Mutagenesis, Vol. 15, No. 3, 195-202,
May 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
In vitro and in vivo evaluation of the antihypertensive drug atenolol in cultured human lymphocytes: effects of long-term therapy
1 Departamento Biología Animal y Genética, Facultad de Ciencias, Universidad del País Vasco, Bilbao, Spain, 2 Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), Oporto, Portugal, 3 Instituto Superior da Maia, Portugal, 4 Departamento de Farmacología Clínica y Dietética, Escuela de Enfermería, Universidad del País Vasco, 5 Departamento Especialidades Médico-quirúrgicas, Facultad de Medicina y Odontología, Universidad del País Vasco and 6 Departamento Química Analítica, Facultad de Ciencias, Universidad del País Vasco, Bilbao, Spain
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Abstract |
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The genotoxicity of atenolol, a ß-blocker antihypertensive drug, both in vitro and in vivo, was cytogenetically tested for its ability to induce sister chromatid exchange (SCE) and micronuclei (MN) in cultured peripheral lymphocytes. Also, fluorescence in situ hybridization (FISH) with a centromeric probe was performed to determine the origin of the induced MN. The in vivo study was carried out, on the one hand, on four patients under antihypertensive treatment with atenolol and, on the other hand, on four matched control individuals taking an oral dose of atenolol. The in vitro study was performed on the control individuals by adding the drug to the culture medium at a final concentration similar to the levels found in plasma. When a comparison was made, the frequency of SCE did not show significant differences in any case. A statistically significant increase in the frequency of MN was detected in patients but not in control individuals either in vitro or in vivo. FISH analysis revealed statistically significant differences between patients and control individuals without the drug with respect to the frequency of centromeric signals in MN. Taking all these observations together, our data suggest that chronic exposure to atenolol resulted mainly in the induction of chromosome loss, so an aneugenic activity could be predicted. Different sensitivity to the compound was observed among control individuals. Nevertheless, all of them responded to the presence of atenolol in the same way in both assays. Interindividual variability was also reported. The intervariability seen in patients suggested an adaptive response to the chemical after long-term therapy.
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Introduction |
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Arterial hypertension is a health problem representing one of the most frequent diagnoses in the population at large in terms of prevalence and incidence. Several studies carried out during the last two decades in Spain have estimated that ~20% of the adult population suffer from arterial hypertension, of whom only 50–60% are aware of it and 40–50% are treated with antihypertensive drugs. Antihypertensive drug consumption has increased from 34.78 daily doses per 1000 inhabitants per day (DID) in 1985 to 103.55 DID in 1995 (Prieto et al., 1998
).
Among the most used antihypertensive drugs, the group of chemicals known as ß-adrenergic blockers or, simply, ß-blockers have been found to have markedly beneficial therapeutic value in treating many cardiovascular symptoms such as hypertension, angina and arrhythmias (Jackson and Fishbein, 1986). Their pharmacological application is due to their ability to bind to ß receptors and prevent their stimulation by catecholamines, resulting in a lowering of the heart rate and a lowering of systemic blood pressure. As a result of the high volume of these drugs used and suggestions that some members of this chemical class may produce chronic toxicological effects, the regulatory agencies have become concerned over the possible long-term adverse effects of ß-blockers (Jackson and Fishbein, 1986).
In their review, Jackson and Fishbein (1986) reported the toxicological data available on 18 ß-blockers. Several of them were reported to have some indication of carcinogenic or tumorigenic activity whereas the results were negative in all cases except for one when ß-blockers were tested in one or more mutagenicity test systems (principally the Ames test and the micronucleus test in CF1 outbred mice). However, apart from this review, the published literature concerning genotoxic studies on antihypertensive drugs in general and ß-blockers in particular is poor. Only a few studies have been published in the last few years (for example Aruna and Krishnamurthy, 1986; Grisolia and Takahashi, 1991; Martelli et al., 1992; Tsutsui et al., 1994; Herzog and Leuschner, 1995; Martelli et al., 1995; Gasiorowski et al., 1997; Andreassi et al., 1999; Nesterova et al., 1999), some of them with positive results.
