Mutagenesis, Vol. 15, No. 2, 143-147,
March 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Cytokinesis-block micronucleus assay in primary human liver fibroblasts exposed to griseofulvin and mitomycin C
Dipartimento Uomo e Ambiente, Universita' di Pisa, Via S.Giuseppe 22, and 1 Istituto di Mutagenesi e Differenziamento del CNR, Via Svezia 10, 56100 Pisa, Italy
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Abstract |
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Primary liver fibroblasts were applied in a cytokinesis-block micronucleus assay in combination with fluorescence in situ hybridization (FISH) using two protocols. In protocol A (Prot. A), cytochalasin B (Cyt B) was added at the end of the treatment time directly to the medium containing the standard compounds, whereas in protocol B (Prot. B) the chemical-containing medium was removed and fresh medium with Cyt B was added. The study was performed using the aneugen griseofulvin (GF) and the clastogen mitomycin C (MMC) as standard compounds. With both protocols GF induced a significant increase in MN frequency over controls in a dose-related manner at the lower concentrations tested (7.5 and 15 µg/ml). At the highest dose (30 µg/ml) the aneugen effect was substantially reduced. MN induction obtained with Prot. A was significantly higher (~3-fold) than with Prot. B at the most effective concentration. The aneugen effect induced by GF did not change when different cell densities were used, but again with Prot. A we obtained the highest effect. MN induced by MMC showed a dose- and time-dependent increase in both protocols. In contrast to GF, the greater clastogenic response induced by MMC in human liver fibroblasts was obtained with Prot. B, ~3-fold higher than Prot. A at the most effective concentration and ~2-fold with 24 h treatment at 0.17 µg/ml MMC. With GF, the FISH data in human liver fibroblasts (80% C+MN) were fairly consistent with those obtained in the rodent cell lines. In human whole blood cultures, the same dose used in our experiment produced a relatively higher percentage of C+MN. FISH analysis showed that MMC induced mainly MN containing acentric fragments rather than whole chromosomes. In conclusion we have demostrated that chemically induced genetic effects are strongly dependent on the cell culture employed, treatment schedule and intra- and post-treatment experimental conditions.
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Introduction |
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The use of primary liver cells in genotoxicity testing, as compared with established cell lines or human peripheral lymphocytes, should ensure a more adequate and reproducible response, more closely reflecting the in vivo situation because they retain most of the differentiated functions expressed in the liver that are involved in the metabolism and repair processes.
To evaluate the feasibility and optimize the culture conditions of primary human liver fibroblasts in the cytokinesis-block micronucleus assay, two protocols (A and B) were applied using the aneugen griseofulvin (GF) and the clastogen mitomycin C (MMC) as standard compounds. GF interacts with the cell structure involved in chromosome segregation, probably by binding to microtubule-associated proteins and thereby inhibiting mitosis (Wehland et al., 1977
). Recently it has been shown that GF induces a 126-fold increase in cytochrome P4502A5 and a 10-fold increase in 7-hydroxylation of coumarin in mouse liver, even if the molecular mechanism(s) responsible for the up- and down-regulation of this isoenzyme is not fully understood at present (Salonpaa et al., 1995). This finding seems to be linked indirectly with hepatic inflammatory events, cell damage and necrosis in mice on a GF-supplemented diet before the appearance of hepatocellular carcinomas (Wastl et al., 1998). GF apparently modulates drug metabolizing enzymes and affects its own metabolism: 6-demethylation, but not 4-demethylation, was enhanced in rats and mice (Lin and Symchowicz, 1975). Up to now, however, no data have been available to demonstrate that GF is biotransformed to an aneuplogenic metabolite(s).
MMC requires metabolic activation to induce genotoxic effects. It is non-exclusively metabolized by two bioreductive mechanisms (one-electron and two-electron reduction) mediated respectively by NADPH-cytochromo P450 reductase and DT-diaphorase (Pan et al., 1984; Siegel et al., 1992). To verify the individual contribution of the reductive pathways under aerobic conditions on the observed clastogenic effects induced by MMC, dicoumarol, a potent and specific inhibitor of DT-diaphorase (Ki = 1–10 nM; Lind et al., 1982), was introduced into the experiments.
