Mutagenesis, Vol. 15, No. 2, 137-141,
March 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Evaluation of antimutagenic effect of todralazine in cultured lymphocytes
siorowski1
Wroc
aw Medical University, Department of Basic Medical Sciences, 14 Kochanowskiego Str., 51-01 Wrocaw, Poland
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Abstract |
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Todralazine, an antihypertensive drug from the hydrazinophthalazine group, significantly decreased the activities of benzo[a]pyrene and mitomycin C in three short-term genotoxicity tests in human lymphocyte cultures. The thioguanine resistance test, the cytokinesis-blocked micronucleus assay and the sister chromatid exchange test were used to demonstrate the antimutagenicity of todralazine. Todralazine lowered the level of free radicals generated by human granulocytes in vitro in the presence of benzo[a] pyrene and also in the presence of the granulocyte activator and tumor promoter phorbol myristate acetate. These results, together with our previous data obtained in the standard bacterial Ames test, strongly suggest that todralazine is a good antimutagen in vitro and deserves further research on its inhibitory action on mutagenesis and carcinogenesis.
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Introduction |
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Identification of antimutagenic compounds and evaluation of the mechanisms of their action deserve special attention for their possible significance in the protection of human health. In previous studies we established that todralazine (TDR), an antihypertensive drug of the hydrazinophthalazine family, markedly decreased the mutagenic activity of several direct and indirect acting mutagens in the standard bacterial Ames test (Gasiorowski et al., 1993, 1994a, 1997). We also noticed that TDR markedly decreased the activation of several promutagens by the liver microsomal S9 fraction (Gasiorowski et al., 1993 et al., 1995). It is commonly accepted that the activation of promutagens to their genotoxic derivatives is a complex pathway mediated by microsomal mixed function oxidases and other enzyme systems that may involve the formation of free radicals (see for example Cavalieri and Rogan, 1992; Frenkel, 1992). The action of TDR on microsomal activation of promutagens could be explained by its inhibition of free radical generation and release in the presence of mutagenic agents.
The general chemical formula of todralazine (TDR) is given in Figure 1
. As suggested by its chemical structure shown in Figure 1, TDR can operate as a radical scavenger, because such an activity is an attribute of phenolic rings.
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The aim of the present study was to verify the antimutagenic activity of TDR against some standard mutagens in human lymphocyte cultures and also to evaluate its influence on free radical generation by granulocytes in vitro. Blood cells were separated from the venous blood of heavy smoker donors, who also suffered from mild primary hypertonia arterialis. We wanted to check whether TDR, often considered as a potential component of antihypertonic treatment of these patients, can also significantly decrease the genotoxic action of mutagens in their blood cell cultures. Higher exposure to genotoxic agents is obviously connected with the smoking habits of the donors.
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Chemicals
Todralazine–HCl (TDR), analytical grade, was kindly supplied by Polfa (Pabianice, Poland). Benzo[a]pyrene (B[a]P), cytochalasin B, mitomycin C, 5-bromo-2'-deoxyuridine, phorbol 12-myristate 13-acetate (PMA) and dimethylsulfoxide (DMSO) were purchased from Sigma (St Louis, MO).
The blood cell separation solutions (Histopaque-1077 and Histopaque-1119), as well as components of the cell culture medium [RPMI 1640, fetal calf serum (FCS) and L-glutamine] were also obtained from Sigma. Phytohemagglutinin (PHA-M) was obtained from Gibco (Gaithersburg, MD). The stains acridine orange, ethidium bromide, azur II, eosin B, trypan blue, Giemsa solution and nitroblue tetrazolium (NBT) were purchased from Sigma. The other reagents used for buffers and culture medium preparation were from POCH (Gliwice, Poland).
Blood cell separation
Heparinized blood was obtained by venipuncture from four male volunteers aged 40–50 who were heavy smokers (>30 cigarettes/day). The blood donors were picked from a group of hypertonic patients, periodically examined at Wrocaw Medical University in the framework of a special health care project. Except for suffering from a mild primary non-complicated hypertonia arterialis, these volunteers were generally healthy. The cells were separated from venous blood by a single-step discontinuous density gradient centrifugation technique with Histopaque-1119 and Histopaque-1077 layers (English and Anderson, 1974). Isolated lymphocytes and granulocytes were washed in phosphate buffered-saline (PBS, pH 7.2).