Human populations exposed in vivo to possible genotoxic agents can be monitored using different chemical and biological end-points. Pharmaceutical companies in Europe, the USA and Japan require tests for gene mutation in bacteria, chromosomal aberrations in mammalian cells and in vivo mammalian cell tests to detect chromosomal damage (Purves et al., 1995). However, there are differences of opinion regarding the most appropriate tests to use in genotoxicity testing of pharmaceuticals (Aaron et al., 1993; Purves et al., 1995).
Cytogenetic end-points such as sister chromatid exchange (SCE) and micronucleus (MN) formation have potential use as indicators of chromosomal damage and have been used during recent decades in a lot of studies to evaluate the induction and persistence of cytogenetic alterations in individuals and populations with medical, occupational or accidental exposure (Tucker et al., 1997).
SCE are events that involve breaks in both chromatids of a single chromosome, at coincident locations, with subsequent interchange and repair (Latt et al., 1981). SCE have been evaluated in numerous studies involving human exposure to pharmaceuticals (Giri et al., 1996; Awara et al., 1998; Migliore et al., 1998; Vlastos et al., 1998; Lanza et al., 1999).
MN are acentric chromosome fragments or whole chromosomes that are left behind during mitotic cellular division and appear in the cytoplasm of interphasic cells as small additional nuclei. There are several genotoxic studies on pharmaceutical compounds using this assay (Migliore et al., 1991, 1998, 1999; Erexson et al., 1995; Gutiérrez et al., 1997; Miele et al., 1998; Vlastos and Stephanou, 1998; Monsieurs et al., 1999). To distinguish between MN derived from acentric fragments and MN containing whole chromosomes, different methods have been proposed (Yamamoto and Kikuchi, 1980; Degrassi and Tanzarella, 1988; Vanderkerken et al., 1989). At present, fluorescence in situ hybridization (FISH) with centromere-specific DNA probes is a valuable approach of increasing interest and it is being increasingly used in monitoring studies (Miller et al., 1994; Eastmond et al., 1995; Parry et al., 1995; Zijno et al., 1996; Ramírez et al., 1997; Tucker et al., 1997; Thierens et al., 1999). Since the presence of a hybridization signal in a MN is a direct measure of the presence of a centromere (Tusell et al., 1996; Migliore et al., 1998), this methodology allows detection of chromosome loss (Kirsch-Volders et al., 1996; Migliore et al., 1997; Ramírez et al., 1997).
To our knowledge, there are no published data using SCE and/or MN assays in humans to assess chromosomal damage of ß-blockers. The treatment of hypertension is long and continuous (years) and, therefore, patients are exposed to prolonged contact with these drugs. So, it would be of interest to evaluate any long-term genotoxic effects of antihypertensive drugs (ß-blockers among them) using the different assays available both in vitro and in vivo in humans.
For this purpose, we chose the ß-blocker atenolol because of its widespread use not only for the treatment of hypertension but for the treatment of angina pectoris, cardiac arrhythmia and heart attack. The chemical structure of atenolol {4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]benzeneacetamide} is shown in Figure 1. The dose levels are between 50 and 100 mg atenolol/day by oral administration. Activity starts 60 min after oral intake and lasts 24 h. Atenolol is rapidly absorbed from the gut and blood levels reach a peak concentration in 2–3 h (Fitzgerald, 1979). Due to its hydrophilic nature, metabolism of atenolol is minimal and almost the total absorbed drug (85–100%) is cleared via excretion in the urine in an unaltered manner (Wadworth et al., 1991).
