Modulation of light-induced skin tumors by N-acetylcysteine and/or ascorbic acid in hairless mice
Francesco D'Agostini,
Roumen M. Balansky1,
Anna Camoirano and
Silvio De Flora2
Department of Health Sciences, University of Genoa, Via A.Pastore 1, I-16132 Genoa, Italy
2 To whom correspondence should be addressed Email: sdf{at}unige.it
 |
Abstract
|
---|
The light emitted by halogen quartz bulbs contains a broad spectrum of UV wavelengths, is strongly genotoxic and is a potent inducer of skin tumors in hairless mice. By using a UVC filter, this light mimics solar radiation and induces a variety of genomic and transcriptional alterations in mouse skin. UV-related carcinogenesis involves depletion of antioxidants and glutathione in skin cells. On this basis, we evaluated modulation of carcinogenicity of UVC-filtered halogen lamps in SKH-1 hairless mice by the antioxidants N-acetyl-L-cysteine (NAC) and ascorbic acid (AsA). Both agents were given in the drinking water, either individually or in combination. The earliest skin lesions were detected after 300 days' exposure to light and became confluent in a number of mice after 480 days. NAC administration prolonged the latency time by 90 days. Moreover, NAC considerably and significantly decreased both incidence and multiplicity of light-induced skin tumors, prevented the occurrence of malignant lesions (squamocellular carcinomas) and reduced the tumor size. In contrast, AsA, which may behave as a prooxidant rather than an antioxidant, increased the multiplicity of total skin tumors, carcinomas in situ and squamocellular carcinomas. Co-administration of NAC with AsA significantly attenuated the negative effect of AsA, presumably due to the ability of this thiol to maintain a reduced environment. Therefore, in agreement with our previous in vitro findings, oral NAC is able to attenuate the detrimental effects of AsA.
Abbreviations: AP-1, activator protein-1; AsA, ascorbic acid; COX-2, cyclooxygenase 2; GSH, reduced glutathione; JNK, Jun N-terminal kinase; NAC, N-acetyl-L-cysteine; ROS, reactive oxygen species
 |
Introduction
|
---|
Solar radiation represents the most ubiquitous mutagen and carcinogen in nature and has been estimated to account for 10% of human cancers (1). The carcinogenicity of sunlight is due to several mechanisms, including genetic predisposition factors, e.g. related to the phototype and DNA repair patterns, formation of pyrimidine dimers and a variety of other DNA photoproducts, generation of reactive oxygen species (ROS) and UVB-related immunosuppression (2,3).
We found that the light emitted by halogen quartz bulbs, an artificial illumination system to which millions of people are exposed, is strongly genotoxic to both bacteria (4,5) and human cultured cells (6) and is a potent inducer of skin tumors in various strains of hairless mice (7,8). DNA damage and carcinogenicity are due to the fact that these lamps emit a broad spectrum of UV radiation, starting from the UVC region (49). Neither genotoxic nor carcinogenic effects were produced when halogen quartz bulbs were covered with a common glass cover (48). This discovery prompted the manufacturers to replace the traditional halogen lamps with UV-block or UV-stop lamps, in which the quartz bulb is doped in order to render it less permeable to UV wavelengths. In fact, the light emitted by these lamps failed to produce either mutations in bacteria (10) or genotoxic effects in human cultured cells (11).
Besides their possible impact on human health, halogen lamps provide a suitable model for simulating solar radiation. In fact, at variance with other experimental models using pulse radiation, it is sufficient to expose the animals, typically hairless mice, every day for a predetermined number of hours fixed with a timer, until the skin tumors become evident. Moreover, by using a UVC filter, the radiation spectrum of halogen lamps mimics the spectrum of solar radiation. Under these conditions UVA- and UVB-related alterations could be detected in skin cells of hairless mice, including biochemical and genomic alterations (12), changes in multigene expression (13) and skin tumors (our unpublished data).