In addition, a fluorescence in situ hybridization technique (FISH) with a pancentromeric probe was introduced into the assay to evaluate chromosome loss quantitatively as a measure of the aneugenic and clastogenic potential of the two standard compounds in metabolically competent human liver cells.
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Materials and methods |
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Chemicals
GF, cytochalasin B (Cyt B) and dicoumarol were provided by Sigma Chemical Co. (St Louis, MO), whereas MMC was purchased from Kyowa Hakko Kogyo Co. (Japan). GF was dissolved in dimethylsulfoxide (DMSO; Merck, Germany) for spectroscopy, Cyt B in a mixture of physiological solution and 50% DMSO (v/v), whereas MMC was dissolved in serum-free Ham's F-12 immediately before treatment.
Cell culture and treatments
Primary human liver fibroblasts (HLF), obtained by enzymatic disaggregation of a male liver surgical fragment, were maintained in Ham's F-12 medium (Sigma Chemical Co.) supplemented with 7.5% fetal bovine serum (Gibco, Paisley, UK) and antibiotics at 37°C in a humidified atmosphere containing 6% CO2 in air. Under these experimental conditions the HLF doubling time was 24 h. Stock cultures were maintained in liquid nitrogen and periodically spot checked for mycoplasma contamination by fluorescent microscopy (Chen, 1977). The experiments were performed using cells in the interval of passages 3–8.
Confluent cultures were trypsinized and 1x105 cells (~17x103 cell/cm2) were seeded onto coverslips in 35 mm Petri dishes. Cells were exposed 24 h later to GF, MMC or DMSO, used as a negative control, never exceeding 1% in the medium. Optimal concentrations and exposure times were chosen according to preliminary cytotoxicity tests. GF exposure was performed for 24 h at 7.5, 15 and 30 µg/ml, whereas for MMC the treatment was carried out for 2, 5, 10 and 24 h, at a concentration of 0.17 µg/ml and at 0.04, 0.085, 0.17 µg/ml for 5 h. At the end of each treatment, Cyt B was added in a final concentration of 3 µg/ml and maintained for 24 h, according to the experimental procedure applied with lymphocytes (Migliore et al., 1996). Cyt B itself does not induce micronuclei (Prosses et al., 1988). In Prot. A, Cyt B was added at the end of the treatment time, directly to the medium containing GF or MMC, whereas in Prot. B the chemical-containing medium was removed, cells were washed twice with cold phosphate-buffered saline (PBS) and fresh medium containing Cyt B was added. In both protocols, cells were harvested 24 h after Cyt B addition.
To evaluate the role of DT-diaphorase in metabolic activation of MMC under aerobic conditions, dicoumarol, a strong inhibitor of this enzyme, was added to the culture medium 2 h before treatment with MMC, at 2 µM final concentration.
The ratio of percent binucleated to mononucleated cells was used as a parameter of cytotoxicity.
Slide preparation
For Giemsa staining, cultures were washed once with PBS and fixed twice with methanol:acetic acid (7:1). Coverslips were then stained with 3% Giemsa (Merck, Germany) in distilled water for 10 min, air dried and mounted on clean slides.
For FISH analysis, slide preparation was performed according to the following procedures. Briefly, cells were trypsinized, gathered in tubes and prefixed with methanol:acetic acid (3:5). Fibroblasts were centrifuged (4 min at 2000 g), the supernatant was removed and cells were fixed twice with cold fixative (methanol:acetic acid 7:1). Cell suspensions were dropped onto clean slides which were allowed to air dry, and finally stored at –20°C.