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In their review, Jackson and Fishbein (1986) reported the toxicity data available on atenolol. In chronic studies with C57B1/10J mice and Alderley Park Strain I rats, it was concluded that there was no evidence that atenolol had any tumorigenic potential (Fitzgerald, 1979
). Studies including the dominant lethal test, in vivo cytogenetics of Chinese hamster bone marrow and the Salmonella typhimurium back mutation test with and without metabolic activation have shown that atenolol does not possess mutagenic potential (Fitzgerald, 1979). Atenolol was also studied in the MN test in adult CF1 outbred mice and no significant, dose-dependent increase in the number of MN was observed (Okine et al., 1983). The effects of atenolol in producing DNA damage in vitro and in vivo in liver nuclei of outbred Wistar rats were studied (Presta et al., 1983) but there was no evidence of DNA damage produced by atenolol. Finally, there was no teratogenic potential exhibited by atenolol in either rats or rabbits (Fitzgerald, 1979). We report here our data from a study of the genotoxic potential of atenolol, which was tested for its ability to induce SCE and MN in cultured human peripheral blood lymphocytes of treated patients and control individuals in vitro and in vivo. The origin of MN was determined by FISH with a probe detecting all human centromeres. We are not aware of other studies done in lymphocytes of hypertensive patients under treatment with antihypertensive ß-blocker drugs.
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Materials and methods |
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Sample
The sample was made up of two different groups: four hypertensive patients (treated by Dr Ortiz-Lastra) and four control individuals, matched with respect to sex, age and smoking habit (in this present study all individuals were non-smokers). Patients (three male and one female) ranged in age from 39 to 79 years with an average age of 52.75 years. All of them received a dose of 50 mg atenolol/day (one tablet) by oral administration. The mean duration of treatment was 3.4 years (8 months–6 years). None of them were undergoing other medical treatments. They did not have a previous history of exposure to genotoxic compounds or recent X-ray examination. Each patient was given a code number (P1–P4) according to the sequence of blood collection. All patients gave informed consent to take part in this study. Control individuals (three male and one female) had an average age of 52.5 years. All of them were healthy individuals not undergoing any pharmaceutical compound intake, X-ray exposure nor alcohol and/or drug consumption in the recent past. Each control individual was given a code number (C1–C4) according to the sequence of arrival at the laboratory. Informed consent was obtained from control individuals to take part in the study.
Blood collection and cell cultures
Several cultures were established depending on the group. For patients, venous blood was taken from each subject and duplicate whole blood cultures were set up. These cultures were numbered P1–P4. For control individuals, three different cultures were performed. On day T–1 (1 day prior to dosing), venous blood was taken from each subject and duplicate whole blood cultures were set up, numbered C1/T–1–C4/T–1 (controls/T–1). Another set of cultures was performed by adding the chemical compound to the culture medium at the start of culture. Previously, atenolol, kindly provided by Dr Ortiz-Lastra, had been dissolved in dimethyl sulphoxide to give a final concentration in the culture medium similar to that found in patients after oral intake (these analyses were performed in the Department of Analytical Chemistry, University of the Basque Country, Spain). These cultures were numbered C1/medium–C4/medium (controls/medium). On day T (day of dosing), each control individual received a therapeutic oral dose of 50 mg atenolol (one tablet). On day T+1 (1 day after dosing), venous blood was taken from each individual and duplicate lymphocyte cultures were set up. These cultures were numbered C1/T+1–C4/T+1 (controls/T+1).
This methodology was based on the study by Kirkland et al. (1992).
Cytogenetic tests
Lymphocyte cultures were set up by adding 0.5 ml of heparinized whole blood to 4.5 ml of RPMI 1640 medium (Gibco), supplemented with 10% foetal bovine serum (Gibco), antibiotics (penicillin and streptomycin) (Gibco), glutamine (Gibco) and HEPES buffer solution (Gibco). Lymphocytes were stimulated with 4% phytohaemagglutinin (Gibco). Atenolol was added to those cultures numbered C1/medium–C4/medium.