Based on these premises, we designed a study aimed at evaluating the efficacy of two chemopreventive agents having antioxidant properties in modulating the yield of light-induced skin tumors. We used, either individually or in combination, the thiol N-acetylcysteine (NAC) and ascorbic acid (AsA, vitamin C). NAC works extracellularly of itself and intracellularly as a precursor of L-cysteine and reduced glutathione (GSH) through a variety of mechanisms, among which is scavenging of ROS (14,15). AsA is a major water soluble antioxidant present in cells and plasma and is a strong reducing agent (16). These properties are particularly relevant to UV-related skin carcinogenesis, due to the contribution of ROS to this process and to the circumstances that cutaneous antioxidants are depleted in UV-exposed cells (17) and sensitivity to UV is inversely related to the GSH content of cells (18). In addition, combination of NAC with AsA has been proposed due to the ability of NAC to counteract possible adverse effects of AsA (19).
We show here that the two chemopreventive agents tested display opposite modulations of UV carcinogenesis in mouse skin. In fact, AsA further enhanced the multiplicity of light-related tumors, while NAC efficiently decreased their incidence, multiplicity and size and prevented the occurrence of squamocellular carcinomas. In addition, when given in combination, NAC attenuated the negative effects of AsA.
 |
Materials and methods
|
---|
Mice
Female SKH-1 hairless mice (150), 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, San Polo d'Enza, Italy) and tap water ad libitum. The housing conditions were as follows: temperature 23 ± 2°C, relative humidity 55%, 12 h day/night cycle. The housing and treatment of mice were in accordance with our national and institutional guidelines.
Exposure to light
One hundred and twenty mice were exposed to the light emitted by halogen quartz bulbs (12 V, 50 W), incorporated into dichroic spotlights, which were supplied by Leuci (File S.p.A., Lecco, Italy). This illumination system emits a broad spectrum of visible light wavelengths and UVA, UVB and UVC radiation. Typical spectra and irradiance values are available in McKinlay et al. (9). The bulbs, which were changed every 6 months, were covered with filters cutting out UVC radiation (WG 280; Schott Optics Division, Mainz, Germany). The distance from the back of the mice was regulated to yield an illuminance level of 10 000 lux. Exposure was for 3 h/day, every day throughout the duration of the experiment.
Design of the study and treatment with chemopreventive agents
After acclimatization for 2 weeks, the mice were divided into seven groups. Three groups, each of 10 animals, included mice that were not exposed to light and were either untreated or treated with either NAC or AsA. The remaining four groups, each of 30 animals, included light-exposed mice that were either untreated or treated with either NAC or AsA or both agents. AsA was purchased from Sigma Chemical Co. (St Louis, MO) and NAC from Zambon (Vicenza, Italy), in the form of a commercially available, lyophilized drug preparation (Fluimucil). Both agents, either individually or in combination, were dissolved in the drinking water to yield a calculated daily intake of 1 g/kg body wt. The solutions were prepared every 2 days. Treatment with NAC and/or AsA started 3 days before the first day of exposure to light and continued throughout the duration of the experiment.
Survival, body weight gain, and skin tumor yield
The mice were inspected daily for survival and general appearance. At monthly intervals they were individually weighed and scored for the presence, morphology and size of skin lesions. The experiment was stopped after 480 days, when the lesions became confluent in a proportion of mice (see Results). Representative skin lesions of various morphological appearance were excised, fixed in neutral buffered formalin and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin. The histopathological analysis of tumors followed the classification proposed by Gallagher et al. (20).
Statistical analysis
Data relative to survival and incidence of tumors were expressed in terms of frequency and differences between groups were evaluated by
2 analysis. Data relative to body weight and multiplicity were expressed in terms of means ± SE within the mice belonging to each experimental group. When the skin lesions became confluent an arbitrary value of 15 lesions/mouse was given. The overall differences between experimental groups regarding time-related increases in body weight and multiplicity of skin lesions were evaluated by ANOVA followed by the Bonferroni/Dunn post hoc test for evaluating differences at individual times. The differences between experimental groups regarding the size of lesions were evaluated by Student's t-test.
