Systemic genotoxic effects produced by light, and synergism with cigarette smoke in the respiratory tract of hairless mice

Roumen M. Balansky1,2, Alberto Izzotti1, Francesco D'Agostini1, Anna Camoirano1, Maria Bagnasco1, Ronald A. Lubet3 and Silvio De Flora1,4

1 Department of Health Sciences, University of Genoa, via A. Pastore 1, I-16132 Genoa, Italy
2 National Centre of Oncology, Sofia 1756, Bulgaria
3 National Cancer Institute, Rockville, MD 20892, USA

4 To whom correspondence should be addressed Email: sdf{at}unige.it


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
No information is available on the interaction between cigarette smoke, the most important man-made carcinogen, and light, the most widespread natural carcinogen. In order to clarify this issue, SKH-1 hairless mice were exposed to environmental smoke and/or to the light emitted by sunlight-simulating halogen quartz bulbs. After 28 days, intermediate biomarkers were evaluated in skin, respiratory tract, bone marrow and peripheral blood. The results showed that, individually, the light produced extensive alterations not only in the skin but even at a systemic level, as shown by formation of bulky DNA adducts in both lung and bone marrow and induction of cytogenetic damage in bone marrow and peripheral blood erythrocytes. Smoke damaged the respiratory tract and produced significant alterations in the skin as well as an evident cytogenetic damage in both bone marrow and peripheral blood. Interestingly, as compared with exposure to smoke only, alternate daily cycles of exposure to both light and smoke significantly increased malondialdehyde concentrations and DNA adduct levels in lung and the frequency of micronuclei in pulmonary alveolar macrophages. The oral administration of sulindac, a non-steroidal anti-inflammatory drug, attenuated several biomarker alterations due to the combined exposure of mice to light and smoke. In conclusion, the light induces a systemic genotoxic damage, which is presumably due to the UV-mediated formation in the skin of long-lived derivatives, such as aldehydes. This damage may mechanistically be involved in light-related hematopoietic malignancies. In addition, the light displayed an insofar unsuspected synergism with smoke in the induction of DNA damage in the respiratory tract.

Abbreviations: COX, cyclooxygenases; CS, cigarette smoke; DRZ, diagonal radioactive zone; ECS, environmental cigarette smoke; LI, labeling index; MDA, malondialdehyde; MN, micronucleated; NCE, normochromatic erythrocytes; 8-OH-dG, 8-hydroxy-2'-deoxyguanosine; PAM, pulmonary alveolar macrophages; PCE, polychromatic erythrocytes; PCNA, proliferating cell nuclear antigen; PN, polynucleated; SBC, sunburn cells; TBA, thiobarbituric acid; TLC, thin-layer chromatography; TUNEL, TdT-mediated dUTP nick end labeling


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is well established that tobacco smoking causes lung cancer, cancer at other sites and several other pathological conditions. Interactions between cigarette smoke (CS) and other agents have been evaluated in experimental studies and/or epidemiological investigations. For instance, CS acts synergistically with arsenic, asbestos, ethanol, silica and radiation (radon, atomic bomb emissions and X-rays), while there is antagonism with chloromethyl methyl ether, bis(chloromethyl) ether and chromium(VI) (14). Collectively, tobacco smoking and sunlight have been estimated to account for 40% of all human cancers (5). Combined exposures to CS, either mainstream or environmental (ECS), and sunlight or UV-emitting artificial light sources are likely to be extremely common in the population. From a theoretical point of view, reciprocal influences might occur via several mechanisms. For instance: (i) exposure to CS affects the immune system (2,6); (ii) the UV radiation present in sunlight or emitted by artificial light sources, especially in the UV-B region, also produces alterations of the immune function (7,8); (iii) CS components deposited onto the skin could undergo photoactivation, resulting in the conversion of procarcinogens into direct-acting carcinogens (9); (iv) inhibition of DNA replication following in vitro exposure of lymphocytes to UV light is less marked in smokers than non-smokers (10); (v) UV-A light and CS extracts act in an additive manner in inducing the expression of matrix metalloproteinase-1 in human fibroblasts (11); (vi) a typical CS component, benzo[a]pyrene, interacts with UV light in inducing DNA damage in vitro (1214); (vii) both UV light and CS are genotoxic and carcinogenic via multiple mechanisms, which may be a premise to a possible interaction between these agents.

