Mutagenesis, Vol. 14, No. 4, 433-436,
July 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Induction and prevention of micronuclei and chromosomal aberrations in cultured human lymphocytes exposed to the light of halogen tungsten lamps
Department of Health Sciences, Section of Hygiene and Preventive Medicine, University of Genoa, Via A. Pastore 1, I-16132 Genoa and 1 Laboratory of Cytogenetics, S.Martino Hospital, Genoa, Italy
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Abstract |
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Previous studies have shown that the light emitted by halogen tungsten lamps contains UV radiation in the UV-A, UV-B and UV-C regions, induces mutations and irreparable DNA damage in bacteria, enhances the frequency of micronuclei in cultured human lymphocytes and is potently carcinogenic to the skin of hairless mice. The present study showed that the light emitted by an uncovered, traditional halogen lamp induces a significant, dose-related and time-related increase not only in micronuclei but also in chromosome-type aberrations, such as breaks, and even more in chromatid-type aberrations, such as isochromatid breaks, exchanges and isochromatid/chromatid interchanges, all including gaps or not, in cultured human lymphocytes. All these genotoxic effects were completely prevented by shielding the same lamp with a silica glass cover, blocking UV radiation. A new model of halogen lamp, having the quartz bulb treated in order to reduce the output of UV radiation, was considerably less genotoxic than the uncovered halogen lamp, yet induction of chromosomal alterations was observed at high illuminance levels.
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Introduction |
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Millions of people are exposed, often at high illuminance levels and for long periods of time, to the light emitted by halogen tungsten lamps, which provide a modern and pleasant illumination system for public and private indoor environments. Unfortunately, the quartz bulb of halogen lamps is permeable to the UV radiation delivered by the incandescent tungsten filament, covering a wide spectrum of wavelengths not only in the UV-A region but also in the UV-B and UV-C regions (McKinlay et al., 1989
).
We discovered that, due to these UV components, the light emitted by uncovered halogen lamps induces a range of mutations in his– Salmonella typhimurium strains (De Flora et al., 1990), is potently genotoxic in DNA-repair deficient Escherichia coli strains (De Flora et al., 1991), as confirmed in another laboratory (Woójcik and Janion, 1997), and increases the frequency of micronuclei (MN) in cultured human lymphocytes (D'Agostini et al., 1993). Moreover, under various experimental conditions, exposure to halogen lamps consistently resulted in a high yield of skin tumours in hairless mice (De Flora and D'Agostini, 1992; D'Agostini and De Flora, 1994). All exposed mice developed a variety of lesions, both benign and malignant, including squamocellular carcinomas carrying p53 mutations (D'Agostini et al., 1994). All investigated genotoxic and carcinogenic effects were completely prevented by shielding the quartz bulb of halogen lamps with UV-blocking glass covers (De Flora et al., 1990, 1991; De Flora and D'Agostini, 1992; D'Agostini et al., 1993; D'Agostini and De Flora, 1994).
Recently, new models of halogen tungsten lamps have become available, in which the quartz bulb is `doped`, e.g. by incorporating titanium and cerium compounds, in order to reduce its permeability to UV radiation. Here we show that exposure of cultured human lymphocytes to the light emitted by uncovered traditional halogen lamps results in a dose-related and time-related induction of MN and chromosomal aberrations (CA), including a variety of chromosome-type and chromatid-type aberrations. These alterations were attenuated but not completely absent when testing a halogen lamp with a UV-blocking quartz bulb, which contrasted with the total protection obtained by shielding a traditional halogen lamp with a silica glass cover.
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Materials and methods |
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Lamps and exposure systems
We used two commercially available models of tungsten halogen lamps. Both models operated at 12 V, had a power of 50 W and were incorporated in a dichroic spotlight lamp. One of them was a traditional lamp having an untreated quartz bulb, whereas the second model had the quartz bulb `doped' in order to reduce its permeability to UV light.
The exposure time (15–120 min) was regulated with a timer and the distance of the lamp from lymphocyte cultures was adjusted to yield illuminance levels between 1000 and 10 000 lux, which were obtained at distances ranging between ~200 and 50 cm. Illuminance was measured by means of a luxmeter (model 1300V; ICE, Milan, Italy). In some experiments in which the traditional halogen lamp having an untreated quartz bulb was tested, a 3 mm silica glass cover was installed 15 mm below the lamp, in order to avoid overheating of the light source. Irrespective of the presence of the cover, the air temperature near the irradiated cultures did not vary to an appreciable extent, a maximum of 30
C being reached after exposure for 120 min at a distance of 50 cm.
