Mutagenesis, Vol. 14, No. 5, 463-472,
September 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Genotoxicity testing of potassium canrenoate in cultured rat and human cells
Department of Internal Medicine, Division of Clinical Pharmacology and Toxicology, University of Genoa, Viale Benedetto XV 2, I-16132 Genoa, Italy
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Abstract |
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Potassium canrenoate (PC), a competitive aldosterone antagonist used as a diuretic and in the treatment of hypertension, was examined for its capacity to produce genotoxic effects in cultured rat and human cells. At subtoxic concentrations (10–90 µM) PC was found to induce a dose-dependent degree of DNA fragmentation, as detected by the Comet assay, and of DNA repair synthesis, as measured by quantitative autoradiography, in primary cultures of hepatocytes from rat and human donors of both genders. In rat hepatocytes both DNA fragmentation and DNA repair were more marked after 3 h than after 20 h exposure and in cultures from females than from males. In human hepatocytes from one male and two female donors, PC caused a similar effect in terms of DNA fragmentation, whereas DNA repair was detected in cultures from only two of the same three donors and was less marked than in rat hepatocytes. A modest but statistically significant increase in micronucleated cells was present in primary cultures of replicating rat hepatocytes exposed to 10 or 30 µM PC for 48 h, the response being, in this case also, more evident in females than in males. In contrast, PC did not induce micronucleus formation in human hepatocytes from two female donors. Any evidence of DNA fragmentation and micronucleus formation was absent in cultured human lymphocytes. Taken as a whole these findings support the hypothesis that hepatocytes activate PC to DNA-damaging reactive species. PC induced the observed genotoxic effects at concentrations close to those produced in humans by the administration of therapeutic doses, but these effects were as a whole more marked in rat than in human hepatocytes. Since PC shares the 17-hydroxy-3-oxopregna-4,6-diene structure with cyproterone acetate, chlormadinone acetate and megestrol acetate, previously found to be genotoxic to both rat and human hepatocytes, the potential carcinogenic hazard of this type of steroids cannot be neglected.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Potassium canrenoate (PC) is a competitive aldosterone antagonist used as a diuretic and in the treatment of hypertension. When this steroid was administered to rats by diet admix for 1 or 2 years at doses ranging from 20 to 270 mg/kg, a dose-related increase in myelogenous leukemia and statistically significant increases in malignant tumors of the liver, thyroid, brain and mammary gland were observed (Cook et al., 1988
). It has been shown (Cook et al., 1988, 1993) that PC is metabolized by rat hepatic S9 to 6
,7- and 6ß,7ß-epoxycanrenone; the ß-epoxide is further metabolized to its 3- and 3ß-hydroxy derivatives; both 3- and 3ß-hydroxy-6ß,7ß-epoxycanrenone are direct acting mutagens in the L5178Y (TK+/TK–) mouse lymphoma assay, whereas 6,7- and 6ß,7ß-epoxycanrenone are not. The chemical structure of PC (Figure 1) is similar to that of three progestins, cyproterone acetate (CPA), chlormadinone acetate (CMA) and megestrol acetate (MGA), which have recently been shown to form DNA adducts (Topinka et al., 1993, 1995; Werner et al., 1997; Feser et al., 1998) and to induce DNA repair synthesis (Martelli et al., 1995, 1996a) in both rat and human hepatocytes. PC shares with CPA, CMA and MGA the keto group at C3 and the two double bonds C4=C5 and C6=C7; moreover, reduction of the keto group at C3 is a step in the activation to reactive species common to PC and CPA (Kerdar et al., 1995; Werner et al., 1996). Taking into account all these findings, we deemed that further information on the genotoxic activity of PC would be useful to better assess its potential carcinogenic activity in humans as well as adding to the knowledge of structural features accounting for the DNA-damaging activity of this type of steroid, which are all derivatives of 17-hydroxy-3-oxopregna-4,6-diene. Our study was carried out to examine the capability of PC at inducing DNA fragmentation, DNA repair synthesis and an increase in the frequency of micronucleated cells in primary cultures of both rat and human hepatocytes and in human peripheral blood lymphocytes. The rationale for the choice of this strategy was the opportunity to use different target cells and different genetic end-points. Primary hepatocytes maintain the metabolic capability of the liver for the first 24 h of culture and therefore possess a comprehensive biotransformation capacity, whereas lymphocytes are essentially deficient of monooxygenases catalyzing the biotransformation of xenobiotics. Testing not only in rat but also in human hepatocytes was supported by the previously recognized species specificity of some carcinogens (Martelli, 1995). The concurrent evaluation of DNA fragmentation and DNA repair synthesis in hepatocytes takes into account that in mammalian cells the type of DNA excision repair depends on the nature of the DNA lesion (Regan and Setlow, 1974); chemicals producing bulky adducts which elicit a long patch type of DNA repair, as inducers of unscheduled DNA synthesis (UDS), are more easily revealed by autoradiographic techniques, whereas chemicals inducing a short patch type of DNA repair produce a high frequency of DNA breaks and are more easily revealed by the Comet assay. Testing for the induction of micronuclei is based on the knowledge that genotoxic agents may be capable of eliciting DNA damage and/or repair but fail to induce changes in chromosome structure and number which can be detected by an increase in the frequency of micronucleated cells.