For the SCE assay, the cultures were incubated at 37°C for 72h in the dark and 5-bromodeoxyuridine (Sigma) was added 24 h after the initiation of culture at a final concentration of 12 µg/ml. One hour prior to harvesting, 0.4 µg/ml of colcemid (Gibco) was added to arrest the cells at metaphase. For the MN assay, the cultures were incubated at 37°C for 72 h. Binucleated cells were accumulated by adding cytochalasin B (Cyt-B) (Sigma) at a final concentration of 6 µg/ml (Surrallés et al., 1992) at 48 h following initiation of culture. At the end of the incubation time, the cells were collected by centrifugation and, for SCE, resuspended in a pre-warmed hypotonic solution (0.075 M KCl) for 10 min and fixed three times in methanol:acetic acid (3:1). Cells were spread on slides, air dried and stained with fluorescence plus Giemsa (Perry and Wolff, 1974). For MN, the cells were washed once in RPMI 1640 medium and then a mild hypotonic treatment (2–3 min in 0.075 M KCl at room temperature) was carried out. The cells were then centrifuged and a methanol:acetic acid (5:1) solution was added. This fixation step was repeated twice. Air dried preparations were made and the slides were stained with 10% Giemsa in phosphate buffer for 20 min.
Fluorescence in situ hybridization (FISH)
For the identification of centromeres in MN, FISH was performed with a digoxigenin-labelled probe for all human centromeres (ONCP 5095; Oncor), following the protocol recommended by the manufacturer. Slides were incubated for 30 min in 2x SSC, pH 7.0, and then dehydrated in a series of ice-cold ethanol washes (70, 80 and 95% for 2 min each) and dried at room temperature. The probe was diluted in Hybrisol VI (Oncor), denatured at 70°C for 5 min, applied to the slides, coverslipped and incubated overnight at 37°C in a humidified chamber. After hybridization, slides were washed in 2x SSC, pH 7.0, at 37°C for 10 min and in phosphate-buffered saline (PBS) at room temperature for 10–20 min. Detection of the digoxigenin-labelled probe was performed by layering of fluorescein-labelled anti-digoxigenin antibody (Oncor) for 10 min at 37°C in a moist chamber. Slides were counterstained with propidium iodide (Oncor) and mounted in antifade solution (Oncor) to preserve fluorescence. To evaluate probe hybridization efficiency, metaphase spreads were also examined.
Slide scoring
For the SCE assay, a total of 50 well-spread metaphases of the second division were examined for each individual and type of culture on coded slides in a blind study. Statistical analysis for SCE was performed using the t-test. One hundred metaphases were also scored to determine the proportion of cells that had undergone one, two and three or more divisions. The proliferative rate index (PRI) was evaluated according to the formula 
, where M1, M2 and M3 indicate those metaphases corresponding to first, second and third or subsequent divisions and n is the total number of metaphases scored (Lamberti et al., 1983).
For the MN assay, 1000 binucleated (BN) cells with well-preserved cytoplasm were examined for each individual and type of culture on coded slides in a blind study. The identification of MN was according to the criteria described in Fenech (1993). The distributions of the BN cells with MN (BNMN) and the total MN obtained among the individuals studied were compared with the normal distribution by means of the Kolmogorov–Smirnov test of goodness of fit. Neither of them departed significantly from normality and therefore parametric tests (the t-test in this case) were adequate for statistical analysis of the results. In addition, a minimum of 500 lymphocytes were also scored to determine the percentage of cells with 1, 2, 3, 4 or >4 nuclei. A nuclear division index (NDI) was calculated according to the formula 
, where M1–M4 represent the number of cells with one to four nuclei, respectively, and n is the number of cells scored (Eastmond and Tucker, 1989).
For the FISH analysis, a total of 50 MN for each individual and type of culture on coded slides in a blind study were analysed for the presence of a fluorescent signal. MN were classified as centromere-negative (CN–) or centromere-positive (CN+) by considering the presence of a hybridization signal in a MN as a direct indication of the presence of a centromere (Tusell et al., 1996). Among CN+ MN, signals of different intensities were found due to variable amounts of 
-satellite DNA on different human chromosomes (Migliore et al., 1996). However, hybridization on metaphase chromosome preparations confirmed the extremely precise location of the signals only at the centromere, varying only in intensity. Statistical evaluation of the results was performed using the 
2 test.
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Results |
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The effect of atenolol on the induction of SCE is summarized in Table I
. In Table I the mean number of SCE per cell (± SE) and the PRI values obtained after the different treatments with atenolol in each subject and in the total sample are reported.
