymes involved in the detoxification of BITC (Doostdar et al., 1988; Kusamran et al., 1998). In contrast to the pronounced effects seen with the E.coli test system, only marginal effects were obtained in plate incorporation assays with strains TA98 and TA100 at substantially higher exposure concentrations (100 µg/plate). The sensitivity of the test system did not increase even with the preincubation protocol described by Yamaguchi (1980).
In vivo differential DNA repair and the SCGE tests enabled a comparison with the in vitro effects of BITC as identical indicator cells and end-points are used. The effects of BITC in laboratory rodents are much lower than might be expected on the basis of the in vitro assays. In the host-mediated differential DNA repair assays with E.coli strains, a clear effect (reduction in the differential survival rate below 50%) was seen upon oral administration of 90–270 mg/kg body wt (Figure 4). It is also notable that the extent of DNA damage measured in bacteria which had been exposed in different organs of mice to BITC was quite similar, whereas pronounced organ differences were seen in earlier studies with genotoxic procarcinogens such as nitrosamines and heterocyclic aromatic amines (Knasmüller et al., 1992a,b, 1994). Likewise, in the in vivo SCGE assays with colon and gastric mucosa cells of rats, DNA damage was detectable only when the animals were exposed to a high dose of BITC (220 mg/kg body wt; Figure 6).
The strong discrepancy between the genotoxic effects of BITC seen under in vitro and in vivo conditions indicated that the compound is detoxified in the living animal. Therefore, we investigated the impact of proteins and body fluids on the DNA-damaging activities of BITC. The results obtained in bacterial tests with rat liver homogenate (Figures 1a and b and 3) and BSA (Figure 7) clearly indicate that the effects of BITC are reduced by non-enzymatic binding to proteins. Note that in this context genotoxic agents whose in vitro activity is decreased by metabolic systems are often not active or are poorly active in vivo (De Flora, 1978). In agreement with these observations, ITCs were reported to bind to egg white proteins accordingly; BITC preferentially reacted with amino groups and sulfhydryl chains of bovine proteins to form thiourea and dithiocarbamate derivatives (Rowel and Kroll, 1995). The results described in Figures 5b and 7 clearly show that body fluids such as saliva and gastric juice are also able to attenuate the DNA-damaging effects of BITC in bacteria and in rat mucosa cells. It is conceivable that protein binding is responsible for these effects; in the case of saliva, enzymatic processes might also be involved, as part of its protective effects against model mutagens could be attributed to peroxidase activity (Nishioka et al., 1981; Nishioka and Nunoshiba, 1986).
In the last part of our study we attempted to elucidate the mechanisms responsible for the genotoxic properties of BITC. Musk et al. (1995) found that this compound causes by far more chromosomal aberrations than sister chromatid exchanges in Chinese hamster ovary cells and stated that this pattern of activity is reminiscent of ionizing radiation and radiomimetic chemicals. To investigate whether free radicals might be involved in the genotoxic effects elicited by BITC, we tested the effect of radical scavengers on the genotoxicity of this compound in bacterial cells. Indeed, we observed protective effects by -tocopherol, sodium benzoate and ß-carotene (Figure 8), but not with mannitol. These dietary antioxidants as well as enzymatic free radical scavengers may contribute to the reduced genotoxicity of BITC under in vivo conditions. The assumption that free radicals might be involved in the genotoxicity of BITC was partly supported by the results of the TBARS assays, which indicated that this compound causes lipid peroxidation (Figure 9). However, TBARS formation was induced only at high BITC concentrations and this might be due to the poor sensitivity of tumor cells towards lipid peroxidation, which was found to be directly related to the degree of differentiation of the cells (Cheeseman et al., 1988).
Brassica vegetables from which BITC is formed contain antioxidant constituents such as polyphenols, flavonoids, carotinoids, chlorophyll and other antigenotoxic substances (Watzl and Leitzmann, 1995). The presence of these dietary protective factors as well as the moderate in vivo genotoxicity of BITC indicates that human exposure to BITC via consumption of vegetables is probably not associated with an increased risk of genetic damage. It has been estimated that the daily intake of glucosinolates (which are partly converted to ITCs) in the UK is ~43 mg/kg/person (Sones et al., 1984). The benzylaglycones formed from these glucosinolates are estimated to be <1% of the total ITCs. Since the amount of BITC required to cause genotoxic effects in rats or mice in the present experiments was ~200 mg/kg body wt, the actual human exposure is about four orders of magnitude lower.
On the other hand, the BITC doses required to reduce the tumor incidence in chemoprevention studies with rodents were relatively high. For example 40–100 mg/kg body wt/day prevented induction of neoplasias by NDMA and B(a)P in mice (Wattenberg, 1977), 32 mg/kg body wt of BITC given daily in the feed inhibited methylazoxy methanol acetate-induced intestinal tumors in rats (Sugie et al., 1994), and the dose of BITC reported to cause inhibition of 7,12-dimethylbenzanthracene-induced mammary tumors in rats by 60% was even higher than 200 mg/kg body wt/day (Wattenberg, 1981). Attempts to elucidate the chemopreventive mechanisms of ITCs have led to the assumption that their protective effects are primarily due to interactions with the metabolism of carcinogens. As in the anticarcinogenicity studies, relatively large BITC doses were required to cause changes in the activities of drug metabolizing enzymes: moderate inhibition of demethylation of nitrosamines in rats was seen after oral administration of 149 mg/kg (Chung et al., 1985); the amount required to cause a slight induction of -GST in rats was 75 mg/kg (Huber et al., 1997) and in a feeding experiment with rats and mice the overall GST activity was elevated after continuous consumption of daily doses 
50 mg/kg body wt (Sparnins et al., 1982; Benson and Barretto, 1985). Since these doses are close to those which caused an induction of DNA damage in mice and rats in the present study, considerations concerning the potential protective effects of BITC in humans should take into account its DNA-damaging properties.
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Acknowledgments |
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The authors are thankful to R.Schulte-Hermann (University of Vienna) for his support and encouragement. The experimental work was sponsored by an EC grant (to S.K.).
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2 To whom correspondence should be addressed. Tel: +43 1 4277 65143; Fax: +43 1 4277 65143; Email: profeka{at}yahoo.com
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Received on May 10, 1999; accepted on July 19, 1999.
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