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Materials and methods |
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Chemicals
Potassium canrenoate (PC), collagenase type IV, Phaseulus vulgaris lectin (PHA-M), cytochalasin B, epidermal growth factor (EGF), Williams' medium E (WME), Eagle's minimal essential medium (MEM), RPMI 1640 medium, mitomycin C and N-nitrosodimethylamine (NDMA) were purchased from Sigma Chimica (Milan, Italy); methyl methanesulfonate (MMS) from Aldrich Chimica (Milan, Italy); insulin and fetal bovine serum from Boehringer Mannheim Italia (Milan, Italy); [methyl-3H]thymidine (sp. act. 23–25 Ci/mmol) from Amersham (Amersham Italia, Milan, Italy). All the other chemicals were of the purest grade available.
Cytotoxicity, DNA-damaging activity and DNA repair induction
Rat hepatocytes were isolated from Sprague–Dawley male and female albino rats (200–250 g) by collagenase perfusion, as described by Williams (1977). The percentage of viable cells, as measured by the trypan blue exclusion method, ranged from 80 to 90%. Human hepatocytes were obtained from fragments of liver discarded during the course of prescribed surgery essentially according to Strom et al. (1982). Donor HH 124 was a 72-year-old female who underwent surgery for hepatic metastasis of colon carcinoma; donor HH 126 was a 68-year-old female who underwent surgery for gall bladder carcinoma; donor HH 128 was a 62-year-old male who underwent surgery for hepatic metastases of colon carcinoma. The proportion of viable cells after perfusion was 81% in donor HH 124, 87% in donor HH 126 and 90% in donor HH 128. Isolated hepatocytes were suspended in WME supplemented with 10% fetal bovine serum and gentamicin (50 µg/ml). Aliquots of these suspensions were plated in 60 mm uncoated plastic dishes (2x106 cells/dish) for the DNA fragmentation Comet assay and in 35 mm dishes coated with rat tail collagen (1x106 cells/dish) for determination of cytotoxicity and DNA repair synthesis. At the end of a 3 h attachment interval at 37°C in a 95% air/5% CO2 atmosphere, hepatocytes were washed and incubated for a further 3 or 20 h in the presence of the test compound in serum-free medium. PC was directly dissolved in the medium immediately before use. NDMA was used as the positive control to verify the capability of hepatocytes to activate procarcinogens. DNA fragmentation and repair were assayed immediately after the end of exposure to the test compound. Human lymphocytes were isolated from samples of peripheral blood from healthy non-smokers aged 25–35 years by Ficoll Paque density gradient centrifugation. These cells were suspended in phosphate-buffered saline (PBS), exposed for 3 h to the test compound and immediately after assayed for DNA fragmentation. MMS was used as the positive control.
The presence of DNA fragmentation was evaluated in both hepatocytes and lymphocytes by the Comet assay. The procedure was essentially that described by Singh et al. (1988). Ten microliters of cellular suspension (>10 000 cells) was mixed with 75 µl of low melting point agarose at 37°C and then added to normal melting point agarose-coated microscope slides. The slides were immersed in cold lysing solution overnight and then placed in an electrophoresis tray with an alkaline solution (300 mM NaOH, 1 mM Na2EDTA, pH 13) for 20 min to allow DNA to unwind. Electrophoresis was conducted at room temperature for 20 min at 25 V and 300 mA (0.83 V/cm). The slides were washed, stained with ethidium bromide and examined at 400x magnification using a fluorescence microscope. Images of 50 randomly chosen cells from two slides were examined and DNA migration was measured with a scaled ocular. The length of DNA migration was taken as a measure of DNA damage (Rojas et al., 1996). The statistical analysis for the comparison of the pooled data between control and each dose level was performed using ANOVA followed by Dunnet's test.
