Mutagenesis Advance Access published online on January 27, 2008
Mutagenesis, doi:10.1093/mutage/gem049
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Pre-irradiation exposure of peripheral blood lymphocytes to glutaraldehyde induces radiosensitization by increasing the initial yield of radiation-induced chromosomal aberrations
Health Physics Laboratory, National Centre for Scientific Research "Demokritos," 15310 Agia Paraskevi, Athens, Greece 1Department of Occupational Hygiene, National School of Public Health, Athens, Greece & Hellenic Institute for Occupational Health and Safety ELINYAE, Athens, Greece 2Department of Forensic Medicine and Toxicology, Medical School, University of Athens, Greece
Glutaraldehyde (GA) is a high production volume chemical that is very reactive with a wide spectrum of medical, scientific and industrial applications. Since human exposure in anthropogenic and occupational environment occurs frequently, GA has been extensively tested for genotoxic activity in vitro and in vivo. However, there are conflicting results in the literature and there is a lack of information concerning the combined effects of exposure to both GA and ionizing radiation in human cells. In the present study, the results obtained using conventional cytogenetic analysis do not suggest a statistically significant clastogenic or genotoxic activity of GA when concentrations in the range of 10–6 to 10–2 mM were applied. However, a 24-h pre-irradiation exposure of human peripheral blood lymphocytes (PBLs) to non-genotoxic doses of GA showed a statistically significant (P > 0.05) increase in chromosomal radiosensitivity. The observed increase may be an effect of GA-induced alterations in the cell-cycle and feedback control mechanisms during the cell-cycle transition points or it may be a consequence of an effect of GA either on the DNA repair capacity of the cells after irradiation or on the initial induction of radiation-induced chromosomal damage. To elucidate the mechanism underlying the obtained radiosensitization, conventional cytogenetics, the G2 chromosomal radiosensitivity assay and premature chromosome condensation methodologies were applied. The results support the hypothesis that pre-irradiation exposure of PBLs to GA induces radiosensitization by increasing the initial yield of chromosomal aberrations following irradiation.
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Introduction |
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Glutaraldehyde (GA) is an aliphatic dialdehyde with a wide spectrum of medical, scientific and industrial applications. It is used as a disinfectant for cold sterilization of medical equipment but also as a fixative in histochemistry and electron microscopy, a leather tanning agent, an ingredient in cosmetics and pharmaceuticals (1
,2) and as a hardener in X-ray developer solution. Radiologists and X-ray technicians, who represent a large segment of the working population exposed to radiation from human-made sources (3), may be exposed both to GA and ionizing radiation (IR). Although the genotoxic action of GA in bacterial tests and mammalian cells has been studied extensively, until now there is no cytogenetic data available concerning the effect of GA in human peripheral blood lymphocytes (PBLs). In addition, no studies in mammalian cells have been reported concerning the combined effect of GA with IR. The need for an in-depth investigation in GA's genotoxic effects arises from the fact that this chemical has been characterized as an irritant and corrosive to the skin, eyes and respiratory track and it has also been associated with the darkroom disease among radiographers (4), with multiple chemical sensitivity among nurses (5) and occupational asthma (1). At present, however, there are conflicting results with regard to GAs' genotoxic activity and genotoxicity studies in rodent systems have given both positive and negative results (1,6). Controversial results have been also reported in studies concerning the carcinogenic potential of this chemical. Low, but statistical significant, levels of leukemia and bone marrow hyperplasia were seen in one chronic drinking water study in rats, but not in a chronic inhalation study in rats or two chronic inhalation studies in mice (7). In humans, there is a lack of genotoxic studies on GA in blood cells and no studies with positive carcinogenic results for GA have been published. However, a recent case report, referring to occupational exposure to GA, may support the possibility of this chemical to be considered as a suspected leukemogen and opens up new questions concerning its carcinogenic potential (8). This finding, together with the fact that IR is associated with acute myeloid leukemia (AML) (9), triggers the interests for further investigation of the impact of the combined action of GA and IR at chromosomal level. Increased risk of AML and cancer predisposition in general is linked with several inherited conditions, such as ataxia telangiectasia (10) and Nijmegen breakage syndrom (11), which possess excessive chromosomal fragility spontaneously as well as following irradiation. The latter can be analyzed using the so-called G2 chromosomal radiosensitivity assay (G2 assay) (12–14). It has been extensively reported that increased G2 chromosomal radiosensitivity is linked with early development of several forms of cancer and, therefore, chromosomal radiosensitivity has been proposed as a biological indicator for cancer proneness (13–16). Even though there is interindividual variation in response to IR (17), not only endogenous factors such as inherited conditions but also environmental factors such as chemicals, which are able to cause increased G2 chromosomal radiosensitivity, may be considered as potential carcinogens.