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The mean frequency of SCE in patients (8.08) was slightly increased with respect to controls/T–1 (7.94), but the differences were not statistically significant (P > 0.05). On the other hand, when the SCE frequency in controls/T–1 (7.94) was compared with those found in controls/medium (9.03) and in controls/T+1 (9.67), it could be observed that the SCE values increased slightly when atenolol was present in the culture medium and they were still higher when individuals had received atenolol by oral administration, but none of the differences were significant (P > 0.05). These data for human lymphocytes demonstrate that atenolol has little effect in inducing SCE in vitro and in vivo.
Concerning its cytotoxicity, measured as cell cycle delay, a decrease in the rate of cell proliferation was found when controls/T–1 were compared with patients, controls/medium and controls/T+1. However, this inhibition in the PRI values was not significant (P > 0.05).
Taking into account the mean frequency of SCE in each subject, a wide variability among individuals can be noted in both groups. Among patients, individual P4 presented the highest SCE frequency in spite of being the patient with the least time under treatment with atenolol (8 months). Among control individuals, C4 was the one with the highest frequencies of SCE and the lowest PRI values (except for culture C4/T–1) of all cultures, whereas individual C3 presented, in general, the lowest SCE frequencies and the highest PRI values. Moreover, all control individuals in general showed an increase in SCE values when atenolol was added to the culture medium and these values were still higher when atenolol was tested in vivo. In spite of this tendency, individual C1 was the only one in which the SCE frequencies obtained in the three different cultures were statistically significant (P < 0.001 comparing both C1/T–1 with C1/medium and C1/T–1 with C1/T+1). All these observations together seem to indicate that, despite the interindividual variability and sensitivity, all individuals responded to the presence of the chemical in the same way.
The results of the MN study are shown in Table II. This table shows the percentage of BN cells, the distribution of MN in the 1000 BN cells scored, the total number of MN, the total number of BNMN and the NDI values obtained after the different treatments with atenolol in each subject and in the total sample.
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When the means of total MN and total BNMN variables obtained in patients (14 and 12.75, respectively) were compared with those obtained in controls/T–1 (3 and 2.75, respectively), the differences were statistically significant (P < 0.01 in both cases). There were significant differences as well in the mean frequencies of MN and BNMN between patients and controls/medium (14 and 4.75 for total MN, respectively, P < 0.05; 12.75 and 4.25 for total BNMN, respectively, P < 0.05) and between patients and controls/T+1 (14 and 3.25 for total MN, respectively, P < 0.01; 12.75 and 2.50 for total BNMN, respectively, P < 0.01). However, when the averages of MN and BNMN obtained in controls/T–1 were compared with those obtained in controls/medium and in controls/T+1, slight increases could be observed, but without statistical significance. These data seem to indicate an effect of the chemical in inducing the significantly higher frequencies of MN and BNMN in patients.
The NDI measure, reflecting the average number of nuclei per cell, did not show significant differences when patients were compared with controls/T–1, with controls/medium and with controls/T+1 (P > 0.05). In the same way, no statistical differences were found when controls/T–1 were compared with controls/medium and with controls/T+1 (P > 0.05). Accordingly, atenolol does not seem to show cellular toxicity in vitro or in vivo.
Variability among individuals can be observed in both groups. Among patients, individual P4 again presented the highest MN and BNMN frequencies. Among control individuals, the MN and BNMN frequencies in general increased when atenolol was added to the culture medium but these values were the same as found in controls/T–1 when atenolol was tested in vivo. Individual C1 showed the greatest differences in the frequencies of MN and BNMN when atenolol was tested in vitro, although they did not reach the significance level in this case, while individual C3 had the lowest MN and BNMN frequencies of all cultures. These observations suggest that all individuals respond to the presence of atenolol in the same way.
The application of FISH with a centromeric probe to determine the nature of atenolol-induced MN gave the results reported in Table III. In Table III the total number and percentage of CN– and CN+ among the 50 MN scored for each subject and for the total sample are reported. Only patients and controls/T–1 were analysed for the presence of centromeric signals.