In order to assess the interaction between CS and light, we designed a study in which hairless mice were exposed to ECS and/or light under well-controlled experimental conditions. Traditional halogen quartz bulbs were used as a source of UV-containing light. Notwithstanding the widespread application of these lamps as a modern illumination system, the light emitted by halogen bulbs contains a broad spectrum of UV wavelengths starting from the UV-C region (15). Accordingly, unless filtered through a common glass cover, this light is strongly genotoxic in both bacteria (16,17) and human cultured cells (18), and is a potent inducer of skin tumors in hairless mice (19,20). In the present study, the halogen quartz bulbs were covered with a UV-C filter in order to mimic more closely the spectrum of solar radiation on earth. The results obtained provide sound evidence that the light induces genotoxic damage not only in the skin but also at a systemic level. In addition, the light was found to interact synergistically with ECS in inducing molecular and cytogenetical alterations in the respiratory tract. The non-steroidal anti-inflammatory drug (NSAID) sulindac inhibited a variety of alterations produced by the combined exposure of mice to light and ECS.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mice
Forty female SKH-1 hairless mice, aged 4 weeks, were purchased from Charles River (Calco, Italy). The mice were housed in MakrolonTM cages on sawdust bedding, and maintained on standard rodent chow (MIL, Morini, S.Polo d'Enza, Italy) and tap water ad libitum. The temperature of the animal room was 23 ± 2°C, with a relative humidity of 55% and a 12 h day–night cycle. The housing and treatments of animals were in accordance with our national and institutional guidelines.

Treatments
After acclimatization for 10 days, the mice were randomly divided into five groups, and treated for 28 days as follows: Group 1, sham-exposed mice kept in filtered air and unexposed to halogen lamps; Group 2, mice exposed to the light of halogen lamps; Group 3, mice exposed to ECS; Group 4, mice exposed to both light and ECS; Group 5, mice exposed to both light and ECS, and additionally treated with oral sulindac.

Exposure to light was obtained by using halogen quartz bulbs, incorporated into dichroic spot light lamps (12 V, 50 W), which were supplied by Leuci (File S.p.A., Lecco, Italy). The lamps were equipped with filters cutting UV-C light (WG 280, Schott Optics Division, Mainz, Germany). The distance from the back of the mice was regulated in order to yield an illuminance level of 10 000 lux. Exposure was 5 days/week, 9 h/day.

ECS was generated by burning Kentucky 2R1 reference cigarettes (Tobacco Research Institute, University of Kentucky, Lexington, KY), having a declared content of 44.6 mg tar and 2.5 mg nicotine each, with a 23 mm butt remaining after smoking. The cigarettes were kept for 48 h before use in a standardized atmosphere, humidified with a mixture of 70% glycerol and 30% water. A whole-body exposure of mice to ECS was achieved by using a smoking machine (model TE-10, Teague Enterprises, Davis, CA). The machine was adjusted to produce a combination of side-stream smoke (89%) and mainstream smoke (11%), mimicking exposure to ECS. Burning five cigarettes at one time yielded on an average a total suspended particulate of 83 mg/m3 in the exposure chambers. Exposure was 5 days/week, 6 h/day divided into two rounds with a 3-h interval.

Exposure of mice to both light and ECS was achieved by following, 5 days/week, the following schedule: 3 h ECS, 3 h light, 3 h ECS and 6 h light. Light and ECS were not simultaneously applied in order to avoid the risk of alterations of ECS components by light (9).

Sulindac or [Z]-5-fluoro-2-methyl-1-[p-(methylsulfinyl)benzylidene]indene-3-acetic acid (Sigma Chemical Co., St Louis, MO) was given daily in drinking water, starting 3 days before the first exposure to light and ECS, and continuing until the end of the experiment. Solutions of the drug in drinking water (150 p.p.m., corresponding to an average intake of 44.7 mg/kg body wt) were prepared daily.

The body weight of each mouse was measured at time 0 and after 5, 8, 14, 18, 23 and 28 days of treatment. At time 0 and at periodical intervals (see Figure 1), peripheral blood was collected from the lateral tail vein of the eight mice from each experimental group, and smeared on duplicate slides. After 4 weeks, all animals were deeply anesthetized with diethyl ether and killed by cervical dislocation. Bronchoalveolar lavage was immediately performed by lavaging the lungs with three 2 ml aliquots of cold (4°C) 0.15 M NaCl infused via a cannula inserted in the trachea. The cells were washed twice with RPMI 1640 and then spun in a cytocentrifuge and fixed with methanol. The left femur of each animal was removed and dissected. Bone marrow was collected, partly smeared on duplicate slides, and partly stored at -80°C. The dorsal skin and lungs were removed, partly stored at -80°C and partly fixed immediately in buffered formalin for 24 h in order to preserve the stability of P53 epitopes, and then embedded in paraffin.