For DNA repair assay, rat or human hepatocytes were simultaneously exposed to the test compound and [methyl-3H]thymidine (10 µCi/ml) and after 20 h incubation DNA repair synthesis was evaluated according to the autoradiographic method developed by Williams (1977) with minor modifications (Brambilla et al., 1989). Silver grains over the nucleus minus the grains over a randomly chosen equal sized area in the cytoplasm was defined as net nuclear grains (NNG). Cytoplasmic labeling was also considered in order to assess a possible effect of the test compound on mitochondrial DNA. Data are the means of 100 counts obtained from two autoradiographs from each donor. According to recommendations for the performance of UDS tests (Madle et al., 1994), the criterion for positive results was the occurrence over at least two consecutive concentrations of a dose-dependent increase in NNG exceeding our laboratory-specific threshold (NNG treated – NNG control = 5).
Micronucleus assays
The induction of micronuclei in rat and human hepatocytes was evaluated essentially as described by Hwang et al. (1993). Hepatocytes, isolated as reported in the preceeding paragraph, were suspended in serum-free MEM (0.4 mM Ca2+) supplemented with non-essential amino acids and 50 µg/ml of gentamicin (medium A) and plated in 60 mm dishes coated with rat tail collagen (104 cells/cm2). At the end of a 3 h attachment interval, the medium was removed and the cultures were refed with medium A supplemented with insulin (10–7 M), dimethyl sulfoxide (2%), EGF (20 ng/ml) and serial concentrations of the test compound. After a 48 h incubation, cultures were washed and refed with medium A supplemented with insulin (10–7 M) and EGF (20 ng/ml). Incubation was stopped 48 h later by washing cultures with cold PBS. Hepatocytes were exposed for 7 min to a hypotonic shock in 0.01 M KCl and then fixed with methanol:formalin:acetic acid (85:10:5). Cells were stained with May Grünwald–Giemsa and scored at 1250x magnification. Micronucleated hepatocytes were counted regardless of the number of micronuclei per cell. Scoring was limited to cells with intact nuclear and cellular membranes: no distinction was made with respect to cellular ploidy. Only small bodies lying in close proximity to the nucleus, with shapes and staining properties like the parent nucleus and a diameter less than one-third of that of the main nucleus were identified as micronuclei. Statistical analysis was performed using the method of Bailey (1959).
Micronucleus induction in human lymphocytes was examined using whole blood cultures. For each culture 0.3 ml of whole blood were added to 4.7 ml of RPMI 1640 medium supplemented with 15% fetal bovine serum, 5 mg/l of PHA-M and 50 µg/l of gentamicin. Test compound dissolved in saline was added 24 h after starting culture. After a 20 h exposure cells were centrifuged (250 g, 8 min), washed with PBS, resuspended in fresh medium containing 5 mg/l of cytochalasin B and incubated for an additional 28 h. At the end of incubation, cultures were centrifuged, washed and treated for 5 min with a weak hypotonic solution (0.075 M KCl) and then fixed in fixative (ethanol:acetic acid 3:1) for 5 min at room temperature. Slides were prepared by centrifuging aliquots of cell suspensions (50 µl) at 30 g for 5 min in a Cytospin 2 (Shandon, UK) centrifuge. The smears were air dried and stained with May Grünwald–Giemsa in Sörensen buffer (pH 6.7). Three slides were examined for each culture and at least 1000 binucleate cells were scored. The criteria used to identify micronuclei and the method employed for statistical analysis were the same as described for hepatocytes.
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Results |
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DNA damaging activity
A preliminary cytotoxicity assay showed that a 20 h exposure to 100 µM PC produced a 24% reduction in the fraction of viable trypan blue-excluding primary hepatocytes from male rats; at 200 µM no viable cells were present. In order to avoid toxicity-induced non-specific effects, PC was tested at 10, 30 and 90 µM; the relative survival values listed in Tables IV and V
indicate that these PC concentrations were subtoxic for both rat and human hepatocytes.