Sister chromatid exchange (SCE) test
Lymphocytes were cultured for 72 h. Plastic dishes (24-well) were seeded at a density of 5x105 cells/ml in complete culture medium (RPMI 1640, 10% FCS, 2 mM L-glutamine) and stimulated to mitogenesis with PHA-M (1% v/v). The promutagen B[a]P was dissolved in dimethylsulfoxide (DMSO) and added to the culture at a volume of 50 µl to obtain the desired final concentration. TDR dissolved in distilled water was added simultaneously to the culture at a volume of 50 µl and at the dose needed to obtain the defined final concentrations in the culture medium. The thymidine analogue bromodeoxyuridine (BrdUrd) was added for the last 48 h of the culture time to a final concentration of 30 µM. The cultures were harvested following a standard cytogenetic method. The cell smears on glass slides were air dried for 3 days. Afterwards, the slides were immersed in 2x SSC solution (0.03 M sodium citrate in aqueous 0.3 M sodium chloride solution) at 62°C for 30 min and, simultaneously, they were illuminated under a UV lamp (Perry and Wolff, 1974). Then the slides were stained with a mixture of dyes (azur II and eosin) to reveal differential chromatid staining (Antoshina and Poriadkova, 1978) and examined under a microscope. The results were expressed as mean SCEs/metaphase among 25 harlequin metaphases examined in each of the analysed slides.
Cytogenetic assessment of lymphocyte proliferation
The replication indices (RI) were estimated by counting the number of metaphases in the first (M1), second (M2) and third (M3) divisions in the presence of BrdUrd and calculated according to the formula RI = (M1 + 2M2 + 3M3)/100. The mitotic indices (MI) were determined on the slide for each culture for 1000 cells found randomly under microscope examination. The proliferation potential (PP) was calculated by multiplying the MI and the RI for each lymphocyte culture, PP = MIxRI.
Cytokinesis-blocked micronucleus assay (CBMN)
The lymphocyte micronucleus assay was performed by means of a cytokinesis-blocked assay in accordance with standard procedures (Fenech, 1993; Lee et al., 1994). Lymphocyte cultures were stimulated with PHA-M (1% v/v) and a standard micronucleus-inducing agent (mitomycin C, MMC) was added to the culture medium to a final concentration of 0.03 µg/ml. To each culture was added 50 µl of TDR in aqueous solution at the dose needed to achieve the desired final concentration. Control cultures contained the same volume of water, instead of MMC and/or TDR. After 24 h, lymphocytes were exposed to the cytokinesis-blocking agent cytochalasin B, added to a final concentration of 4.5 µg/ml culture medium, for 48 h. The MMC and TDR were present in the culture medium for the whole period of culture, i.e. for 72 h. The cultures were harvested by means of a mild hypotonic treatment (0.075 M KCl for 10 min) followed by fixation with cold methanol:acetic acid (3:1) (Lee et al., 1994). The cell suspension was gently spread on microscopic slides, dried and stained with 10% Giemsa solution (in phosphate buffer, pH 6.8) for 5 min. The slides were examined under a microscope and the number of micronuclei was counted per 1000 binucleated lymphocytes.
Thioguanine resistance (TG-R) assay
Lymphocytes were suspended in the culture medium and stored in a plastic culture bottle for 18 h at 4°C to prevent phenocopies. Afterwards, the cells were suspended in fresh medium and cultured for 48 h following the assay protocol (Ostrosky-Wegman et al., 1987; Montero et al., 1993), with small modifications. Briefly, 0.8x106 cells/ml were cultured in 24-well plastic dishes and stimulated with PHA-M (1% v/v) in the presence of 2x10–4 M thioguanine (TG). Then B[a]P and/or TDR were added to obtain the desired final concentrations. After 24 h, 30 µM BrdUrd was added to the cultures for the next 24 h. The harvesting procedure comprised centrifugation of the cultures, fixation of pellets with 75% ethanol for 30 min, treatment with lysis solution (containing 0.2 mg/ml RNase, 0.5 mM disodium versenite and 0.5% non-ionic detergent Nonidet NP-40) for 30 min at 10°C. For partial denaturation of DNA, the cell pellets were suspended in 0.5 N HCl and stored for 30 min at room temperature. Then the cells were washed twice with PBS and smears were prepared on microscope slides. Cells which had incorporated BrdUrd into the DNA were detected by means of an immunocytochemical procedure with a monoclonal mouse antibody able to recognize BrdUrd in single-stranded DNA (clone Bu20a; DAKO, Denmark). Subsequently, visualization and staining were performed following the alkaline phosphatase–anti-alkaline phosphatase (APAAP) technique (Cordell et al., 1984; Vanderlaan and Thomas, 1985; Magaud et al., 1988), in which TG-resistant lymphocytes stain intensively red. A total of 5000 cells randomly found under the microscope were counted.