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Fig. 1. Examples of autoradiographic patterns of DNA adducts detected by 32P post-labeling in lung and bone marrow of unexposed controls and light-exposed mice.

 
Determination of total thiobarbituric acid reactive aldehydes
Malondialdehyde (MDA) and other thiobarbituric acid (TBA) reactive aldehydes were measured in skin and lung using the thiobarbituric acid method (21). Tissue homogenates (100 µl) were added to a mixture of 8.1% sodium dodecylsulfate, 20% acetic acid and 0.8% TBA, pH 3.5. The samples were heated at 95°C for 1 h, cooled in ice and extracted with n-butanol/pyridine (15/1, vol/vol). Absorbance at 532 nm of the organic phase was measured in a spectrophotometer (U-3200, Hitachi, Tokyo, Japan), referred to an MDA standard curve, and expressed as nmol TBA reactive aldehydes/grams wet tissue. The results are means ± SE of three replicate analyses.

DNA extraction
Samples of skin, lungs and bone marrow (5 mg each) were thawed, pooled within each experimental group, and homogenized in a Potter-Elvehjem apparatus at 4°C in 250 mM sucrose, 5 mM 1,4-dithiothreitol, 50 mM Tris HCl, pH 7.6. DNA was purified by a phenol–chloroform procedure (22) using an automatic DNA extractor (Genepure 341, Applied Biosystems, Foster City, CA) working under helium atmosphere. Homogenates were incubated with proteinase K (Boehringer Mannheim, Mannheim, Germany) in the presence of sodium dodecylsulfate, and sequentially extracted with 1 vol of a 25/24/1 vol/vol/vol phenol–chloroform–water mixture and 1 vol of chloroform. DNA was precipitated with absolute isopropanol, collected on a nitrocellulose filter, washed in 80% ethanol, dried under helium flow and dissolved in sterile water. DNA purity was examined by evaluating the absorbance at 230, 260, 270 and 280 nm (23).

Bulky DNA adducts
Bulky lipophilic DNA adducts were detected, after butanol extraction and 32P post-labeling (24), in skin and lung samples pooled from the five experimental groups and in bone marrow samples pooled from sham-exposed and light-exposed mice. 32P binding to DNA adducts was catalyzed by T4 polynucleotide kinase (Rockland, Gilbertsville, PA, USA), using [{gamma}-32P]ATP (64 µCi, sp. act. >=6000 Ci/mmol) (ICN, Irvine, CA, USA) as 32P donor. 32P labeled DNA adducts were separated by multidirectional thin layer chromatography (TLC) on a 10 x 8 cm cellulose sheet coated with polyethylenimine (Macherey and Nagel, Düren, Germany). Four chromatographic developments (D1, D2, D3, D4) were as described previously (24). DNA adducts were detected by means of a 32P imager (InstantImager, Packard, Meriden, CT). Adduct levels were quantified by calculating the ratio between c.p.m. detected in DNA adducts and c.p.m. detected in normal nucleotides, and reported as adducts/108 normal nucleotides. The results are means ± SE of either triplicate analyses (skin) or quintuplicate analyses (lungs and bone marrow).

Oxidative DNA damage
8-Hydroxy-2'-deoxyguanosine (8-OH-dG) was evaluated in skin and lung samples pooled from the five experimental groups and in bone marrow samples pooled from sham-exposed and light-exposed mice. A 32P post-labeling procedure was used, as described previously (25,26). The 32P post-labeling reaction was performed by polynucleotide kinase and [{gamma}-32P]ATP (16 µCi/µl, sp. act. 325 Ci/mmol). 32P-labeled 8-OH-dG molecules were purified by monodirectional TLC on 3 x 18 cm polyethyleneimine-coated cellulose sheets (Macherey and Nagel) in formic acid and detected by means of a 32P imager. Adduct levels were quantified by calculating the ratio between c.p.m. detected in the 8-OH-dG-related spot and c.p.m. detected in normal nucleotides. The results were expressed as 8-OH-dG molecules/105 normal nucleotides. A DNA-free sample was used as a negative control. An 8-OH-dG positive reference sample was obtained by incubating at 37°C calf thymus DNA with 1 mM CuSO4 plus 50 mM hydrogen peroxide, as reported previously (27). The results are means ± SE of either three replicate analyses (skin) or seven replicate analyses (lungs and bone marrow).