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Among control individuals, approximately half of the observed MN were CN+, whereas among patients, this percentage increased to 70% of the total MN scored. When the percentage of CN+ MN in patients (70%) was compared with the same percentage in controls/T–1 (48%), the differences were statistically significant (P < 0.01). These results seem to suggest an ability of atenolol to induce chromosome loss in vivo.
Slight variability among individuals was observed in both groups. Among patients, P1, the only female and the oldest individual, presented the highest percentage of CN+, whereas the other patients' frequencies were similar. Among control individuals, all of them presented similar rates of CN– and CN+ MN.
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Discussion |
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Unlike the results reported by Jackson and Fishbein (1986) in their review, the present study revealed a variety of cytogenetic effects of atenolol in cultured human lymphocytes.
Recent studies point to important structural differences among different eukaryote organisms (yeast, rat, mouse and man) (Aardema et al., 1998). Toxicological data from animal experiments can only be used to a limited extent for human risk assessment (Hagmar et al., 1998) because, as Aardema et al. (1998) pointed out, `to date, there is no information on the relevance of the data obtained in experimental animals for human beings and this is an area requiring further investigation'. Another critical factor that is often difficult to gauge is the likely exposure of humans to the drug. The disease entity and factors related to bioavailability (the fraction that will reach the circulation following oral exposure), potency (plasma concentration required to produce the desired therapeutic effect) and biological persistence (half-life in the body) often determine the exposure (Aaron et al., 1993). On the other hand, in vitro analysis alone is insufficient to identify hazardous effects and in vivo analyses also have to be carried out (Vlastos et al., 1998). One of the key factors leading to discrepancies between the results in bacterial or cell culture assays and the expected outcome in humans is differences in metabolism of the compound in vivo, since in vitro assays do not reflect the human body's absorption, distribution and elimination of the compound (Aaron et al., 1993).
In an attempt to clarify whether chronic exposure to the antihypertensive drug atenolol has any in vitro and/or in vivo genotoxic effects on humans, this work reports the results of cytogenetic analyses of four patients following treatment with atenolol and four matched control individuals (controls/T–1, controls/medium and controls/T+1, according to the treatment followed; see Materials and methods), as determined by SCE and MN assays in peripheral blood lymphocytes. FISH was also performed to determine the origin of the induced MN.
Peripheral blood lymphocytes represent a good indicator of exposure to genotoxic agents, carrying information on both doses and genotoxic effects (Morimoto et al., 1993; van Asten et al., 1998; Anderson, 1999). As they circulate throughout the body, this provides an estimate of average whole body exposure (Tucker and Preston, 1996). In the case of in vivo assays, two arguments are frequently presented for the decision to use human lymphocytes: on the one hand, cytogenetic tests with established cell cultures tend to give more false positives than with human lymphocytes and, on the other hand, for the human situation human lymphocytes are more relevant than non-human cell cultures (Müller et al., 1991).
Also, the above-mentioned assays have been widely used to evaluate the genotoxic potential of physical and chemical agents in vitro and in vivo (see for example Latt et al., 1981; Carrano and Natarajan, 1988; Tucker et al., 1997; Migliore et al., 1998; Monsieurs et al., 1999). The use of multiple end-points in the same population can provide valuable information relative to the sensitivity of each end-point as well as to the potential hazard for the population (Carrano and Natarajan, 1988).
The SCE test provides a powerful tool to evaluate genetic damage deriving from exposure to genotoxic agents (Lambert et al., 1976; Morimoto et al., 1993; Tucker and Preston, 1996; Tucker et al., 1997) and it assesses the impact of clastogens on chromosomes (Latt et al., 1981; Carrano and Natarajan, 1988; Morimoto et al., 1993; Wolff, 1998).