 |
Results
|
---|
Survival and body weights
Of the 150 mice under study, 138 (92.0%) were still alive 480 days after the start of exposure, when the experiment was terminated. The survival rate ranged between a minimum of 83.3% in light-exposed mice and a maximum of 96.7% in light-exposed mice treated with both NAC and AsA, without any significant difference related to treatment. The body weights (mean ± SE) at the start of the experiment were 25.2 ± 0.11 g in control mice, 24.9 ± 0.18 g in light-exposed mice, 25.6 ± 0.25 g in light-exposed mice treated with NAC, 25.3 ± 0.22 g in light-exposed mice treated with AsA and 25.5 ± 0.22 g in light-exposed mice treated with both NAC and AsA. At the end of the experiment the body weights were 36.4 ± 0.15, 36.9 ± 0.15, 36.8 ± 0.13, 37.1 ± 0.19 and 37.0 ± 0.30 g, respectively. Thus, there was no significant difference among the experimental groups in body weight gain.
Yield of skin tumors
Table I summarizes the data relative to the yield of skin tumors, in terms of incidence and multiplicity, as related to treatment of mice and to the time elapsed since the start of exposure to light. No skin lesions were observed throughout duration of the experiment in the 30 control mice that were not exposed to light, 10 of which were untreated, 10 were treated with NAC and 10 were treated with AsA. Therefore, these mice were grouped together and served as negative controls.
View this table:
[in this window]
[in a new window]
|
Table I. Yield of skin tumors in variously treated SKH-1 hairless mice at various times after the start of exposure to UV-containing light
|
|
The earliest light-related skin lesions were detectable, at a monthly score, after 300 days' exposure. In particular, lesions were detected after 300 days in light-exposed mice, irrespective of treatment with AsA, after 330 days in light-exposed mice treated with both NAC and AsA and after 390 days in light-exposed mice treated with NAC. Thus, the oral administration of NAC delayed the appearance of light-induced skin tumors by 3 months. The incidence and multiplicity of tumors grew with time, until the lesions became confluent in a number of mice. When the experiment was stopped, after 480 days, the lesions were confluent in 7 of 28 surviving light-exposed mice, 22 of 28 light-exposed mice treated with AsA and 10 out of 29 light-exposed mice treated with both NAC and AsA. The lesions did not become confluent in any of the light-exposed mice treated with NAC.
As shown in Table I and Figure 1, administration of NAC considerably and significantly decreased both incidence and multiplicity of light-induced skin tumors. In contrast, administration of AsA increased the multiplicity of light-related tumors, with significant overall differences between the two time coursemultiplicity curves (P < 0.001) and at individual times after 480 days (P < 0.001). This negative effect was attenuated by the combined administration of AsA and NAC. In fact, there were significant differences between light + AsA and light + NAC + AsA regarding the overall time coursemultiplicity curves (P < 0.001) and at individual times after 390 days (P < 0.05) and 480 days (P < 0.001). However, the combined treatment with NAC and AsA was not as effective as NAC alone in decreasing the multiplicity of light-induced tumors.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 1. Multiplicities (means ± SE) of skin tumors in variously treated mice, as related to treatment and to the time elapsed since the start of exposure to light.
|
|
Histopathology and size of skin tumors
The light-induced skin lesions had various morphological appearances, which were macroscopically categorized into four main types (Figure 2). Intermediate lesions were also detectable. Some of the type A lesions spontaneously regressed, but in general there was a trend to a time-related evolution into lesions of larger size and higher malignancy. At the end of the experiment 10 lesions per morphological type were subjected to histopathological analysis. As shown in Figure 2, type A lesions had the appearance of small pink warts and were microscopically classified as epidermal hyperplasias. Type B lesions were of a larger size, tended to be reddish, were more or less protruding with a central excrescence and were microscopically classified as papillomas. Type C lesions had the appearance of outgrowths, with a knobbly surface, often hyperemic and with a keratinized surface. Most of them were microscopically classified as carcinomas in situ, but one keratoacanthoma-like tumor and an appendage/basal tumor were also detected. Type D lesions were large, irregularly shaped tumors and were often ulcerated. They were microscopically classified as squamocellular carcinomas.