DNA photoproducts
DNA photoproducts were detected by DNA depolymerization to trinucleotides, 32P labeling and selective TLC, according to Bykov and Hemminki (28). DNA (10 µg) was depolymerized by sequential incubation with Snake Venom Phosphodiesterase (Sigma) and Prostatic Acid Phosphatase (Sigma). The enzymes were inactivated by heating (90°C for 15 min) and removed by centrifuge filtration using cartridge mounted membranes which collect molecules with a molecular weight >5000 (Ultrafree-MC Filter Units NMWL 5000, Sigma). The samples were vacuum evaporated and labeled with [{gamma}-32P]ATP by T4 polynucleotide kinase reaction. 32P labeled DNA photoproducts were purified by multidirectional TLC on 19 x 10 cm cellulose sheets coated with polyethyleneimine (Macherey and Nagel). Three chromatographic developments (D1, D2, D3) were performed by rotating the sheet 90° clockwise during D2. The following chromatographic buffers were used: D1, 1 M ammonium formate, 1% acetic acid, pH 5.4; D2, 1.75 M ammonium formate, 0.5% acetic acid, pH 5.4; D3, 1 M ammonium formate, 0.5% acetic acid, pH 5.4. DNA photoproducts were detected by electronic autoradiography and quantified by calculating the ratio between c.p.m. detected in DNA photoproduct-related spots and c.p.m. detected in normal nucleotides. The background value recorded in negative samples in the area corresponding to photoproducts was subtracted from the intensity of radioactive signals. A positive reference standard was obtained by exposing calf thymus DNA to an UV-C monochromatic source at 254 nm, while unexposed calf thymus DNA was used as a negative control. The results, expressed as photoproducts/106 nucleotides, are means ± SE of three replicate analyses.

Immunohistochemical analyses
P53 oncoprotein was detected in sections of skin and lung from each mouse by using CM5 polyclonal rabbit antibody (NCL-P53 CM5p, Novocastra Laboratories Ltd, Newcastle upon Tyne, UK), which detects both over-expression and mutation of P53 gene. Formalin-fixed paraffin-embedded sections (5 µm) were pre-treated with 0.01 M sodium citrate buffer (pH 6.0) in a microwave oven at high temperature for 10 min. The sections were routinely processed using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA), following the manufacturer's instructions. A slide of the same tissue, incubated with normal serum without CM5 antibody, was used as negative control. Slides of mouse T3T3 cells were used as positive controls. Slides were scored at a magnification of x400 and 1000 cells/mouse were examined. Any nucleus showing a partial or total brown staining was defined as ‘positive’. The results, expressed as percent of P53-positive cells (LI or labeling index) in either skin cells or bronchial epithelial cells, are means ± SE of the data obtained in the eight mice belonging to each experimental group.

For the evaluation of proliferating cell nuclear antigen (PCNA) immuno reactivity in skin and bronchial epithelium, 5 µm sections were cut and placed onto slides treated with poly-L-lysine (Poly-PrepTM Slides, Sigma Diagnostics, St Louis, MO). PCNA was detected by using the NCL-PCNA Kit (Novocastra Laboratories), following the manufacturer's instructions. This detection kit is based on an anti-PCNA monoclonal antibody (clone PC10) and employs avidin–biotinylated horseradish peroxidase complex technology (ABC technique). Slides were scored at a magnification of x400 and 1000 cells/mouse were examined. The results, expressed as percent of PCNA-positive cells, are means ± SE of the data obtained in the eight mice belonging to each experimental group.

Apoptosis
Apoptotic cells were identified both by morphological analysis (skin) and by TUNEL (TdT-mediated dUTP nick end labeling) method (skin and bronchial epithelium). For morphological analysis in skin, the slides were stained in hematoxylin and eosin. Identification of apoptotic sunburn cells (SBC) was based on cell shrinkage and nuclear features. Apoptotic cells have condensed, darkly stained nuclei, which often are smaller in size and occasionally may be fragmented. Previous studies (29) demonstrated that SBC are indeed apoptotic cells. The percentage of apoptotic SBC was detected in the whole epidermis (basal plus suprabasal layers). For TUNEL method applied to skin sections we used the DermaTacsTM In Situ Apoptosis Detection Kit (Trevigen, Gaithersburg, MD), following the manufacturer's instructions. Apoptotic cells in mouse bronchial epithelium were identified by TUNEL method by using the Tacs XL Blue Label In Situ Apoptosis Detection Kit (Trevigen), following the manufacturer's instructions. With all methods the slides were scored at a magnification of x400 and 1000 cells/animal were examined. The results, expressed as percent of apoptotic cells, are means ± SE of the data obtained in the eight mice belonging to each experimental group.