In the present work, the genotoxic effects of GA were studied and, particularly, the potential of this chemical to enhance radiosensitivity in human PBLs at the chromosome level was investigated. The experimental data show that GA, at doses that are not acutely toxic to PBLs, enhances G2 chromosomal radiosensitivity that is quantitated as excess chromosomal fragility in the subsequent metaphase. The molecular mechanisms underlying such chromosome radiosensitization may be a consequence of an effect of GA either on the initial induction of radiation induced DNA damage or on the DNA repair capacity of the cells after irradiation. Furthermore, the observed radiosensitization may be linked to GA-induced alterations in the cell-cycle and feedback control mechanisms during the cell-cycle transition points (18–22). To elucidate the mechanism involved, conventional cytogenetic analysis, the G2 assay and premature chromosome condensation (PCC) methodologies (23) were applied. The results obtained support the hypothesis that pre-irradiation exposure of PBLs to GA induces radiosensitization by increasing the initial yield of radiation-induced chromosomal aberrations.
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Materials and methods |
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Cell culture, irradiation conditions and chemicals
Peripheral blood samples were taken by venipuncture from healthy non-smoking individuals and collected in heparinized tubes. A 0.5 ml of whole blood was added to each culture tube containing 5 ml of McCoy's 5A medium, supplemented with 10% foetal calf serum (FCS), 1% l-glutamine, 1% antibiotics [penicillin (100 U/ml)–streptomycin (100 µg/ml)] and 0.2% phytohemagglutinin (PHA). All incubations were at 37°C for 72 h in a humidified incubator (5% CO2, 95% air). Human lymphocytes were obtained from freshly drawn blood, separated by Ficoll-Paque sedimentation and suspended in McCoys's 5A medium supplemented with 10% FCS. Chinese hamster ovary (CHO) cells were grown in McCoy's 5A culture medium supplemented with 10% FCS and antibiotics. PCC experiments described here were performed using M-phase hamster cells obtained from exponentially growing cells after a 4-h treatment with 0.2 µg/ml colcemid. All culture media and chemicals were obtained from Biochrom-AG, Germany unless stated otherwise. GA was prepared in PBS. Stimulated (with PHA) and non-stimulated lymphocytes were treated for several time intervals at a range of 10–6 to 10–2 mM. Calyculin-A (Wako, Japan) was prepared as a 1 mmol/l solution in ethanol. All chemicals were purchased from Sigma–Aldrich (Germany) unless stated otherwise. Irradiations were carried out at room temperature with a GammaCell 220 irradiator (Atomic Energy of Canada Ltd, Ottawa, Canada) at doses ranging from 1 to 6 Gy. To test the genotoxic activity of GA, whole-blood cultures were exposed to GA for 24 h, at final concentrations ranging from 10–6 to 10–2 mM, before stimulation of PBL with PHA to enter mitosis 48–50 h latter on. Furthermore, the genotoxic activity of GA was tested at chromatid level in PHA-stimulated PBLs by exposing blood cultures to GA, 24 h after stimulation. In addition, the effect of GA on G2 lymphocytes was tested by exposing blood cultures 72 h after PHA stimulation to GA for 1 h before harvesting cells at metaphase. To test the effect of GA on radiation-induced initial chromosomal damage, isolated unstimulated G0-phase PBLs were exposed to GA for 24 h followed by exposure to 2, 4 and 6 Gy of
-radiation. Cells were fixed 48 h after irradiation of the culture. For each experimental point, standard deviations of the mean values from three independent experiments were calculated. Data were evaluated statistically by Student's t-test. All P values were considered statistically significant at P < 0.05.
