Mutagenesis, Vol. 16, No. 6, 517-521,
November 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Chronic long-term nitrate therapy: possible cytogenetic effect in humans?
CNR Institute of Clinical Physiology, via Moruzzi, 1, 56100, Pisa and 1 G.Pasquinucci Hospital, Via Aurelia Sud-Montepepe, 54100 Massa, Italy
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Abstract |
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Nitrates act as donors of nitric oxide (NO), a molecule with a recognized potential for genotoxicity. In order to assess whether chronic long-term nitrate therapy may increase genotoxicity, we evaluated chromosomal damage in peripheral lymphocytes of 27 ischaemic patients undergoing chronic nitrate treatment for
4 years (7.9 ± 3.1, mean ± SD) and 18 age- and sex-matched subjects without any previous nitrate treatment. At the same time, after treatment in vitro with 0–20 µM sodium nitroprusside as NO donor, micronucleus induction and cell proliferation were also evaluated using blood from six different healthy donors. The results showed that the frequency of structural chromosomal aberrations was not significantly higher in the drug-treated group than the control [2.1 ± 1.4 versus 1.6 ± 1.2 (mean ± SD); P = 0.23]. The frequency of micronucleated lymphocytes was higher in the nitrate group than in the control group (6.5 ± 4.6 versus 3.5 ± 2.9, P=0.01). In vitro treatment indicated a dose-dependent increase in the frequency of micronucleated lymphocytes with increasing SNP concentrations. Cytotoxicity and cell cycle delay, with a statistically significant difference with respect to control culture, were also observed. Our results suggest a possible genotoxic activity of nitrate therapy. Further studies focusing on the possible link between nitrate therapy and genotoxicity are warranted at this point. |
Introduction |
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Organic nitrates, or nitrovasodilators, represent a widely-used class of extremely effective antianginal medications capable of causing systemic and coronary vasodilation whether or not the endothelium is intact. Although chronic nitrate therapy is not primarily anti-hypertensive, it is a common concomitant therapy in the hypertensive patient. As anti-hypertensive drugs, nitrates have multiple actions that improve large artery stiffness and early wave reflection and are considered especially useful in treating isolated systolic hypertension in the elderly (Franklin, 2000
).
These agents all lead to the formation of the reactive free radical nitric oxide (NO), which activates intracellular guanylate cyclase to produce cyclic guanosine monophosphate, which in turn triggers smooth muscle relaxation (Robertson and Robertson, 1995).
Recently, it has been suggested that nitric oxide produced during an inflammatory response could become an endogenous cytotoxin, with mutagenic and/or carcinogenic properties (Victorin, 1994; Liu and Hotchkiss, 1995; Felley-Bosco, 1998; Wink et al., 1998; Burney et al., 1999).
Studies exploring the potential link between chronic nitrate administration in humans and their potential genotoxicity are lacking to date. The most extensively employed method for assessing the genetic effects of exposure to potential mutagens has been the analysis of chromosome alterations in stimulated peripheral blood lymphocytes of exposed individuals (Carrano and Natarajian, 1988; Tucker and Preston, 1996).
Chromosomal aberrations in peripheral lymphocytes are considered indicators of exposure to mutagens/carcinogens, and several studies suggest—albeit do not conclusively demonstrate—a strong association between frequency of chromosome aberrations in peripheral lymphocytes and subsequent cancer morbidity (Hagmar et al., 1998; Bonassi et al., 2000).
Micronucleus formation has become another important endpoint in genotoxicity testing. Fragments originating from chromosome breakage as well as whole chromosomes which are not correctly distributed during mitosis give rise to micronuclei visible in the next interphase. It has been shown that micronucleus induction may assist in the prediction of breast cancer risk (Rothfuss et al., 2000) and in the estimation of the potency of a chemotherapeutic agent for induction of secondary lymphomas (Stopper et al., 1999).