Cytogenetic parameters
The cytogenetic damage was evaluated in pulmonary alveolar macrophages (PAM) and bone marrow polychromatic erythrocytes (PCE) of mice killed at the end of the experiment, and in peripheral blood normochromatic erythrocytes (NCE) collected at periodical intervals, as described previously (30). Briefly, slides of bronchoalveolar lavage cells were fixed with methanol and stained with a Giemsa 10% solution. A total of 2000–4000 PAM/mouse were scored for micronucleated (MN) and polynucleated (PN) cells. Bone marrow smears were air-dried and stained with May-Grünwald-Giemsa, and 5000 PCE per mouse were scored for the presence of MN erythrocytes. The PCE/NCE ratio, calculated by scoring 200 cells, was taken as an indicator of toxicity to bone marrow cells. Peripheral blood smears were air-dried and stained with May-Grünwald-Giemsa, and 30 000 NCE per mouse were scored for the presence of MN erythrocytes.

Statistical analyses
Depending on the end-point, the results were expressed as means ± SE of the results obtained in the eight mice belonging to each experimental group, thus reflecting the inter-individual variability, or of replicate analyses performed on samples pooled from eight mice within each experimental group, thus reflecting the variability among replicates. Comparisons between groups were made by Student's t-test for unpaired data and by means of ANOVA for repeated measurements.


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Survival and body weights
All 40 mice remained alive and apparently in good health during the 4 weeks of the experiment. The body weights (mean ± SE) at the start of the experiment were 21.7 ± 0.2 g in sham-exposed mice, 21.6 ± 0.3 g in light-exposed mice, 22.4 ± 0.5 g in ECS-exposed mice, 22.0 ± 0.3 g in mice exposed to both light and ECS, and 22.2 ± 0.3 g in mice additionally treated with oral sulindac. At the end of the experiment, the body weights were 25.8 ± 0.7, 25.5 ± 0.4, 25.9 ± 0.8, 24.3 ± 0.5 and 23.8 ± 0.3 g, respectively. Since day 14 onwards, the body weight gain was slightly but significantly lower (P < 0.05) in mice exposed to both ECS and light, irrespective of treatment with sulindac.

Multiple biomarkers in the skin
The results of the analyses evaluating intermediate biomarkers in the skin are summarized in Table I. TBA reactive aldehydes were significantly increased in mice exposed individually to either light (1.5x) or ECS (1.4x). Exposure to both agents resulted in an antagonistic effect, and administration of sulindac caused a further, significant loss of TBA reactive aldehydes.


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Table I. Alterations of biomarkers in the skin, respiratory tract and bone marrow of SKH-1 hairless mice, either unexposed (sham) or exposed for 28 days either to the light emitted by UV-C-covered halogen quartz bulbs (Light) or to ECS or to both agents, with or without oral administration of sulindac

 
The total levels of bulky DNA adducts were not affected in light-exposed mice, while they were significantly increased (2.6x) in ECS-exposed mice, in which we detected a diagonal radioactive zone (DRZ) and an individual spot upper end of the DRZ, similar to the patterns that had been detected previously in various tissues of ECS-exposed rodents (24). The simultaneous exposure to light significantly attenuated the formation of ECS-related DNA adducts, and their levels were further decreased by administration of sulindac.

8-OH-dG levels in the skin were significantly increased in both light-exposed mice (1.9x) and ECS-exposed mice (1.7x). These levels were not further increased by combining light and ECS (2.0x) or by additionally administering sulindac (2.1x).

Two main radioactive spots were detectable in the central part of the chromatographic area in UV-C-exposed calf thymus DNA used as a positive control but not in the corresponding unexposed control (not shown). Other authors identified these photoproducts as T = T dimers attached either to a purine (A-T = T) (low migrating spot) or pyrimidine nucleotide (T-T = T; C-T = T) (fast migrating spot) (28). The presence of similarly migrating spots was detected in DNA pooled from the skin of light-exposed mice. The levels of photoproducts in light-exposed animals were not appreciably modified by co-treatment with ECS and sulindac.