The MN test, after the improvement introduced by Fenech and Morley (1985) in using Cyt-B to arrest cytokinesis, has been adopted by many laboratories world wide as a sensitive and reliable method for assessing chromosomal damage (Duffaud et al., 1997; Surrallés and Natarajan, 1997; Fenech, 1998) because of its ability to detect both chromosome breakage (clastogenicity) and loss (aneuploidy), two mechanisms involved in genetic and carcinogenic risk (Heddle et al., 1991; Marzin, 1997; Fenech, 1998; Miller et al., 1998; van Delft et al., 1998). This test combined with FISH with a centromeric probe is considered a useful screen to distinguish between clastogenic and aneugenic agents by analysing the chromosome content of MN (Migliore et al., 1996; Ramírez et al., 1997; Thierens et al., 1999).
While SCE are indicative of an early biological effect that may not be permanent and may not have further consequences, MN represent early but irreversible biological effects (van Delft et al., 1998).
Concerning the results obtained in our study, chronic exposure to atenolol did not seem to have clastogenic effects in vivo because no significant increases in the frequency of SCE were found in patients as compared with controls/T–1. Atenolol did not induce changes in the frequency of SCE in control individuals in any treatment. Although slight increases could be observed in vitro (controls/medium) and in vivo (controls/T+1) as compared with controls/T–1, these increases were far from the level of significance. Thus, atenolol was incapable of inducing SCE in peripheral lymphocytes after long and short exposure to the chemical in vivo or after exposure in vitro.
As regards the MN assay, the results indicate that chronic exposure to atenolol led to a statistically significant enhancement of the two variables studied, total number of MN and total number of BNMN. These increases in the total frequency of MN and BNMN were also statistically significant when patients were compared with controls/medium and with controls/T+1, whereas there were no differences when comparing the different treatments made among control individuals. This seems to suggest that the effect of atenolol is observed only in vivo and after continuous exposure.
In this context, our results showing significant increases in MN and BNMN frequency in hypertensive patients after treatment with atenolol indicate that the MN assay is sensitive enough to detect the chromosomal damage resulting from treatment. This is in good agreement with previous reports on drugs treatment (Erexson et al., 1995; Gutiérrez et al., 1997; Miele et al., 1998; Migliore et al., 1998; Vlastos and Stephanou, 1998).
In this assay, the spontaneous frequencies of MN were slightly lower than those reported by Surrallés et al. (1992) and Di Giorgio et al. (1994), but they are in agreement with those reported by Parry et al. (1995) for human lymphocytes.
Taking the results obtained in both assays together, our data suggest that chronic exposure to atenolol results mainly in the induction of chromosome loss, suggesting a possible aneugenic potential of the compound. This suggestion was confirmed by FISH analysis using an -satellite probe for all human centromeres. The spontaneous frequency of MN containing centromeric signals is in good agreement with those reported previously (Cimini et al., 1996; Tusell et al., 1996; Migliore et al., 1997; Vlastos and Stephanou, 1998). The statistically significant differences in CN+ MN between patients and control individuals indicate more frequent involvement of aneuploidy (specifically chromosome loss) phenomena in the origin of atenolol-induced MN in vivo. These results agree with those previously reported by the SCE assay. From our results we can deduce that atenolol after long-term treatment in vivo induces MN that contain mainly lagging chromosomes and, therefore, an aneugenic effect is supported.
Individual P1, the only female and the oldest individual, presented the highest frequency of CN+ MN. Chromosome loss is known to increase with increasing age and it is also known to be higher in females (Nowinski et al., 1990; Migliore et al., 1991; Guttenbach et al., 1994; Stone and Sandberg, 1995; Scarpato et al., 1996; Migliore et al., 1997). Our data agree with this.
Concerning cytotoxicity, measured as PRI in the SCE test and NDI in the MN technique, atenolol did not seem to show cellular toxicity in vitro or in vivo in any group. For the MN assay, both BN (%) and NDI were calculated, as seen in Table III
, but statistical calculations were only applied to NDI since, as Surrallés et al. (1994) reported, `the NDI is a better parameter for measuring toxicity, or cell cycle delay, than % BN'.
The present study has revealed interindividual variation in the frequency of all end-points studied. The variability observed among individuals may be associated with different factors, one being a particular individual's responsiveness to drugs, but apparently not with differences in experimental protocols nor with age, sex and/or smoking habit.