View larger version (120K):
[in this window]
[in a new window]
|
Fig. 2. Macroscopic and histopathological appearance of the main skin lesions induced by light in hairless mice. (A) Epidermal hyperplasia; (B) papilloma; (C) carcinoma in situ; (D) squamocellular carcinoma.
|
|
The morphology and histopathology of light-induced skin lesions, when detectable, were not affected by treatment with AsA and/or NAC, but treatment with the chemopreventive agents affected the frequency of the different morphological and histopathological types as well as their size. Table II shows the yield of skin lesions of various types and their size 420 days after the start of exposure of mice to light, when the lesions were not yet confluent. All four types of skin lesions were detectable in light-exposed mice, including the occurrence of squamocellular carcinomas in 21.4% of mice. Treatment with NAC significantly decreased the incidence and multiplicity of all types of light-induced lesions, and no squamocellular carcinoma was observed. Treatment with AsA significantly increased the multiplicity of light-induced carcinomas in situ and squamocellular carcinomas. As compared with AsA alone, treatment with both NAC and AsA significantly increased the incidence and multiplicity of light-induced epidermal hyperplasia but significantly decreased the multiplicity of carcinomas in situ and the yield of squamocellular carcinomas. Indeed, as in light-exposed mice treated with NAC, the combined treatment with NAC and AsA abolished the occurrence of squamocellular carcinomas.
View this table:
[in this window]
[in a new window]
|
Table II. Yield of skin tumors of different morphology and histopathological type and size of lesions in variously treated SKH-1 hairless mice, 420 days after the start of exposure to UV-containing light
|
|
In parallel, both NAC and its combination with AsA significantly reduced the size of light-induced skin lesions, which was not affected by AsA alone (Table II).
 |
Discussion
|
---|
The first result emerging from the present study is that exposure of hairless mice to the light of UVC-filtered halogen lamps provides a suitable experimental model for assessing the cutaneous carcinogenicity of UVA- and UVB-containing wavelengths mimicking sunlight exposure. A short daily exposure (3 h) was chosen in order to allow a slower growth of skin lesions and to better evaluate their modulation by chemopreventive agents. Under these conditions the earliest tumors appeared on the exposed mouse backs after 10 months and progressively grew until they becam confluent in a proportion of mice after 16 months. The nature of light-induced skin lesions ranged from preneoplastic lesions, such as epidermal hyperplasia, to benign tumors, such as papillomas, evolving towards keratoacanthoma-like tumors, appendage/basal tumors, carcinomas in situ and squamocellular carcinomas.
This long-term effect parallels earlier alterations of intermediate biomarkers, detected after 4 weeks of exposure under identical exposure conditions, excepting a longer daily exposure (9 h), in particular light-induced increases in lipid peroxidation products, DNA photoproducts, 8-hydroxy-2'-deoxyguanosine, P53 protein, proliferating cell nuclear antigen, cytogenetic damage and apoptosis (12). Moreover, 55 of the 746 cancer-related genes (7.4%) analyzed were overexpressed in the skin of light-exposed mice, including oncogenes, genes encoding for metalloproteinases and cytoskeleton proteins and genes involved in xenobiotic metabolism, oxidative stress, the stress response, protein repair, DNA repair, apoptosis, cell cycle regulation, signal transduction, inflammation and immune response (13).
The oral administration of NAC was quite efficient in inhibiting the formation of light-induced skin lesions and in totally preventing the occurrence of squamocellular carcinomas. The latency time of the earliest tumors was prolonged by 3 months and both incidence and multiplicity of lesions as well as their size were remarkably reduced. The dose tested (1 g/kg body wt) has previously been shown to exert a variety of protective effects in animal models (reviewed in 14,15). Although rather high, this dose is not toxic, as shown not only by the unchanged survival and body weight gains of the mice used in the present study but also by the lack of alterations at the molecular level. In fact, the same oral dose of NAC had a negligible influence on the basal profiles of gene expression in organs of rodents, including the lung of A/J mice (15), the liver of transplacentally exposed Swiss mouse fetuses (21) and both liver and lung of SpragueDawley rats (A.Izzotti et al., unpublished data). These experimental findings are consistent with the safety of this molecule, as assessed in 40 years of extensive clinical use (14,15).