The mutated or over-expressed P53 oncoprotein, showing a typical ‘nuclear’ pattern, was mainly localized in the cells of the basal layer of epidermis. The LI for P53 was considerably enhanced (18x) in the skin of light-exposed mice. Exposure to ECS did not affect P53 levels, irrespective of treatment with sulindac.

PCNA was significantly enhanced (2.9x) in light-exposed mice. Exposure to ECS had no effect on this marker, whereas treatment with sulindac significantly attenuated the light-related proliferation stimulus.

The proportion of SBC, as assessed by morphological analysis of skin sections, was significantly enhanced (3.4x) by exposure to light. Exposure to ECS enhanced the proportion of SBC (1.6x) and, irrespective of treatment with sulindac, increased the effect of light (4.8x), but these differences were not statistically significant as compared with light alone. All effects appeared to be more pronounced when apoptosis was evaluated by TUNEL analysis. In fact, exposure to light enhanced 4.7 times the apoptotic LI, and exposure to ECS resulted in a borderline increase (1.8x). The two agents had a roughly additive effect (5.5x), which was not further modified by administration of sulindac.

Multiple biomarkers in the respiratory tract
As shown in Table I, ECS produced a significant increase of MN PAM (1.8x). Light was per se ineffective but, interestingly, potentiated the genotoxic effect of ECS to such an extent that the frequency of MN PAM in mice exposed to both ECS and light (3.4x) was significantly higher than that observed in mice exposed individually to either agent. Sulindac significantly attenuated the synergistic effect of ECS and light. In addition, ECS increased the frequency of PN PAM (1.4x). Sulindac attenuated the effect of the two combined agents.

The LI for PCNA in the bronchial epithelium was significantly enhanced in ECS-exposed mice (2.3x). Exposure to light did not modify the effect of ECS, whereas administration of sulindac produced a significant attenuation.

Evaluation of apoptosis in the bronchial epithelium by TUNEL analysis showed that exposure to ECS resulted in a considerable stimulation of apoptosis (11.9x), which was not further affected by exposure to light or administration of sulindac.

Exposure of mice to ECS resulted in a significant increase of TBA reactive aldehydes in lung homogenates (1.3x). The combined exposure to ECS and light strongly enhanced TBA reactive aldehydes concentrations, which were significantly higher as compared not only with sham-exposed mice but also with mice exposed individually to either agent. Administration of sulindac significantly inhibited this effect.

ECS produced a significant formation of bulky DNA adducts in the lung mixed cell population (8.0x), in the form of a DRZ and an individual spot, like in the skin (not shown). Even exposure to light produced a moderate but significant increase of lipophilic DNA adducts in the lung (1.6x), in the form of slowly migrating and poorly resolved radioactive spots (Figure 1). Moreover, co-exposure of mice to ECS and light resulted in synergistic effects, with DNA adduct levels (15.2x) which were significantly higher than those recorded not only in sham-exposed mice but also in either light-exposed or ECS-exposed mice. Both DRZ and the individual spot observed in mice treated with light plus ECS were significantly attenuated by sulindac.

8-OH-dG levels in the lung were significantly enhanced in ECS-exposed mice (1.8x). Exposure to light did not affect this end-point, while administration of sulindac totally prevented the oxidative DNA damage produced by ECS.

Multiple biomarkers in bone marrow
Bulky DNA adducts and oxidative DNA damage were only evaluated in sham-exposed mice and in light-exposed mice (Table I). Exposure to light resulted in a remarkable increase (3.0x) of bulky DNA adducts in bone marrow (Figure 1). Conversely, 8-OH-dG was not increased in light-exposed mice.

Both light (1.5x) and ECS (1.6x) produced a significant increase of MN PCE. Combination of these two agents had less than additive effects, and was not further modified by administration of sulindac. ECS produced a significant decrease of the PCE/NCE ratio.

Micronuclei in peripheral blood erythrocytes
The time-course of MN frequency in NCE was evaluated by periodically collecting peripheral blood during the first 4 weeks of exposure (Figure 2). Exposure to the light of halogen lamps progressively increased the frequency of MN NCE from day 11 onwards, with a statistically significant effect on days 11 and 15 (P < 0.05), and 20, 22 and 27 (P < 0.001). Irrespective of co-exposure to light, a significant early increase of MN frequency occurred in ECS-exposed mice. The increase was significant since the earliest collection time (day 2, P < 0.05) and grew with time (P < 0.001 thereafter). ECS and light had less than additive effect, and administration of sulindac did not further affect its genotoxic response.