Chromosome/chromatid aberration analyses
Blood samples (10 ml) were collected by venipuncture from three male healthy donors aged 30–38 years. One millilitre aliquots of whole blood were incubated at 37C in plastic flasks containing 10 ml RPMI-1640 medium supplemented with 10% fetal calf serum (Biochrom Beteiligungs, Berlin, Germany) and stimulated with purified phytohaemagglutinin (Murex Biotech, Dartford, UK) at a concentration of 10 µg/ml. After the first two experiments, in which whole blood was light exposed immediately after collection, all remaining experiments were carried out by exposing cultured lymphocytes. In particular, after 72 h incubation, the cell cultures were removed from the flasks, distributed into plastic Petri dishes and exposed to the light emitted by the halogen lamps. Immediately after exposure, colcemid (Sigma, St Louis, MO) was added at a final concentration of 0.05 µg/ml for 45 min. Hypotonic treatment and cell spreading were performed according to standard metaphase analysis methods. Slides were air dried, aged at room temperature for 3 days and then stained by the Q-banding and G-banding techniques. Metaphases were photographed with a Zeiss Photomicroscope III (Zeiss, Oberkochen, Germany) and then examined by two different readers with a high resolution Sony PHV 7E4 photo video system (Sony, Tokyo, Japan). A total of 100 metaphases/donor were analysed.
Micronucleus analyses
Immediately after collection of blood by venipuncture, lymphocytes were separated by sedimentation in a TC Chromosome Blood Separation Vial (Difco, Detroit, MI). Aliquots of separated blood (0.5 ml) were diluted with an equal volume of phosphate-buffered saline (120 mmol/l NaCl, 2.7 mmol/l KCl and 10 mmol/l phosphate buffer, pH 7.4), distributed into plastic Petri dishes and exposed to the light emitted by the halogen lamps. Irradiated lymphocyte suspensions were collected and added to 5 ml of complete culture medium (Chromosome Medium 1x; Boehringer Mannheim, Mannheim, Germany), containing phytohaemagglutinin. After incubation at 37C for 44 h in sealed vials, cytochalasin B (Sigma) was added at a final concentration of 6 µg/ml. After a further 28 h incubation, cells were recovered by centrifugation at 200g for 10min, washed twice with saline solution and fixed in methanol:acetic acid (3:1). Slides were prepared using a cytocentrifuge (Heraeus Sepatech, Osterode, Germany). After air drying, the slides were stained with 10% Giemsa solution for 10 min, cleared with xylene and mounted with Eukitt. Coded slides were scored by two different readers, each one examining at least 1000 binucleated lymphocytes at 1000x magnification. Induction of MN by halogen lamps, under the experimental conditions reported in Results, was evaluated in three separate experiments using lymphocytes from a male, 34-year-old donor.
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Results |
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In the first two experiments, whole blood was light exposed immediately after collection, after which lymphocytes were stimulated with phytohaemagglutinin and cultured for 72 h. These experiments did not yield satisfactory results. In fact, even at 10 000 lux for 120 min, the number of chromosomal aberrations in irradiated samples was hardly doubled as compared with unirradiated controls (data not shown). Therefore, all subsequent experiments were carried out by exposing the lymphocytes at the end of the 72 h incubation period.
A traditional lamp having an untreated quartz bulb enhanced the frequency of both CA (Table I) and MN (Table II). In particular, exposure of cultured human lymphocytes to the light emitted by the traditional lamp resulted in a significant enhancement of chromosome-type aberrations, such as breaks, and of chromatid-type aberrations, such as isochromatid breaks, exchanges and isochromatid/chromatid interchanges, all including gaps or not. The frequency of MN was also enhanced to a significant extent. At equivalent exposure times, the genotoxic response was significantly related to illuminance levels in the 1000–10 000 lux range for chromosome-type aberrations (r = 0.974, P = 0.027), chromatid-type aberrations (r = 0.983, P = 0.017) and MN (r = 0.996, P = 0.0037). The equations for the regression lines relating illuminance level (x) to either chromosome-type aberrations, chromatid-type aberrations or MN (y) were y = 1.46 + 0.001x, y = 6.6 + 0.004x and y = 1.35 + 0.004x, respectively. Likewise, at equivalent illuminance levels, the genotoxic response was significantly related to exposure time (0, 30, 60 and 120 min for CA and 0, 15, 30 and 60 min for MN) for chromosome-type aberrations (r = 0.976, P = 0.024), chromatid-type aberrations (r = 0.984, P = 0.016) and MN (r = 0.955, P = 0.045). The equations for the regression lines relating the exposure times (x) to each one of the three types of genotoxic effects (y) were y = 0.62 + 0.071x, y = 1.2 + 0.328x and y = 2.28 + 0.177x, respectively.
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Shielding the same type of traditional lamp with a 3 mm thick cover of silica glass protected the exposed lymphocytes from genotoxic effects, as shown by a lack of increase in chromosome-type and chromatid-type aberrations at an illuminance of 10 000 lux for 120 min (Table I
) and by a lack of increase in MN at either 1000, 5000 or 10 000 lux for 15, 30 or 60 min (Table II).