NAC has previously been shown to inhibit the formation of chemically induced skin tumors. In fact, administration of NAC in the diet reduced the multiplicity of skin tumors resulting from the topical administration of benzo[a]pyrene in cancer-prone p53 haploinsufficient Tg.AC (v-Ha-ras) transgenic mice (22). However, in this model NAC did not inhibit the progression towards malignancy (22), while in the present study no squamocellular carcinomas developed in light-exposed mice treated with NAC. The ability of NAC to inhibit light-induced skin tumors is likely to be in part due to NAC itself and in part to the generation of L-cysteine and GSH in skin cells. In fact, GSH plays a central role in the endogenous defense against UV radiation (23) and the formation of UVB-induced skin tumors in hairless mice has been shown to be inhibited by administering GSH isopropyl ester prior to each UV irradiation (24).
A major mechanism of NAC in UV-mediated skin carcinogenesis may be related to its ability to scavenge ROS, especially hydroxyl radicals generated in Fenton-type reactions (25,26). Studies in cultured skin cells showed that NAC inhibits the UVB-induced expression of cyclooxygenase-2 (COX-2) (27), which is up-regulated in the skin of light-exposed hairless mice (13). Moreover, NAC inhibited the activation by UV radiation of factors involved in signal transduction, such as growth arrest and DNA damage-inducible protein 45 (28), Jun N-terminal kinase (JNK), nuclear factor
B and activator protein-1 (AP-1) (17). Note that activation of AP-1 and overexpression of COX-2 are involved in UVB-induced promotion of skin tumors (29). Although in a number of studies available in the literature NAC behaved as an inhibitor of apoptosis, due to its ability to attenuate the DNA damage triggering this process (14,15), in human C8161 melanoma cells NAC enhanced UV radiation-mediated apoptosis (30). A further protective mechanism of NAC in UVB-related skin carcinogenesis is the immunomodulating properties of this thiol. In fact, independent of GSH synthesis, NAC inhibited UVB-induced immunosuppression in mice, consisting of suppression of the T cell-mediated immune response, both local and systemic (31). However, NAC had no effect on UV-B-induced DNA damage, suggesting that NAC exerts its photoprotective effect during the post-initiation phase of photoimmunosuppression (32).
In contrast to the beneficial effect of NAC, in the present study the oral administration of AsA, at the same dose, resulted in a further increase in light-induced skin tumor multiplicity. Previous studies had shown that the local application of AsA protects animal skin against the harmful effects of UV radiation (33,34). Moreover, in cultured skin cells AsA mediated cell damage and cell death by interfering at multiple levels with the activity of the JNK/AP-1 pathway and by modulating the expression of AP-1-related genes (35). However, our negative finding was not totally unexpected, since under certain conditions AsA has already been shown to work as a prooxidant rather than as an antioxidant. Several studies provided evidence that AsA may enhance both spontaneous and chemically induced mutations in prokaryotes and eukaryotes and increase the yield of chemically induced tumors in rodents (reviewed in 36,37). From a mechanistic point of view, AsA has been proposed to act as a reducing agent for chelate-complexed Fe3+, thereby favoring production of hydroxyl radicals from hydrogen peroxide through a Fenton-type reaction (38). Interestingly, UVA radiation mobilizes iron from intracellular stores and labile iron acts as a catalyst to exacerbate the generation of secondary lipid messengers in skin cell membranes (39). It has also been shown that AsA supplementation generates reactive aldehydes in vivo (40). In cultured keratinocytes AsA 6-palmitate, a synthetic lipophilic compound of AsA located at the cellular membrane, was effective in scavenging intracellular ROS but strongly promoted UVB-induced lipid peroxidation, JNK activation and cytotoxicity (41). Other studies showed that UV-induced AsA oxidation in murine skin homogenates is caused by photoactivated reactions rather than by ROS reactions (42). Even in humans, the dietary intake of AsA was inversely associated with the UV-induced minimal erythema dose among 335 volunteers (43).