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Fig. 2. Time-course variation in the frequency of MN normochromatic erythrocytes (NCE) in peripheral blood from SKH-1 hairless mice, either sham-exposed (Sham) or exposed to the light of halogen lamps (Light) and/or ECS, and treated with oral sulindac (SUL). The results are the means ± SE of the results obtained in eight mice (average of 30 000 NCE scored per mouse). See Statistical analyses.

 

    Discussion
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 Abstract
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 Materials and methods
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The results of the present study led to several new findings, which may bear relevance in the pathogenesis of cancer at various sites. Table II shows at a glance all significant effects produced, individually or in combination, by exposure of mice to light and ECS, and by administration of sulindac. Interestingly, some of the evaluated end-points, such as bulky DNA adducts and cytogenetic damage, exhibited parallel trends, while other end-points varied among the investigated targets.


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Table II. Summary of the effects of light, ECS, sulindac, and their combinations on biomarkers in organs, tissues, and cells of SKH-1 hairless mice

 
Summing up, the exposure of mice to the UV-containing light of the halogen lamps resulted in several alterations in skin cells, including increases in TBA reactive aldehydes, DNA photoproducts, 8-OH-dG, P53 protein, PCNA and apoptosis. In addition, the light produced an unexpected elevation of bulky DNA adducts in both lung and bone marrow, and an evident cytogenetic damage in both bone marrow and peripheral blood. Although the frequency of MN cells provides limited information regarding the type of chromosomal aberration, this end-point convincingly indicates the occurrence of a genotoxic damage. The observed molecular alterations in the skin of light-exposed mice are consistent with the notion that both solar irradiation (8) and artificial light sources (19,20,31) induce skin cancer. It was shown previously that repeated exposures of hairless mice to UV-B increase the epidermal levels of 8-OH-dG, which is a mutation-prone DNA base modified product generated by reactive oxygen species (ROS) or photodynamic action (32). In addition, exposure of hairless mice to UV-B resulted in significant increases of 8-OH-dG levels not only in skin but also in brain, liver and spleen (33). A significant formation of 8-OH-dG was confirmed, in the present study, in the skin of hairless mice exposed to UV-C-covered halogen quartz bulbs. Like solar radiation reaching the earth, this kind of lamps delivers both UV-A and UV-B wavelengths (15), which are known to have various effects on DNA, including the formation of cyclobutane pyrimidine dimers (28) and 8-OH-dG (31), which is able to induce G:C to T:A transversions (32). This mutation is detectable in the P53 gene of sunlight-induced skin cancers in humans (31), which is consistent with the observed accumulation of P53 oncoprotein in the light-exposed mice used in the present study.

The whole-body exposure of mice to ECS significantly increased TBA reactive aldehydes, bulky DNA adducts, and 8-OH-dG in both skin and lung mixed cell population, apoptosis in skin and bronchial epithelium, PCNA in bronchial epithelium and cytogenetic damage in PAM, bone marrow and peripheral blood erythrocytes. Although the respiratory tract is the main target of carcinogenicity for active tobacco smokers, CS behaves as a systemic carcinogen by affecting a variety of organs (2). Involuntary smoking due to inhalation of ECS is now classified by IARC as a Group 1 carcinogen, but the evidence for carcinogenicity to humans is consistent only for the lungs (34). However, this target specificity is presumably due to the relatively low doses of ECS to which passive smokers are exposed. It is apparent, as shown in the present study and in previous studies using animal models (24,35), that high-dose ECS has the potential capability to affect multiple organs due to the systemic distribution of genotoxic components.

Exposure of mice to daily cycles of light and ECS had, roughly, additive or less than additive effect on most investigated parameters. However, there were some significant antagonistic or synergistic interactions. In fact, presumably due to UV-induced transformations of ECS components deposited on mouse skin, the light antagonized the ECS-induced elevation of bulky DNA adduct levels, and there was an antagonism between light and ECS in the accumulation of TBA reactive aldehydes in skin cells. These findings correlate with the conclusion that repetitive UV irradiation can systematically prevent skin tumorigenesis in mice treated according to a two-stage protocol with 7,12–dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate (36). On the other hand, the light enhanced the ECS-induced accumulation of TBA reactive aldehydes and the elevation of DNA adducts in lung cells as well as the cytogenetic damage produced in PAM.