Cell harvesting
Two hours before the total incubation period, cells were arrested in the metaphase stage of their mitosis by the addition of colcemid (final concentration 0.1 µg/ml). The cultured cells were harvested by speed centrifugation (1450 r.p.m.) and treated with hypotonic potassium chloride (0.075 M; Sigma–Aldrich), fixed with freshly prepared 3:1 methanol–acetic acid (v/v) (Fluka and Baker) and 20 µl of cell suspension was dropped on wet slides. The slides were air-dried and stained in a 2% solution of Giemsa dye (Merck KGaA, Germany) for 15 min and rinsed with water. Air-dried slides were embedded with cover slips and coded for analysis to avoid bias. Chromatid-type aberrations and chromosome-type aberrations were scored according to standard criteria (24). Only chromatid breaks being defined as misaligned discontinuities and gaps longer than a chromatid width were considered for scoring. A response was considered positive if the percentage of cells with structural chromosomal aberrations was statistically higher (P < 0.05) compared to the control.
G2 assay
In order to test the effect of GA on G2 chromosomal radiosensitivity, exponentially growing PBL cultures were irradiated with 1 Gy in the presence of a subgenotoxic dose of GA. The cell cultures were then incubated for 30 min at 37°C and subsequently treated with 0.2 µg/ml colcemid for 1 h to arrest cells at metaphase using a modified G2-assay protocol (14). Cells were fixed and coded for analysis to avoid bias. Chromosomal breaks were visualized and quantified as chromatid breaks in the subsequent metaphase. For each experimental point, three independent experiments were carried out. In each case, 100 metaphase cells were analyzed for radiation-induced chromatid damage.
PCC using PEG-mediated cell fusion
To test whether the increased chromosomal damage after exposure to IR and GA is linked either to the radiation-induced initial chromosomal damage or to DNA repair mechanisms, G0 isolated human lymphocytes were treated with GA (24-h treatment) and IR (1–6 Gy) and fused with mitotic CHO cells following the basic protocol for PCC induction (19) first described by Pantelias and Maillie (25). For each experimental point, three independent experiments were carried out. In each case, 100 lymphocytes in G0 phase were scored for induced chromatid breaks.
PCC using chemical induction
To test whether the increased chromosomal damage after exposure to IR and GA is linked to cell-cycle and feedback control mechanism during the cell-cycle transition points (G1/S and/or G2/M checkpoint), the PCC methodology was applied by means of calyculin-A (50 nM) (18,26,27). About 3000 cells per each experimental point were classified according to their chromatin morphology in G1, S, G2 and M phase of the cell cycle. Standard deviations from three independent experiments from six different donors were calculated.
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Results |
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The genotoxic activity of GA in G0 lymphocytes was investigated using conventional chromosome aberration analysis at metaphases of cultured PBLs as described in Materials and Methods. Under these experimental condition, GA did not show any increase in chromosome aberrations (CAs) such as dicentrics, centric rings, acentric fragments and chromatid type of aberration when analyzed at metaphase.
To study the effect of GA on G2 chromosomal radiosensitivity, PBLs from six different donors were treated with GA at a range of 10–6 to 10–2 mM for 24 h before their exposure to 1 Gy of -rays in G2 phase and the G2 assay was applied as described in Materials and Methods. The results obtained are presented in Table I. A statistically significant increase was observed in the mean number of chromatid breaks in all the six donors, after exposure to non-genotoxic doses of GA combined with IR at concentrations of GA 
10–4 mM compared with the samples exposed to IR only. In particular, the effect of 10–2 mM of GA on PBL G2 chromosmal radiosensitivity, which is expressed as an increase in the number of chromatid breaks after exposure to 1 Gy of -rays, is shown in Figure 1.
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To elucidate the mechanism of the increased G2 chromosomal radiosensitivity induced after pre-irradiation exposure to GA, three different sets of experiments were carried out. In the first set of experiments, the effect of GA on G1/S or G2/M checkpoints following exposure to IR was evaluated using chemically induced PCC methodology to study uniquely the cell-cycle progression after exposure both to GA (10–2 mM) and IR (1 Gy in G2). This methodology was applied to visualize, classify and quantitate the progression stage and the percentage of interphase cells at the various phases of the cell cycle (G1, S and G2 phase) (18
,19). Calyculin-A-induced PCC has been used alone or in combination with other cytogenetic techniques in numerous cytogenetic and biological dosimetry studies (18,28–31). In the present work, calyculin-A-induced PCC was used as a cytogenetic tool for the classification of the lymphocytes in the different phases of the cell cycle (i.e. single chromatid per chromosome in G1 phase, non-centromeric chromosomes in G2 and pulverized chromosome regions in S phase) (Figure 2). Using this method, any delay or acceleration of transition from G1 (pre-DNA synthetic phase) to S phase (DNA synthesis) and from G2 phase (post-DNA synthetic) to mitosis (M phase) can be estimated. The results demonstrate that the presence of GA in irradiated cells does not affect the number of cells in the different cell-cycle phases (Figure 3).