As to modulation by AsA of UV-induced skin tumors in hairless mice, the results available in the literature are rather controversial. When given topically on the dorsal surface 2 h prior to each UVB irradiation, AsA (0.1 ml of a 5% solution) slightly but significantly delayed tumor onset and decreased the tumor yield. In parallel, topical AsA inhibited UVB-induced skin wrinkling. However, no inhibition of UV-induced skin wrinkling was afforded by oral AsA and topical AsA failed to protect against UVA-induced skin sagging (33). Linus Pauling's papers (44,45) reported that supplementation of the diet with AsA resulted in a pronounced decrease in the incidence and delayed the onset of malignant lesions. Actually, no multiplicity data were given and an analysis of the results reported in these studies shows that an oral dose corresponding to 0.55 g/kg body wt/day almost doubled the incidence of all lesions >2 mm, while the incidence was decreased at the very high doses of 8.3 and 16.7 g/kg body wt/day. A similar trend was observed by Robinson et al. (46), who found that very high doses of AsA in the diet, corresponding to 8, 16 and 32 g/kg body wt/day, decreased the multiplicity of UV-induced skin tumors, while relatively lower doses (0.5, 1 and 2 g/kg body wt) increased it, with almost a doubling of tumors at the lowest dose. Therefore, both the previous studies (4446) and the present study agree in the conclusion that at a dose of 0.51.0 g/kg body wt/day, which would be quite high in humans, AsA exacerbates UV-related skin carcinogenicity. Only at exceedingly high doses (832 g/kg body wt/day) was there a protective effect (4446).
The combined administration of NAC and AsA attenuated the adverse effect of the latter agent. In fact, compared with treatment with AsA alone, co-administration of NAC delayed the onset of tumors by 30 days, significantly attenuated skin tumor multiplicity, abolished the occurrence of squamocellular carcinomas and reduced the size of lesions. Previously we showed that NAC antagonizes the enhancing effect of AsA on spontaneous mutagenicity in the Salmonella typhimurium his strains TA102 and TA104, which are sensitive to oxidative mechanisms (19). Recycling of AsA from its oxidized form is required to maintain intracellular stores of this vitamin (47) and GSH has been shown, both in vitro and in vivo, to maintain a reducing milieu in the cell which can reduce dehydroascorbic acid (4751). It is likely that NAC exerts similar functions both of itself and as a precursor of intracellular L-cysteine and GSH.
There is evidence that NAC protects towards the adverse effects not only of AsA (19, this study), but also of other chemopreventive agents, such as green tea polyphenols (52) and isothiocyanates (53), and attenuates the side-effects of cytotoxic drugs, such as cyclophosphamide-induced urotoxicity (54) and doxorubicin-induced mutagenicity (55), clastogenicity (56), cardiotoxicity (57) and alopecia (56). NAC and AsA have already been tested as a topical co-application on pig skin and shown to protect against UVA-induced decomposition of the non-steroidal anti-inflammatory drug suprofen by scavenging radicals and ROS (58). The results obtained in the present study provide evidence that NAC can also attenuate the detrimental effects of AsA when given by the oral route.
 |
Notes
|
---|
1 Permanent address: National Centre of Oncology, Str. Plovdivsko pole 6, Sofia 1756, Bulgaria 
 |
Acknowledgments
|
---|
This study was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) and by the Bulgarian Ministry of Education and Science.
 |
References
|
---|
- Higginson,J. and Muir,C.S. (1979) Environmental carcinogenesis: misconceptions and limitations to cancer control. J. Natl Cancer Inst., 63, 12911298.[ISI][Medline]
- IARC (1992) IARC Monographs on the Evaluation of the Carcinogenic Risk to Humans, Vol 55, Solar and Ultraviolet Radiation. IARC, Lyon, France.