In our work and among control individuals, C1 showed the highest differences in the frequency of all variables studied when atenolol was tested in vitro and in vivo. The increases observed were highly significant, suggesting the high sensitivity of this individual to the compound. Thus, it can be said that under these experimental conditions, for each control individual there was a different susceptibility to the chemical. This differential response to atenolol could be attributed to differences in genetic background among individuals that could influence metabolism of the compound, repair of DNA damage and also the percentage of cells that respond to the chemical. Different individual sensitivity is not surprising and significant interindividual variations have been reported in the frequencies of SCE and MN following exposure to a large number of agents (Migliore et al., 1991; Grummt et al., 1993; Duffaud et al., 1997; Vlastos and Stephanou, 1998; Vlastos et al., 1998). Despite this different sensitivity to the chemical, all control individuals responded to the presence of atenolol in the same way in both assays.
As regards the interindividual variation in patients, it was noted that individual P4 presented the highest frequencies for all variables studied in the two assays applied, in spite of being the patient with the least time under treatment with atenolol [8 months as compared with 3 (P3), 4 (P2) and 6 (P1) years]. The fact that this behaviour was repeated in all the assays led us to think of a non-casual event.
Dr Wolff's work on the induction of chromosomal aberrations (CA) in human lymphocytes has shown that low doses of radiation trigger the induction of new proteins that are involved in the repair of molecular lesions. Once induced, these repair enzymes can be effective in cells for up to three cell cycles and cells `adapted' with a low dose of radiation or chemical show much less damage after a subsequent exposure to a high dose of radiation or even chemical mutagens than observed in cells not pre-exposed (adapted) to the low doses. This phenomenon has been termed an `adaptive response' to low doses. The adaptive response can also be induced in vivo with low, or chronic, doses (Wolff, 1998). So it seems that low and chronic doses of atenolol could induce mechanisms whereby cells became somewhat refractory to the induction of damage by subsequent exposure. Migliore et al. (1991) also found this behaviour in patients under antileukaemic therapy. Similar results in which duration of exposure was not correlated with genetic effects analysed have been reported (McDiarmid et al., 1992; Grummt et al., 1993; Farmer et al., 1996).
The phenomenon of adaptive response has now led to a world wide explosion of research in this area to see if the current risk estimated for the effects of mutagens are appropriate (Wolff, 1998). Our findings require further investigation in order to determine the health significance of this event.
The present work is the first report on monitoring the genotoxic potential of the antihypertensive drug atenolol following in vivo treatment in humans. Our results obtained using different biological end-points are interesting since it is shown that an aneugenic effect of the drug can be observed after long exposure to the chemical. Taking into consideration that this drug is administered to a wide spectrum of patients and that the treatment is for prolonged periods, the results obtained reiterate the need to both extend this type of study and to make a careful evaluation of the cost–benefit ratio of this long-term treatment, as suggested by Bigatti et al. (1998) for psychopharmacological treatment.
An important point to address is the Basque origin of all the individuals analysed in this study. The Basques constitute an ancient population now living in the west of the Pyrenees Mountains. Their origin is not exactly known. They speak an ancient language with very distinct characteristics different from the surrounding populations. Many studies, using different traits, have shown peculiarities in Basques as compared with other populations (Arrieta et al., 1992, 1995; Cavalli-Sforza and Piazza, 1993). Since there are no previous studies about the incidence of chromosomal changes in Basque populations, a possible influence of a genetic component in the results observed could not be ruled out.
Although further investigations are needed as regards the influence of the Basque origin on the aneugenic potential and adaptive response observed, these preliminary results should be borne in mind in future studies.
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M.T. holds a doctoral fellowship from the University of the Basque Country. We thank Drs F.Barquinero and J.Surrallés for their technical help and great kindness. This study was supported by the Department of Education, Universities and Research of the Basque Government (PI96/85).
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* To whom correspondence should be addressed. Tel: +34 4 601 5409; Fax: +34 4 464 8500; Email: ggbtesem{at}lg.ehu.es
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Received on August 20, 1999; accepted on January 7, 2000.
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