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The DNA-damaging activity of PC was evaluated in primary hepatocytes from rats of both genders in order to identify a possible sex specificity. Table I
lists separately for the two sexes the average length of migration of nuclear DNA observed in three independent experiments, as well as the means obtained from the pooled data; Figure 2 shows the distribution of DNA migration. Migration was measured for 50 nuclei/experiment and the statistical analysis was performed using the pooled 150 nuclei of the three experiments for each concentration level. After a 3 h exposure to PC concentrations ranging from 10 to 90 µM a statistically significant increase in the migration of DNA was observed in hepatocytes from both male and female rats at all the three doses tested, the frequency of DNA breaks being dose dependent. After a 20 h exposure the increase in DNA migration in hepatocytes from both male and female rats was consistently lower than that observed after 3 h exposure. A comparison of the degree of DNA fragmentation induced by PC in hepatocytes from male and female rats indicates that after 3 h exposure the genotoxic effect was to a limited extent more marked in females than in males. Under the same experimental conditions PC induced a degree of DNA fragmentation substantially similar to that observed in rat hepatocytes in primary hepatocytes from three human donors (Table II and Figure 3), however, taking into account that DNA fragmentation was measured in hepatocytes from two female donors after 20 h exposure and in hepatocytes from one male donor after 3 h exposure, no conclusions can be drawn about the possible influence of length of treatment or of sex of donor.
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The results of experiments performed with cultured human lymphocytes from a male and a female donor in order to evaluate the DNA-damaging effect of PC in cells essentially devoid of the enzyme systems catalyzing biotransformation of xenobiotics are shown in Table III
and Figure 4. Taking into account that in primary rat hepatocytes the greatest amount of DNA fragmentation was present after a 3 h exposure, human lymphocytes were exposed to PC for the same time interval and the same concentrations were used to allow correct comparison. In contrast to rat hepatocytes, human lymphocytes did not exhibit any increase in the migration of nuclear DNA. NDMA and MMS, used as positive controls, produced the expected positive response in hepatocytes and lymphocytes, respectively.
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Induction of DNA repair synthesis
The data in Table IV
indicate that a 20 h exposure to PC concentrations ranging from 10 to 90 µM elicited DNA repair synthesis in primary hepatocytes from both male and female rats. A dose-dependent increase in both the number of NNG and the percentage of cells carrying out repair was detected in six independent experiments performed with cells from three different donors of each sex. Consistent with the results of the Comet assay, induction of DNA repair was to some extent more marked in hepatocytes from female than from male rats. Under the same experimental conditions, PC induced DNA repair in primary hepatocytes from one female and one male human donor, but in both cases the increase in NNG was lower than that observed in rat hepatocytes (Table V). The response of the hepatocytes from a second human female donor (HH 126) should be considered equivocal due to the high degree of DNA repair displayed by the control cultures. The constant occurrence of DNA repair in rat and human hepatocytes exposed to the procarcinogen NDMA, used as a positive control, demonstrates their metabolic competence.
Micronucleus assays
Induction of micronuclei after 48 h exposure to 10, 30 or 90 µM PC was examined in primary rat hepatocytes from donors of both genders stimulated to proliferate by a low calcium concentration, insulin and EGF. As shown by the data listed in Table VI, a modest but statistically significant increase over corresponding controls in the frequency of micronucleated cells was present at 30 µM PC in two of three independent experiments on male rat hepatocytes and at 10–30 µM PC in all three experiments on female rat hepatocytes. The lower or no increase in micronucleus frequency observed at the 90 µM concentration should be considered as the consequence of a PC-induced reduction in cell proliferation. Pooled data show that the clastogenic effect of PC was more marked in hepatocytes from female than in those from male rats. Under the same experimental conditions PC did not induce a statistically significant increase in micronucleated hepatocytes in cultures prepared from two female human donors (Table VII). NDMA, used as a positive control, produced the expected positive effect in both rat and human hepatocytes.
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Any statistically significant increase in micronucleus frequency was absent in cytokinesis-blocked human lymphocytes exposed for 20 h to the same PC concentrations tested on rat and human hepatocytes (Table VIII
), but a reduction in the mitotic index was observed. Mitomycin C, used as a positive control, produced the expected clastogenic effect.