Estimation of granulocyte superoxide radicals
The influence of TDR on superoxide radical generation by human granulocytes in vitro was evaluated using the routine NBT reduction test (Metcalf et al., 1986). Granulocytes were suspended to a density of 2x106 cells/ml in PBS, pH 7.2, containing NBT (final concentration 0.2%), TDR and B[a]P or PMA. After incubation of the samples for 45 min at 37°C in a shaking water bath, the reaction was terminated and the cells were centrifuged at 4°C. The pellets were then lysed with dimethylformamide at 56°C in a shaking bath and absorption (A515 nm) was measured with a spectrophotometer in relation to cell-free blank samples.
The impact of TDR on the level of superoxide radicals generated by granulocytes in the presence of the standard mutagen, B[a]P (16 µM), or the standard granulocyte stimulator, PMA (100 ng/ml), was assessed.
Statistical analysis
Regression equations were calculated and statistical evaluation of regression coefficients was carried out with Statistica for Windows 97 PL (StatSoft, Krakow, Poland).
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Results |
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The influence of TDR on the in vitro viability of lymphocytes in 4 and 18 h cultures was assessed with the standard trypan blue exclusion test and, separately, with the acridine orange–ethidium bromide fluorescence assay. We established that TDR was not cytotoxic to human lymphocytes in vitro in the range of concentrations tested (1.25–80.0 µg/ml culture medium) and that it was also non-cytotoxic to the cells when present in the culture together with the tested mutagens, B[a]P (16 µM) and MMC (0.03 µg/ml) (data not shown).
The initial status of lymphocytes was measured by means of four short-term in vitro tests and the results (negative controls) are presented in Table I, together with results obtained with the standard mutagens.
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The blood donor volunteers were four healthy male heavy smokers (
30 cigarettes/day), aged 40–50 years. As may be seen in Table I
, lymphocytes from the negative control cultures (without mutagen and TDR) exhibited a relatively high level of background genotoxic damage in the TG-R, CBMN and SCE tests. The addition of 16 µM B[a]P to the culture caused a decrease in lymphocyte PP of 40%. Exposure of the cultures to the standard mutagens brought about a marked increase in genotoxically damaged cells. In the SCE and CBMN tests, the data presented in the last column of Table I were ~2.5 times as high in the presence of the mutagens. In the case of the TG-R test, we estimated a 4.5-fold increase in mutant cells, although a relatively high standard deviation was observed. We took the results obtained in positive control cultures (containing the standard mutagens) as the reference value (E0) with which all the results of TDR impact on genotoxicity induced by the standard mutagens were compared.
The impact of TDR on the mutagenicity in vitro of the standard agents, B[a]P (16 µM) and MMC (0.03 µg/ml), is shown in Figure 2.
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The antimutagenic effect of TDR was assayed in three standard short-term lymphocyte tests: the SCE test, the CBMN assay and the TG-R test. For direct comparison, the influence of TDR on the PP of the B[a]P-treated lymphocyte cultures is also shown in Figure 2
. Herein the data are expressed as the ratio E/E0, i.e. the proportion of the results estimated in the cultures where TDR and the genotoxic agent were both present (E) to those where only the genotoxic agent was present (positive control, E0, as given in Table I). The regression equations were calculated and included in Figure 2 together with regression line plots. As may be seen in Figure 2, TDR lowered the proliferation rate of B[a]P-treated lymphocyte cultures in a dose-dependent manner; the slope of this regression line was significantly different from 0 and the PP was lower by ~25% in cultures with B[a]P together with TDR (80 µg/ml) as compared with cultures where only B[a]P was present.