Under the same illuminance and exposure time conditions, the light of a lamp having the UV-blocking quartz bulb significantly enhanced the frequency of MN in lymphocytes, but only when tested at 10 000 lux for 60 min (Table II). The frequency of both chromosome-type and chromatid-type aberrations, tested only at 10 000 lux for 120 min, was also higher than in controls, but this difference was not statistically significant (Table I). This was affected by the higher SD values recorded in the CA analyses, in which the results are means of data obtained with lymphocytes from three donors, as compared with the MN analyses, in which the results are means of three experiments performed with lymphocytes from a single donor.
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Discussion |
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The results of the present study confirm that the light emitted by uncovered, traditional halogen lamps induces a significant increase in MN in cultured human lymphocytes, which is in agreement with our previous data (D'Agostini et al., 1993
). In addition, evidence is provided that the light of these lamps induces a rather high frequency of chromosome-type aberrations, such as breaks, and even more of chromatid-type aberrations, such as isochromatid breaks, exchanges and isochromatid/chromatid interchanges, all including gaps or not. The frequencies of MN and CA were significantly correlated with both illuminance level and exposure time.
The complete avoidance of MN and CA induction obtained by covering the same lamp with a silica glass screen and the attenuation of these effects observed when using a halogen lamp having a `doped', UV-blocking quartz bulb clearly indicate that the genotoxic effects produced by the light of the halogen lamp are due to its UV components. Traditional halogen tungsten lamps, similar to those used in the present study, have been shown to emit a broad spectrum of UV wavelengths, starting from the UV-C region (McKinlay et al., 1989), which is likely to be the range of wavelengths inducing melanoma (Swerdlow et al., 1988).
UV light is known to generate a variety of DNA alterations, including cyclobutane-type pyrimidine dimers, pyrimidine–pyrimidone(6–4)photoproducts, thymine glycols, cytosine damage, purine damage, DNA strand breaks and DNA–protein crosslinks (IARC, 1992). Extensive data are available in the literature concerning the induction of chromosomal effects by UV light in cultured mammalian cells (IARC, 1992). However, cultured human lymphocytes have not been used frequently for this purpose, probably because UV-induced DNA lesions are readily repaired in quiescent lymphocytes. In fact, a high frequency of CA can be induced when UV irradiation of human lymphocytes in the G0 stage is followed by treatment with DNA repair inhibitors, such as ara-C (Holmberg and Gumauskas, 1990). Due to the poor induction of CA in our preliminary experiments using lymphocytes in the G0 stage, we decided to irradiate the lymphocytes after a 72 h incubation. A large proportion of the cells being in the M phase at that time, cellular DNA was expected to be more susceptible to UV-induced damage and very little time was allowed for its repair. The more clearcut increase in MN frequency in lymphocytes irradiated in the G0 stage may be ascribed to the fact that this cytogenetic damage is a consequence not only of chromosome breaks but also of spindle disturbances. Another tentative explanation is that the MN frequency was assessed by irradiating separated lymphocytes rather than whole blood, which may partly shield UV radiation.
Although we do not pretend to extrapolate these data to the in vivo situation from a quantitative point of view, the observed yield of chromosomal alterations in cultured human lymphocytes from healthy donors, together with the recognized ability to cause mutations (De Flora et al., 1990) and non-reparable DNA damage in bacteria (De Flora et al., 1991; Woójcik and Janion, 1997) and to produce a high multiplicity of skin tumours in hairless mice (De Flora and D'Agostini, 1992; D'Agostini and De Flora, 1994) leave little doubt that the light emitted by uncovered, traditional halogen lamps potentially poses a health hazard to humans.
The availability of new models of halogen lamps having treated quartz bulbs represents a step forward in the safety of this illumination technology. However, the results obtained in our experiments show that at high illuminance levels even these lamps can produce an enhancement of chromosomal alterations, which contrasts with the complete prevention of genotoxic effects obtained by simply covering a traditional halogen lamp with a silica glass screen. These conclusions are in agreement with the findings of a parallel study, in which the bacterial genotoxicity of a large number of lamps having treated quartz bulbs, from different producers, was compared with the effects of traditional lamps, either uncovered or covered (Camoirano et al., 1999).
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Acknowledgments |
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This study was supported by grants from the Italian Ministero del Lavoro e della Previdenza Sociale (Fondo Speciale Infortuni) and the Genoese Atheneum.
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Notes |
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2 To whom correspondence should be addressed. Tel: +39 010 353 8500; Fax: +39 010 353 8504; Email: sdf{at}unige.it
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References |
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Received on January 25, 1999; accepted on March 22, 1999.
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