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Discussion |
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In spite of sufficient evidence for a carcinogenic activity of PC in rats, data on the genotoxic activity of this drug are, to our knowledge, limited to the observation that it is metabolized by rat hepatic S9 to reactive species acting as mutagens in the L5178Y TK+/– mouse lymphoma cell assay (Cook et al., 1988
). PC shares the 17-hydroxy-3-oxopregna-4,6-diene structure (Figure 1) with three other steroids, CPA, CMA and MGA, previously found to form DNA adducts (Topinka et al., 1993, 1995; Werner et al., 1997; Feser et al., 1998) and to induce DNA repair synthesis (Martelli et al., 1995, 1996a) in both rat and human hepatocytes. In this study we examined whether PC is genotoxic to rat and human hepatocytes and to human lymphocytes; this was in order to acquire further information on its genetic effects and to identify possible qualitative or quantitative differences in activity between rats and humans. In primary rat hepatocytes PC produced dose-dependent DNA fragmentation and DNA repair synthesis. After a 3 h exposure DNA fragmentation was slightly more marked in hepatocytes from females than from males and in cultures from both genders the frequency of DNA breaks decreased when the exposure was prolonged to 20 h. This reduction in the amount of DNA damage might be tentatively ascribed to a rapid repair of DNA lesions. Consistent with these findings, PC elicited dose-dependent DNA repair synthesis in primary hepatocytes from rats of both genders and this effect was also more marked in cultures from females than in cultures from males. Under the same experimental conditions PC induced a similar effect in terms of DNA fragmentation in primary hepatocytes from three human donors, whereas the degree of DNA repair was lower than in rat hepatocytes. Due to the limited number of donors it was not possible to establish whether the DNA-damaging activity is influenced in human hepatocytes by length of exposure and sex of the donor, as was found to occur in rat hepatocytes. A comparison of these results with those previously obtained with CPA, CMA and MGA (Martelli et al., 1995, 1996a) indicates that PC is more active as an inducer of DNA repair and, unlike the three progestins, also produces a positive response in hepatocytes from male rats. In rat hepatocytes a promutagenic character of PC-induced DNA lesions is suggested by the 3.3-fold increase in the frequency of micronucleated cells observed in cultures from female donors which, as for DNA damage, displayed a sensitivity to this steroid greater than that of cultures from males. This effect was quantitatively similar to those observed in the liver of female rats with CPA, CMA and MGA (Martelli et al., 1995, 1996b), which produced 6.6-, 4.5- and 2.3-fold increases in the average frequency of micronucleated hepatocytes, respectively. In contrast to rats, any substantial evidence of micronucleus formation was absent in human hepatocytes from two female donors. This negative response, which is consistent with the lower degree of DNA repair observed in human hepatocytes, might be considered as suggesting that rats are more susceptible than humans to the genotoxic activity of PC. However, this hypothesis should be interpreted with caution because of the limited number of human donors so far examined. The absence of both DNA fragmentation and micronucleus induction in human lymphocytes exposed to PC supports the hypothesis of a metabolic activation pathway similar to that carrying out the biotranformation to reactive species of CPA, CMA and MGA.
In conclusion, our findings indicate that PC is genotoxic to rat hepatocytes and, even if probably to a lower extent, to human hepatocytes. In this respect, it is worth noting that the concentrations of PC found to induce dose-dependent genotoxic effects (10–90 µM) are close to the 8 µM concentration produced in humans by the therapeutic dose of 200 mg (Ramsay et al., 1976). Taking into account that PC differs from CPA, CMA and MGA, previously found to be genotoxic to both rat and human hepatocytes, in the absence or different nature of some substituent groups but shares with these molecules the 17-hydroxy-3-oxopregne-4,6-diene structure, all steroids of this type should be examined for potential genotoxic activity.
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Acknowledgments |
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This research was supported by a grant from MURST (Italy) targeted project `New Assessment Approaches in Toxicology' (1996–1997) and by funds from Genoa University.
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1 To whom correspondence should be addressed. Tel: +39 010 353 8800; Fax: +39 010 538232; Email: farmdimi{at}unige.it
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Received on December 16, 1998; accepted on June 1, 1999.
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