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In the second set of experiments, the effect of GA on the initial induction of radiation-induced chromosome aberrations in G0 lymphocytes isolated from peripheral blood was investigated. For this purpose, G0 lymphocytes were irradiated at doses 2, 4 and 6 Gy. The chromosomal damage in the lymphocytes was evaluated immediately after irradiation in the presence or absence of GA (10–2 mM, 24-h exposure), using cell fusion with mitotic CHO cells and PCC induction (25
) (Figure 4). The linear dose response curves obtained show that exposure to GA affects the yield of radiation induced chromosomal damage in a statistically significant manner (Figure 5). Following this protocol, it is possible to study the chromosomal damage induced by IR and GA immediately after cellular exposure to genotoxic agents preventing the cellular DNA repair mechanism to recover the damage.
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The third set of experiments was designed to study the effect of GA on the repair kinetics of G0 lymphocytes that were exposed to 10–2 mM GA for 24 h and subsequently irradiated with a dose of 6 Gy. The effect of 10–2 mM GA on the repair kinetics of radiation-induced chromosomal damage was evaluated at various time intervals (0, 2, 4 and 6 h) after irradiation exposure, using cell fusion and PCC methodology. Under these experimental conditions, the exposure to GA resulted in an increase of the initial yield of the IR-induced chromosomal aberrations without affecting, however, the repair rate of chromosomal damage induced by IR as analyzed for up to 6 h after IR exposure (Figure 6).
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The results obtained from the three sets of experiments suggest that GA increases radiosensitivity by affecting the initial yield of radiation-induced chromosomal damage. In order to further validate this observation, a fourth set of experiments was carried out. For this purpose, non-stimulated G0-phase PBLs were exposed to GA for 1 and 24 h before IR exposure (2, 4 and 6 Gy) and PHA stimulation, as described in Materials and Methods. Chromosome aberrations such as dicentric chromosomes, centric rings, acentric fragments and acentric rings were scored for each sample. A statistically significant increase was observed in chromosome-type aberrations (dicentric chromosomes and centric rings) (Figure 7), in acentric fragments, as well as in acentric rings per cell in all samples exposed to both IR and 10–2 mM GA (1- and 24-h treatment), when compared to the samples exposed to IR only (Table II).
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Discussion |
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In the present work, the genotoxic effects of GA and the potential of this chemical to enhance radiosensitivity at the chromosome level were investigated in human PBLs. The outline of the overall experimental design and the results obtained are presented in Figure 8. Our studies have shown that a 24-h exposure to GA alone does not induce a statistically significant increase in chromosomal aberrations in any of the concentrations tested. Although there are no other available cytogenetic studies concerning the genotoxic profile of GA in human lymphocytes, an absence of GA's clastogenic potential was also notified by Vergnes and Ballantyne in rat bone marrow cells (6
). In another study, a weak increase in chromosome aberrations was observed with a statistically significant response only in the higher concentrations tested (32). Other scientific groups have shown that chromosome aberrations in bone marrow cells were reported in only one of eight studies using rats and mice and that micronuclei were not induced in bone marrow cells of mice (33,34).
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The lack of genotoxicity does not necessarily prove that GA has a risk-free profile. Taking into consideration results from previous studies, non-genotoxic doses from widely used chemical agents may have a potentiation effect when combined with other harmful agents. For example, hydroquinone, another constituent of the developers used in X-ray film processing, has been recently shown to enhance chromosomal radiosensitivity in irradiated lymphocytes of healthy donors (19
). With respect to GA, there are limited data available related to its combined effect with other chemical agents (35,36) and, so far, no data on the potentiation of IR-induced chromosomal aberration by this chemical in eukaryotic or mammalian cells have been reported.