In three short-term tests of genotoxicity, we observed that TDR decreased the genotoxic effect of the standard mutagen and the effects were strongly dependent on the concentration of TDR; the slopes of the regression lines were negative and were significantly different from 0. It may be seen in Figure 2 that the antimutagenic effect of TDR was strongest in the TG-R test and weakest in the SCE test. For instance, in the TG-R test the presence of a submaximal TDR concentration (40 µg/ml) caused a decrease in the mutant frequency generated by B[a]P (16 µM) of 65% in comparison with the culture in which only B[a]P was present. At the same TDR concentration, the number of micronuclei induced by the standard clastogen, MMC (0.03 µg/ml), was lower by 40% than in the positive control culture (MMC alone). In the SCE test, at a TDR concentration of 40 µg/ml the mean frequency of chromatid exchanges per mitotic cell was by 15% lower in the presence of B[a]P and TDR, when compared with that estimated in the positive control culture (B[a]P alone).
The regression analyses illustrated in Figure 2, therefore, consistently show a dose-dependent, statistically significant inhibitory influence of TDR on the action of the standard genotoxic agents.
The impact of TDR on the level of of free radicals generated in vitro by human granulocytes is shown in Figure 3.
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The effect of TDR on free radical levels was compared with linear dose–response relations, calculated with regression equations. As may be seen in Figure 3
, TDR decreased the free radical level in granulocyte samples both in the case of spontaneously released radicals and after stimulation with PMA and with B[a]P. At the highest of the tested TDR concentrations (80 µg/ml), the free radical level was lower by ~40% in the samples where only TDR was present than in the control samples (spontaneous release, without TDR). In the samples in which granulocytes were stimulated by a standard radical cascade activator (PMA, 100 ng/ml), the free radical level was lower by almost 45% in the presence of TDR (80 µg/ml) than in the samples in which only PMA was present; whereas in the presence of B[a]P (16 µM), TDR decreased the free radical level by ~25% in comparison with the relative control. The regression equations proved that the inhibitory action of TDR is strongly dose-dependent; the slopes of the regression lines were significantly non-zero. |
Discussion |
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It has been well documented that some hydrazinophthalazines (e.g. hydralazine, dihydralazine and endralazine) are genotoxic in several short-term mutagenicity testing systems (Williams et al., 1980
; De Flora et al., 1982; Chlopkiewicz et al., 1995a). We found previously that hydralazine exhibited a strong mutagenic activity in the Ames test, dihydralazine also exerted a significant genotoxic influence, whereas todralazine had no mutagenic activity on Salmonella typhimurium tester strains both in the presence and in the absence of the S9 activating fraction (Gasiorowski et al., 1993). Our results were confirmed by an independent study which showed that todralazine was not genotoxic, whereas hydralazine was strongly mutagenic in both the Ames test and in the mouse micronucleus test (Chlopkiewicz et al., 1995b). We have also shown that todralazine markedly decreases the mutagenic activity of several direct and indirect acting mutagens in the Ames test (Gasiorowski et al., 1994a,b, 1995, 1997). It was interesting to determine if the results obtained in the standard bacterial tests could be confirmed in human blood cell cultures.
In this paper we present evidence of an influence of TDR on the genotoxic effect in human lymphocyte cultures of two standard mutagens: an indirect acting one (B[a]P) and a direct acting one (MMC). The concentrations of the tested B[a]P (16 µM) and MMC (0.03 µg/ml) were chosen as being non-cytotoxic to lymphocytes in culture and generating a >2-fold increase (2.5–4.5 times) in genotoxicity, as measured with three short-term tests (Table I). In the experiments with B[a]P, we did not apply an external promutagen activating fraction, because human lymphocytes possess the microsomal enzymatic system necessary to in vitro activate polycyclic aromatic hydrocarbon promutagens (including B[a]P) to mutagenic derivatives (Guengerich, 1992; Goldstein and Faletto, 1993). It should be stressed that we used lymphocytes obtained from heavy smoker blood donors which may explain, at least partly, the high level of background mutations.
A significant decrease in the PP of lymphocytes was noticed in cultures containing B[a]P together with TDR. The relations between proliferation and genotoxicity appear complex, although a commonly accepted opinion is that a decrease in the cell proliferation rate would facilitate detoxification and repair of damage caused by mutagenic agents (Ames et al., 1993; Kaston and Kuerbitz, 1993; Tomatis, 1993).