The aim of this work was to assess whether chronic long-term nitrate therapy can have genotoxic effects. To this purpose, 27 patients undergoing chronic nitrate therapy for 
4 years and 18 age- and sex-matched subjects were studied using cytogenetic techniques to evaluate the frequency of chromosome aberrations (CA) and micronuclei (MN) in peripheral blood lymphocytes. In addition, by means of the in vitro MN test, we simultaneously assessed genome mutations and cell cycle delay in peripheral human blood lymphocytes after in vitro treatment with sodium nitroprusside, a potent vasodilator for the treatment of hypertensive emergency and acute pulmonary edema. It has been established that sodium nitroprusside acts by immediately interacting with sulphydryl groups in blood to produce nitric oxide (Meisheri, 1994).
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Materials and methods |
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Patients
The study included 45 subjects divided in two groups matched for age, sex and life-style habits such as smoking and alcohol, which could be confounding factors for cytogenetic analysis (Carrano and Natarajan, 1988). The exposed group included 27 hypertensive and/or ischaemic patients (21 men and 6 women, 63.3 ± 8.9 years) undergoing chronic nitrate therapy (mean treatment duration: 7.9 ± 3.1 years) by oral or transdermal administration. All patients were free of exogenous vitamins. Other medications used by the patients included oral aspirin, calcium antagonists and ACE inhibitors. Control group consisted of 18 healthy subjects (12 men and 6 women, mean age 60.3 ± 9.8 years) not undergoing any drug treatment and free of exogenous vitamins.
Each subject was interviewed about smoking habits, occupational exposure to potential carcinogens and mutagens, and dietary history. The final groups consisted of 6 smokers (2 controls and 4 patients) consuming at least three cigarettes per day at the time of analysis and 39 individuals (16 controls and 23 patients) who had never smoked. Ex-smokers were not included in the study. The demographic and clinical characteristics of patients and controls are reported in Table I.
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The blood samples were blind coded and read by the same two investigators. Two microscopists scored two slides, each from two different cultures, for a total of 50 metaphases and 500 binucleated cells, respectively. Expert observers have previously found this analysis to be reproducible (Barale et al., 1998
; Andreassi et al., 1999).
In vivo study
Peripheral blood was collected using heparin as an anticoagulant; for evaluation of chromosome aberration and micronucleus frequency, two separate cultures (each in duplicate) from each sample were set up by mixing 0.3 ml whole blood with 4.7 ml Ham's F12 medium (ICN, Irvine, USA), supplemented with 10% fetal calf serum (ICN), 1.5% phytohaemagglutinin (Glaxo-Wellcome, Uxbridge, UK) and antibiotics (100 IU/ml penicillin and 100 mg/ml streptomycin; Sigma, St Louis, MO). All cultures were incubated at 37°C in a humidified atmosphere of 5% CO2. For evaluation of chromosome aberrations, the cultures were fixed after 48 h of incubation, following a terminal 2 h treatment with 4 µg/ml colchicine (Sigma) to arrest the lymphocytes in metaphase. For micronucleus frequency determination, cultures were maintained for 72 h and cells were blocked in cytokinesis by adding 3 µg/ml cytochalasin B (Sigma), after 44 h of incubation. Harvesting of cells, hypotonic treatment, fixation and slide preparation were performed according to the previously described method (Barale et al., 1998; Andreassi et al., 1999). For each sample, 100 metaphases per subject were scored, screening for chromosome aberrations and 1000 binucleated cells were scored for the evaluation of micronucleus frequency. Chromosome aberrations frequency without gaps was expressed as percent of aberrant cells.
In vitro study
Six separate experiments were performed each with a blood sample from a different healthy donor. For each culture, 0.3 ml whole blood was added to 4.7 ml Ham's F12 medium, supplemented with 10% fetal calf serum, 1.5% phytohaemagglutinin and antibiotics. Cultures were kept at 37°C during the entire incubation time (72 h). Sodium nitroprusside was dissolved in H2O and 100 µl of solution was added to the culture after 24 h of incubation to reach final concentrations ranging from 0 to 20 µmol/l. These concentrations are lower than those commonly infused in patients for the treatment of cardiovascular disease (i.e. 50–200 mg/l, corresponding to 170–680 µmol/l) (Oates, 1996). Control cultures with 100 µl H2O were performed. Cells were blocked in cytokinesis by adding 6 µg/ml cytochalasin B after a 44 h incubation and then, after a 72 h incubation, were treated with hypotonic solution (0.075 mol/l KCl), fixed with 5:1 (v/v) methanol:acetic acid and dropped onto a clean microscopic slide. Air-dried slides were stained and scored blind for SNP treatment, microscopically; 1000 binucleated cells (BN) were scored for the evaluation of MN frequency. Furthermore, the nuclear division index (NDI) was calculated according to the formula:
Nuclear division index (NDI) = (M + 2B + 3TR + 4TE)/(M + B + TR + TE)
where M, B, TR and TE represent the numbers of cells with one (mononucleated), two (binucleated), three (trinucleated) and four (tetranucleated) nuclei, respectively.