The results obtained in the present work demonstrate that a 24-h pre-irradiation exposure of human PBLs to non-genotoxic doses of GA sensitizes cells to radiation-induced chromosomal damage. The endogenous sensitivity to IR, as evaluated using the G2 assay, varies among individuals (12,14,15,37,38) and the experimental data obtained show that pre-irradiation exposure to GA enhances G2 chromosomal radiosensitivity in all cases tested. Since increased chromosomal fragility in G2 phase has been linked with cancer proneness (13–15,18,37), these results and the mechanism involved may be of particular interest. Recently, it was reported that disturbances in checkpoint control mechanisms, especially during G2 to M-phase transition, can be associated with an increase in chromosomal fragility (14,19,39) and cancer risk (18,40,41). The experimental data obtained in the present study using a chemically induced PCC methodology indicate, however, that the increased chromosomal radiosensitivity induced by non-genotoxic doses of GA is not mediated by an effect of the chemical on cell-cycle transition point mechanism. In particular, the percentage of cells in the different phases of the cell cycle of irradiated cells is not affected after exposure of cells to 10–2 mM of GA. Consequently, such a mechanism cannot be directly related to the increased G2 chromosomal radiosensitivity observed in our studies.
The potential of GA for radiosensitization by increasing the initial yield of radiation-induced chromosomal aberrations was also investigated using the PCC methodology. A major benefit of the PCC assay, compared to the conventional analysis in metaphase, is that it does not require cells to divide for the evaluation of cytogenetic damage. Therefore, PCC has been applied successfully in direct observation of radiation-induced cytogenetic damage in non-stimulated, interphase human lymphocytes (42–44), CHO (25,45) or HeLa cells (46). This application is of particular importance since it allows the visualization and scoring of radiation-induced chromosome damage in G1 cells immediately after exposure to genotoxic chemical agents and IR (47). The linear dose–response curves obtained using polyethylene glycol-mediated cell fusion and PCC induction demonstrate that a 24-h pre-irradiation exposure to 10–2 mM GA increases the yield of CAs after 2, 4 and 6 Gy of IR. This radiosensitization effect of GA could also be the result of its action on the repair process of radiation-induced DNA damage. However, the results obtained using the PCC methodology via cell fusion to test this possibility demonstrate that subgenotoxic doses of GA do not affect the repair rate of chromosomal damage as analyzed up to 6 h after irradiation.
The finding in the present work that GA increases the initial chromosomal damage induced by IR was further validated using conventional cytogenetic analysis. The rationale is that if the presence of GA enhances the initial radiation damage in G0 lymphocytes, an increased yield of dicentrics should be expected at the subsequent metaphase after PHA stimulation. In fact, a statistically significant increase in chromosome-type aberrations (dicentric chromosomes and centric rings), acentric fragments and acentric rings per cell were observed under these experimental conditions. However, the exact biochemical pathway by which GA interacts with IR is still unclear. It has been reported that its ability to produce DNA–histone protein cross-links in vitro (48) may be the mechanism underlying its mode of action. The molecule of GA is fairly small with two aldehyde groups separated by a chain of three methylene bridges. These aldehyde groups (-CHO) can combine with protein nitrogens resulting in cross-linking. Furthermore, GA has the ability to react with deoxyribonucleosides to form reaction products with deoxyadenosine, deoxycytidine and deoxyguanosine (49). These alterations at the chromatid level, although not fully characterized, may in part explain the ability of the chemical to enhance fragility of PBL chromatin following IR exposure. The observed increase in radiation-induced damage as a result of lymphocyte pre-exposure to GA may have important implications for current assessments of safe levels of exposure to this chemical. Even though many organizations for human health and safety have determined peak concentrations and threshold limit values for GA exposure, the results presented here may open new questions concerning the hazard risk of GA for human health, especially when combined with genotoxic environmental factors.
In summary, our results suggest that acute non-toxic doses of GA enhance G2 chromosomal radiosensitivity in PBLs of healthy donors. Conventional cytogenetics and PCC methodologies were applied and the experimental data obtained support the hypothesis that GA exerts its action by increasing the initial yield of radiation-induced chromosomal damage without affecting cell-cycle kinetics or the repair rate of chromosomal damage.
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Hellenic Institute for Occupational Health and Safety (EPAN 1.1.5.2 [EC] ); European Commission (FIGH-CT-2002-00218).
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Conflict of interest statement: None declared.
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* To whom correspondence should be addressed. Tel: +30 210 6503865/210 6529615; Fax: +30 210 6534710; Email: georgia{at}ipta.demokritos.gr
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Received on October 12, 2007; revised on November 23, 2007; accepted on November 26, 2007.
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