TDR markedly decreased the genotoxic action of the standard mutagens in three short-term tests in human lymphocyte cultures. Despite the marked differences in the TDR effectiveness in the three tests, it should be noted that all results demonstrate a statistically significant, dose-dependent inhibitory action of TDR on the genotoxicity of the standard mutagens (Figure 2). The tests applied measure different end-points of genotoxicity: the TG-R test, point mutations; the CBMN assay, clastogenic and aneuploidogenic activities; the SCE test, chromatid rearrangements. The inhibition by TDR of genotoxic effects in all three tests strongly suggests that the mechanism of TDR action could be common for them. The mechanism could involve, for example: (i) inhibition of mutagen processing and activation to nucleophilic derivatives; (ii) stimulation of cellular repair systems; (iii) activation or enhancement of apoptotic events in genotoxically damaged cells. The detailed elucidation of the mechanisms (probably multiple) of the TDR antimutagenic action remains to be investigated. At present we are able to confirm the first of the suggested mechanisms, i.e. that inhibition of free radical pathways is an important component of its antigenotoxic activity. This conclusion can be drawn both from the observed influence of TDR in decreasing free radical levels in human granulocyte samples (Figure 3) and from the strong inhibitory effect of TDR on the activation of B[a]P by the liver microsomal S9 fraction, which was documented in our previous research (Gasiorowski et al., 1993 et al., 1995).
It is a well-known fact that free radicals play an important role in mutagenesis and in carcinogenesis (Troll and Weisner, 1985; Marnet, 1987; Frenkel, 1992). Free radical transformation of mutagens into nucleophilic genotoxic forms has been reported. It was described, for example, that in rat liver microsomes in vitro and in mouse skin, a significant part of B[a]P was transformed into a free radical form, which caused DNA adducts (Dix and Marnet, 1981; Cavalieri and Rogan, 1992). The B[a]P-derived free radicals may add their genotoxic action to that of the majority of adducts caused in vivo by B[a]P metabolites, a bay region diol epoxide and non-bay region diol epoxide (MacLeod et al., 1994; Nesnow et al., 1995). Several mutagenic compounds can also activate cellular free radical-generating systems, which may yield an abundance of oxygen-derived free radicals in the cell (Marnet, 1987; Frenkel, 1992; Li and Trush, 1994). This may add intracellular damage to the main genotoxic action of the mutagen. Since impairment of DNA molecules by free radicals is an important aspect of mutagenicity and carcinogenicity, there is a need to test putative antimutagenic compounds for their action upon the intracellular free radical systems.
We established an inhibitory influence of TDR alone and TDR plus B[a]P or PMA upon the level of human granulocyte radicals, as assayed with the NBT reduction test. The effect was marked and statistically significant, as shown in Figure 3.
It has been documented that activation of granulocyte oxidative bursts by PMA and other phorbol esters is a consequence of phorbol ester binding to and activation of protein kinase C (Blumberg, 1988; Tauber et al., 1989). Protein kinase C binds phorbol esters with high affinity to its cysteine-rich region in the regulatory domain stabilized by Zn2+ (zinc fingers) (Kazanietz et al., 1995). Protein kinase C (a mediator of the major cellular signal transduction pathway) being the main target for phorbol esters enables us to explain a broad range of phorbol ester biological activities, such as interference with cellular growth control, carcinogenesis and tumour promotion (Blumberg, 1988; Weinstein, 1988). One can hypothesize that the inhibitory action of TDR on the granulocyte free radical system stimulated by PMA may suggest either an interference of TDR with this signal transduction pathway and/or its hindering of PMA binding to protein kinase C. This hypothesis remains to be tested in future.
In general, the results presented in this paper complete our previous observations made in the standard bacterial Ames test. They confirm the antimutagenic action of TDR on direct and indirect acting mutagens, prove the conclusions valid in three different end-point lymphocyte tests, and suggest that an inhibitory influence on free radical pathways could be an important feature of the antimutagenic and chemopreventive activities of TDR. We conclude that TDR is an antimutagenic compound in vitro and it is worth further study with regard to its inhibitory action on mutagenesis and carcinogenesis in vivo. At the moment, we can recommend that physicians consider including TDR in antihypertonic therapy (in view of its pharmacological properties), especially in the case of hypertonic patients such as, for example, heavy smokers, who have been excessively exposed to genotoxic agents (in view of its antigenotoxic activities reported in this paper).
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1 To whom correspondence should be addressed. Tel: +48 71 3484310; Fax: +48 71 3479211; Email: kaz{at}basmed.am.wroc.pl
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Received on August 9, 1999; accepted on November 4, 1999.
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