Statistical analysis
All data were expressed as mean ± SD. A non-parametric test was used to compare patient and control populations (Mann–Whitney test).
Multiple regression analysis was also applied in order to investigate the effect of each variable in determining the frequency of micronuclei and chromosome aberrations (Bonassi et al., 1994). Age was included as covariate. Because CA and MN values are not normally distributed, the logarithmic transformation was used for statistical analysis. One-way analysis of variance and Scheffe's multiple range test were used to compare the mean CA and MN frequencies among three or more groups. For all sodium nitroprusside concentrations the induction of micronucleated binucleated cells (MNBN), the frequency of BN and the value of NDI were statistically compared in both treated and control cells by means of Bonferroni test after repeated ANOVA measures.
The statistical analyses were performed with BMDP Statistical Software (BMDP Statistical Software, Los Angeles, CA, USA, 1990) and Statview 4.0 (Abacus Concepts, Berkeley, CA, USA).
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Results |
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In vivo study
Patients had a non-significant increase of CA frequency and a higher value of MN frequency when compared with control subjects (2.1 ± 1.4 versus 1.6 ± 1.2 and 6.5 ± 4.6 versus 3.5 ± 2.9; P = 0.23 and 0.01, respectively). Multiple regression analysis on structural chromosome aberration is shown in Table II
. The frequency of structural chromosome aberrations (excluding gaps) was not affected significantly by therapy or by confounding factors. Comparison between groups for frequency of chromatid- or chromosome-type total aberrations was also not significant (Table III).
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The results of multiple regression analysis on MN data are presented in Table IV
. In the case of MN analysis, the effect of the therapy was clearly evident (P = 0.023), whereas the confounding factors did not seem to significantly influence the frequency of micronuclei. When data were assessed by analysis of variance (ANOVA) non-significant differences in smoking habit and any gender effect were observed in the two groups. None of the cytogenetic parameters considered were found to be correlated with age. A non-significant correlation was found between the frequency of micronuclei and length of exposure to nitrate treatment (P = 0.51).
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In vitro study
Treatment of human lymphocytes with sodium nitroprusside resulted in a clear dose-dependent increase in the frequencies of MNBN throughout the dose range studied (y = 7.2 + 0.617x, R2 = 0.942); moreover, MN frequency significantly increased at all tested doses, compared with basal value (Table V
). Sodium nitroprusside treatment manifested cytotoxicity and cell cycle delay with a statistically significant decrease in the proportion of BN and NDI within the dose range 5–20 µmol/l with respect to basal values. Moreover, we observed on average 0.1% of seriously damaged cells with heavily fragmented nuclei dispersed into the cytoplasm (karyorrhexis) of lymphocytes treated with SNP in all six experiments.
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Discussion |
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To the best of our knowledge, these observations are the first evidence of a possible genotoxic activity of nitrate therapy in human lymphocytes.