DNA probes and in situ hybridization on binucleated cells
In situ hybridization was performed using an alphoid centromere-specific DNA digoxigenated probe (P5095-DG 5; Oncor, USA). Slides were pretreated with a 10% pepsin solution (Sigma Chemical Co.) in 10 mM HCl for 10 min at 37°C. Slides were then washed twice in PBS and PBS containing 50 mM MgCl2, post-fixed with 1% formaldehyde in PBS containing 50 mM MgCl2 for 10 min at room temperature, rinsed in PBS, dehydrated in a 70–80 and 100% ethanol series at –20°C and air dried. Denaturation of slides was performed by immersion in 70% formamide, 2x SSC, pH 7.0, at 70°C for 2 min, followed by a second dehydration. After heating at 70°C for 5 min, 30 µl of probe was placed on the denaturated slides under a glass coverslip and incubated overnight at 37°C in a moist chamber.
Post-hybridization washes were performed twice in 2x SSC, pH 7.0, for 4 min each and then in 4x SSC, 0.05% Tween 20 (Sigma Chemical Co.) for 5 min, both at 37°C. To minimize the background, slides were preincubated for 10 min at 37°C in 4x SSC, 5% non-fat dry milk as immunological buffer (IB).
For detection of the probe we used a mouse anti-digoxigenin antibody (Boerhinger, Mannheim, Germany) followed by a TRITC-conjugated anti-mouse digoxigenin antibody and by a TRITC-conjugated anti-rabbit digoxigenin antibody (Sigma Chemical Co.). The antibodies were diluted in IB and alternately incubated for 30 min at 37°C. Each incubation step was followed by three washes in 4x SSC, 0.05% Tween 20 for 2 min at 37°C. After dehydrating through an ethanol series, slides were counterstained with 24 µl DAPI (Sigma Chemical Co.) dissolved in glycerol + DABCO (Sigma Chemical Co.) as antifade solution.
Slide scoring and statistical analysis
For each experimental point, 2000 (when possible) Giemsa stained binucleated cells from two independent cultures were scored on coded slides in order to determine micronucleus (MN) frequency according to the criteria proposed by Fenech (1993). MN frequency was expressed as the mean of the two experiments. The proportion of binucleated cells was estimated over 1000 scored fibroblasts as a parameter of cell toxicity.
In FISH experiments MN were classified for the presence of a fluorescent signal by considering a TRITC-labeled MN as a centromere-positive MN (C+MN). In order to evaluate the frequency of C+MN a total of 50 MN were scored for each experimental point.
For each treatment, Fisher's exact test was used to determine statistically significant differences between treatment and control, for both MN frequencies and FISH analysis.
First and second order regression analyses between chemical concentration, exposure, time and MN frequency were drawn.
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Results |
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Influence of the experimental protocol on the frequency of MN
The induction of MN after 24 h exposure of human liver fibroblasts to three concentrations of GF (7.5, 15 and 30 µg/ml) plus 24 h of Cyt B following Prot. A and Prot. B are summarized in Table I
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With both protocols GF induced a significant increase in MN frequency over controls, accompanied, at the lower doses, by a decrease in the percentage of binucleated cells. At the highest dose (30 µg/ml) the aneugen effect was substantially reduced, whereas, interestingly, the percentage of binucleated cells remained constant. MN induction obtained with Prot. A was significantly higher (~3-fold) than that obtained with Prot. B at the most effective concentration.
Experiments carried out to verify the influence of cell density (7x103, 12x103 and 17x103 cells/cm2) on the aneugenic effect induced by GF were negative (data not shown), but again we demonstrated that the presence of GF in the medium (Prot. A) gave rise to a significantly higher effect compared with Prot. B.
MN were induced by MMC (Prot. A and Prot. B) with a dose- and a time-dependent increase accompanied by a significant percentage decrease in binucleated cells (Tables II and III). In contrast to GF, the major clastogenic response induced by MMC in human liver fibroblasts was obtained with Prot. B (139.8 BN MN/1000 against 73.5 at 0.17 µg/ml for 24 h, ~2-fold). The relationships for both dose- and time-dependent increases were first order with Prot. A (r2 = 0.9974 and 0.9942, respectively), whereas these obtained with Prot. B showed a quadratic pattern (second order), whose equations were respectively y = 0.6797x2 + 22.015x + 3.306, r2= 0.9811 and y = 3926x2 + 1289.1x + 10.834, r2= 0.9955.