The promutagenic and mutagenic alterations observed in the lung, bone marrow and peripheral blood of light-exposed mice, and the synergism of light with ECS in the respiratory tract are of more difficult interpretation. In fact, all of the energy of UV radiation is absorbed in uppermost layers of the skin, and cannot directly penetrate to the underlying tissue (37). Most likely, UV-containing light induces the generation in the skin of ROS, reactive nitrogen species, lipid peroxidation products and cytokines such as the keratinocyte-derived cytokines interleukin-1 and tumor necrosis factor (38), and other products that may sufficiently be long-lived to travel via the blood circulation and induce genotoxic damage in distant tissues. We tested MDA and other TBA reactive aldehydes as candidate intermediaries of light-induced systemic genotoxicity. Like trans-4-hydroxy-2-nonenal, MDA is a naturally occurring product of lipid peroxidation and PG biosynthesis that forms promutagenic etheno-adducts (39) and is mutagenic and carcinogenic (40). Indeed, the observed variations of TBA reactive aldehydes concentrations in skin and lung were roughly in agreement with the alterations of molecular biomarkers induced by light, ECS and their combination. However, this parallelism was not perfect, which suggests the possible involvement of intermediate products other than or in addition to TBA reactive aldehydes. It is also more reasonable that multiple factors may account for the observed systemic effects of light.

Thus, it appears that the light, beyond its dominant role in the etiology of skin cancers, may additionally play an insofar underestimated role in cancers at other sites. Although the epidemiological data are controversial, there is some suspicion that the solar radiation may be involved in the genesis of human hematolymphopoietic malignancies, and especially of non-Hodgkin's lymphoma (reviewed in ref. 37). It has been shown that, also in healthy humans, solar radiation caused a significant enhancement of frequency of both hprt mutation in T-lymphocytes and bcl-2 t(14;18) translocations in B- lymphocytes (41,42), which could be mediated by the UV-related induction in the skin of cytokines, such as interleukins-6 (IL-6), stimulating the proliferation of different subsets of B or T cells (42). In addition, both DNA strand breaks in human mononuclear cells (43) and mRNA levels DNA repair excision mechanisms in whole human blood (44) were found to be correlated with solar radiation. Recently, it was demonstrated that the UV-induced enhancement of incidence of T cell lymphoid tumors in the spleen or liver of P53 heterozygous C57BL/6 mice was not due to the potential immunosuppressive activity of UV radiation (37). The results of the present study provide clear evidence that, without disregarding the recognized role of immunosuppression (7), the light induces a systemic genotoxic damage that could mechanistically be related to cancers at sites other than the skin.

Sulindac exerted a variety of protective effects towards the molecular and cytogenetical alterations observed in mice exposed to both light and ECS. In fact, not only sulindac inhibited accumulation of TBA reactive aldehydes, formation of bulky DNA adducts, and proliferation in skin cells, but this NSAID also attenuated the cytogenetic damage in PAM and proliferation of bronchial epithelial cells as well as levels of TBA reactive aldehydes, bulky DNA adducts and oxidative DNA damage in lung. Although sulindac may induce apoptosis (45), in the present study the drug did not affect the apoptosis induced either by light in skin cells or by ECS in bronchial epithelial cells. Sulindac is ingested as the inactive prodrug sulfoxide that is either reduced to sulfide or oxidized to sulfone in the liver (45). Sulindac sulfide is an inhibitor of cyclooxygenases (COX-1 and COX-2), whereas the sulfone works via COX-independent mechanisms (46). UV exposure of the skin induces COX-2 expression in keratinocytes (47) and stimulates PG production by increasing the synthesis and activating the cytosolic phospholipase A2.

The multiplicity of the biological effects observed in variously treated mice is supported by the findings of a parallel study evaluating, by cDNA array, the expression of 746 genes in the skin and lungs of the same mice (A.Izzotti et al., manuscript in preparation).

In conclusion, the results of the present study provide evidence that exposure of hairless mice to UV-A- and UV-B-containing light produces a variety of alterations not only in the skin but also in lungs and bone marrow. This mechanism may play a role in the pathogenesis of cancers at sites other than the skin. Moreover, it appears that the light enhances the molecular and cytogenetical alterations induced by ECS in the respiratory tract, which may be involved in the pathogenesis of lung cancer and perhaps of other chronic pulmonary diseases.


    Acknowledgments
 
This study was supported by the National Cancer Institute (Master Agreement N01-CN-75008), the Associazione Italiana per la Ricerca sul Cancro (AIRC), and the Bulgarian Ministry of Science and Education.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 29, 2003; revised June 16, 2003; accepted June 17, 2003.