There are at least three mechanisms by which NO formed within cells could exert genotoxic effects after reacting with O2 (Victorin, 1994; Liu and Hotchkiss, 1995; Felley-Bosco, 1998; Wink et al., 1998; Burney et al., 1999). The first results from the formation of N-nitroso compounds which can, in turn, alkylate DNA (Liu et al., 1991, 1992; Wu et al., 1993). The second is by direct reaction with the primary amino groups of DNA, resulting in deamination. This pathway leads to base conversions (e.g. cytosine to uracil and 5-methylcytosine to thymine); if unrepaired, these types of conversions would lead to G:C
A:T transitions which have been reported to occur in vitro following NO exposure (Wink et al., 1991; Nguyen et al., 1992). The third results from the formation of highly oxidative species such as ONOO· and HO· which can cause oxidative damage. Oxidative damage to DNA produces a complex mixture of damaged bases as well as the generation of apurinic/apyrimidinic sites and strand breaks (Beckman et al., 1990; Ames and Gold, 1991; Tannenbaum et al., 1994; Juedes and Wogan, 1996).
The potential mutagenicity of nitric oxide, nitrates and nitrites was assayed by in vivo and in vitro experiments. Compounds which release NO· and nitroglycerin have been shown to be mutagenic in the Ames bacterial mutagenicity test (Wink et al., 1991). Induction of mutation and chromosomal aberration was observed in experimental models following exposure to nitrogen oxides, nitrate and nitrite (Isomura et al., 1984; Luca et al., 1985, 1987; Nayak et al., 1989) and an increase of micronucleus formation by nitric oxide was observed in mammalian cells using sodium nitroprusside as a drug donor of NO (Lin et al., 1998). In human studies, consumption of drinking water with high nitrate levels can lead to genotoxic risk (Tsezou et al., 1996; van Maanen et al., 1996). An increase in micronucleus frequency was observed in nurses with occupational nitrous oxide exposure (Chang et al., 1996), while no detectable increase in chromosome aberrations in human lymphocytes could be observed after short time inhalation of nitric oxide (Luhr et al., 1998).
The results presented in this study provide evidence for an association between nitrate therapy and DNA damage in human lymphocytes. Nitrates are an unequivocally beneficial drug therapy. The clinical relevance of the observed effect on MN frequency remains unclear. In particular, we cannot assess whether our finding is a non-specific observation of cellular epiphenomena or rather represents a specific cytotoxicity, potentially accounting at least in part for some epidemiological observations, such as the increase in mortality `paradoxically' associated with long-term nitrate use in ischaemic heart disease, as shown by the Multicenter Myocardial Ischaemia Research Group (Nakamura et al., 1999), and usually explained on the basis of the `tolerance' effect that plagues the clinical use of these drugs (Thadani, 1997).
Our data indicate a higher frequency of MN in patients' cells compared with those of healthy controls, while a non-significant increase of CA frequency was observed. A possible explanation for the results observed in CA frequency lies in the smaller number of cells analysed as compared with MN analysis. This difference in the number of observations might have influenced the power of the statistical analysis.
On the other hand, since the chemistry of DNA damage from nitric oxide is considerably complex, the mutagenic consequences are not well understood. The possibility of aneugenic activity of nitrate therapy might be important after long-term chronic exposure. In view of this evidence, since MN can contain either a fragment or a whole chromosome, it would be of interest to verify the presence of centromeric DNA (Becker et al., 1990) in micronuclei of patients undergoing long-term nitrate therapy. In addition, in vitro effects might be limited in vivo by potent antioxidant defences. Thus, studies of metabolic activation with liver membrane fraction (S9 fraction) could demonstrate whether the antioxidant enzymes present in S9 fractions protect the cells from the genotoxic activity of the sodium nitroprusside.
Finally, it is noteworthy to underline that studies on DNA damage in heart disease are lacking and other factors (e.g. reactive oxygen compounds and viruses) might influence the accumulation of DNA damage in these diseases (Andreassi et al., 2000).
These limitations encourage additional work in this area; our results suggest a possible genotoxic activity in human lymphocytes for nitrate therapy, that could be associated with the production of free radicals induced by nitrate tolerance (Munzel et al., 1995) and with the potential adverse effects of long-acting nitrate therapy in chronic coronary disease (Nakamura et al., 1999).
More direct experimental data are needed at this point to investigate if lifelong nitrate therapy has potential mutagenic activity, contributing in particular to the accumulation of DNA damage in coronary heart disease.
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2 To whom correspondence should be addressed, andreas{at}ifc.pi.cnr.it
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Received on February 13, 2001; accepted on July 16, 2001.
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