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In both protocols, pretreatment of human liver fibroblasts with dicoumarol led to a reduction in frequency of MN of 50% compared with the maximum effect obtained at 0.17 µg/ml (Table IV
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Pattern of chromosome segregation in cultured human liver fibroblasts after GF and MMC treatment
The application of FISH with a pancentromeric probe gave the results summarized in Table V
. In these experiments the protocol and the dose used for each compound was the most effective in MN induction, namely Prot. A for GF and Prot. B for MMC. The highest concentration of GF (30 µg/ml) was omitted due to the low frequency of MN induced. Compared with the control, the two concentrations of GF used significantly increased the frequency of C+MN.
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In comparison with the control value, MMC showed elevated and significantly different C–MN frequencies associated with increasing dose (P < 0.01).
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Discussion |
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GF, a mycotoxin produced by various species of Penicillum (Brian et al., 1946
), has been used extensively since 1965 for the treatment of dermatomycoses in humans (Bennett, 1995). Several findings indicate that the compound does not induce point mutations but is genotoxic, carcinogenic and teratogenic in rodents (Klein and Beall, 1972; IARC, 1976; Siracusa et al., 1980). Few data from human studies are at present available to exclude the possibility that the therapeutic use of GF poses a carcinogenic risk to humans (Schmaehl, 1981; Hoover and Fraumeni, 1981). Therefore, attemps have been made to elucidate the mechanisms responsible for the toxic effects of GF and to address the question whether such effects might occur in humans undergoing GF therapy.
It is well documented that GF is a spindle poison, so that induction of numerical chromosome aberrations and of MN in somatic cells may result from interference with tubulin polymerization and microtubule functions (Raimondi et al., 1989; Aardema et al., 1996). In particular, GF binding to microtubule-associated proteins (MAPs) seems to consistently affect the mode of action of the drug (Wehland et al., 1977).
In human liver fibroblasts the co-presence of GF and Cyt B in the culture medium (Prot. A) gives rise to a higher aneugen response (~3-fold) in comparison with Prot. B, where the chemical-containing medium was removed and the cultures were refed with fresh medium containing Cyt B.
GF arrests cells entering mitosis (Mullins and Snyder, 1979; Whittaker et al., 1993), therefore the presence of the drug in the medium for an additional 24 h with Cyt. B may damage a greater number of cycling cells with respect to Prot. B, where the drug-containing medium is removed after the first 24 h, so that the cells dividing in the absence of GF could dilute the aneugen response.
The exact mechanism of action of Cyt B is not yet clear and, so far, contradictory results have been obtained. It is known that Cyt B inhibits the polymerization of actin (MacLean-Fletcher and Pollard, 1980) and induces laggards in the first meiotic division of crane fly spermatocytes, suggesting a role of actin in chromosome segregation (La Fountain et al., 1992). In human lymphocytes Cyt B changes the balance between MN containing fragments and whole chromosomes (Norppa et al., 1993; Surralles et al., 1996; Falck et al., 1997) whereas in human fibroblasts Cyt B in combined treatment with the spindle poison colchicine consistently reduces MN induction (Antoccia et al., 1993).
A possible role of GF metabolism could be invoked to explain these results, but so far the few studies carried out on GF biotransformation in vivo and in vitro have failed to identify an aneugenic metabolite(s), whereas the known microbiologically inactive 6- and 4-hydroxylate forms, which represent the major metabolites in rodents and human (Riegelman et al., 1970; Chiou and Riegelman, 1971; Lin and Symchowicz, 1975), should be preferentially detoxified via transferase conjugation reactions.
An interesting finding is the observation of a substantial reduction in MN frequency at the highest tested dose of GF (30 µg/ml; Table I
) in the absence of cytotoxicity. Our data seem to suggest that another mechanism(s) could more effectively prevail than chromosome loss. However, the frequency of 80% C+MN obtained in our experiments is fairly consistent with those obtained by Stopper et al. (1994) at a dose of 100 µg/ml in mouse L5178Y cells (87% C+MN) and by Seelbach et al. (1993) in V79 cells (85% C+MN). In human whole blood cultures, the same dose of 15 µg/ml used in our experiment produced a relatively higher percentage (94%) of C+MN (Migliore et al., 1996). In addition, we found that GF at 7.5 and particularly at 15 µg/ml induced, in a dose–effect response, a substantial number of signal-negative micronuclei (20 and 34%, respectively).
MMC is a genotoxic anticancer drug which is used as a primary chemotherapy agent for anal, lung and superficial bladder cancers and as a secondary agent in breast, colon, gastric and pancreatic cancers (Powis, 1987).
MMC requires metabolic activation to produce reactive metabolites. Two pathways are involved in this reductive bioactivation. The first, catalyzed by NADPH:cytochrome P450 reductase, xanthine oxidase (Pan et al., 1984; Bligh et al., 1990) and cytochrome b5 reductase (Hodnick and Sartorelli, 1993), is the one-electron reduction of MMC to its semiquinone radical, leading in a successive reductive round to a `relatively' stable hydroquinone which may interact with DNA (depending on its alkylating potency) and/or be conjugated and excreted. The unstable semiquinone radical rearranges to generate two electrophilic centers that interact almost exclusively with the N2 of guanines to form either monoadducts (mono- or bi-reduced) or crosslinks at either CpG (interstrand) or GpG (intrastrand) sites (Pan et al., 1986; Tomasz, 1994). In addition, the semiquinone radical may also enter into futile redox cycling with the regeneration of MMC and the production of clastogen radical species.
The second pathway, mediated by NADPH:quinone oxidoreductase, which is also known as DT-diaphorase (Siegel et al., 1992; Pius et al., 1994), is the obligate two-electron reduction of MMC to form the corresponding hydroquinone, bypassing generation of the semiquinone radical. The hydroquinone metabolite also possesses potent DNA-alkylating activity (Rockwell et al., 1988; Siegel et al., 1990).
MMC has been detected as a potent mutagen and clastogen in many in vitro short-term tests (van Pelt et al., 1991; Turchi et al., 1992; Tafazoli et al., 1995). However, the degree of genotoxic effects of MMC varies considerably in the different systems (Ishidate et al., 1988; Whong et al., 1990; Matsuoka et al., 1993). In our cell system, MMC dose-dependently induces MN formation with both protocols used (A and B) and causes cell cycle delay due to cell accumulation in early and mid S phase (Ma et al., 1992; Tamura et al., 1992). In contrast to GF, MMC is more effective with Prot. B than with Prot. A.
MN induction shows (Prot. B) a quadratic dose and time response that could take into account both enhanced cell proliferation and the multienzyme system involved in MMC activation. This latter hypothesis is also supported by the experiments where dicoumarol, a strong inhibitor of DT-diaphorase, is introduced into the assay. MN frequency is drastically reduced by ~50%, demonstrating that MMC is metabolized competitively under aerobic conditions in human liver fibroblasts by cytochrome P450 reductase and DT-diaphorase (Lee et al., 1993; Koji et al., 1996).
FISH analysis showed that MMC, as a consequence of chromosome breakage, induced predominantly MN containing acentric fragments rather than whole chomosomes. However, at the highest dose tested we detected 82 C–MN and 18% C+MN. Similar results were found by other authors in human fibroblasts (Hennig et al., 1988; Ruud et al., 1991). This relatively large number of signal-positive MN would seem to indicate a preferential, but not exclusive, clastogenic property.
In this study we have added further evidence that cultured primary human cells can be succesfully employed in genotoxicity testing and that the induction of aneugenic and clastogenic effects are strongly dependent on the cell system and the experimental conditions used, including the treatment schedule, and also on the chemical compound tested. Further studies are necessary to understand the role of metabolism under the experimental conditions used.
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Acknowledgments |
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We thank Dr Francesca Degrassi for critical reading of the manuscript.
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Notes |
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2 To whom correspondence should be addressed. Tel: +39 050 574161; Fax: +39 050 576661; Email: g.turchi{at}imd.pi.cnr.it
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Received on August 9, 1999; accepted on November 4, 1999.
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