- Kripke,M.L. (1990) Effects of UV radiation on tumor immunity. J. Natl Cancer Inst., 82, 13921396.[ISI][Medline]
- De Flora,S., Camoirano,A., Izzotti,A. and Bennicelli,C. (1990) Potent genotoxicity of halogen lamps, compared to fluorescent light and sunlight. Carcinogenesis, 11, 21712177.[Abstract]
- De Flora,S., Camoirano,A., Izzotti,A. and Bennicelli,C. (1991) A bacterial DNA-repair test evaluating the genotoxicity of light sources. Toxicol. Methods, 1, 116122.
- D'Agostini,F., Izzotti,A. and De Flora,S. (1993) Induction of micronuclei in cultured human lymphocytes exposed to quartz halogen lamps and its prevention by glass covers. Mutagenesis, 8, 8790.[Abstract]
- De Flora,S. and D'Agostini,F. (1992) Halogen lamp carcinogenicity. Nature, 356, 569.[Medline]
- D'Agostini,F. and De Flora,S. (1994) Potent carcinogenicity of uncovered halogen lamps. Cancer Res., 54, 50815085.[Abstract]
- McKinlay,A.F., Whillock,M.J. and Meulemans,C.C. (1989) Ultraviolet radiation and blue-light emission from spotlights incorporating tungsten halogen lamps. In NRPB-R228. National Radiological Protection Board, Chilton, UK, pp. 113.
- Camoirano,A., Bennicelli,C., Bagnasco,M. and De Flora,S. (1999) Genotoxic effects in bacteria of the light emitted by halogen tungsten lamps having treated quartz bulbs. Mutat. Res., 441, 2127.[ISI][Medline]
- D'Agostini,F., Caimo,A., De Filippi,S. and De Flora,S. (1999) Induction and prevention of micronuclei and chromosomal aberrations in cultured human lymphocytes exposed to the light of halogen lamps. Mutagenesis, 14, 433436.[Abstract/Free Full Text]
- Balansky,R.M., Izzotti,A., D'Agostini,F., Camoirano,A., Bagnasco,M., Lubet,R.A. and De Flora,S. (2003) Systemic genotoxic effects produced by light and synergism with cigarette smoke in the respiratory tract of hairless mice. Carcinogenesis, 24, 15251532.[Abstract/Free Full Text]
- Izzotti,A., Cartiglia,C., Longobardi,M., Balansky,R.M., D'Agostini,F., Lubet,R.A. and De Flora,S. (2004) Alterations of gene expression in skin and lung of mice exposed to light and cigarette smoke. FASEB J., 18, 15591561.[Abstract/Free Full Text]
- De Flora,S., Izzotti,A., D'Agostini,F. and Balansky,R.M. (2001) Mechanisms of N-acetylcysteine in the prevention of DNA damage and cancer, with special reference to smoking-related end-points. Carcinogenesis, 22, 9991013.[Abstract/Free Full Text]
- De Flora,S., Izzotti,A., Albini,A., D'Agostini,F., Bagnasco,M. and Balansky,R.M. (2004) Antigenotoxic and cancer preventive mechanisms of N-acetyl-L-cysteine. In Kelloff,G.J., Hawk,E.T. and Sigman,C.C. (eds) Cancer Chemoprevention, Vol. 1, Promising Cancer Chemopreventive Agents. Humana Press, Totowa, NJ, pp. 3767.
- Frei,B., England,L. and Ames,B.N. (1989) Ascorbate is an oustanding antioxidant in human blood plasma. Proc. Natl Acad. Sci. USA, 86, 63776381.[Abstract]
- Saliou,C., Kitazawa,M., McLaughlin,L. et al. (1999) Antioxidants modulate acute solar ultraviolet radiation-induced NF-kappa-B activation in a human keratinocyte cell line. Free Radic. Biol. Med., 26, 174183.[CrossRef][ISI][Medline]
- Tyrrell,R.M. and Pidoux,M. (1988) Correlation between endogenous glutathione content and sensitivity of cultured human skin cells to radiation at defined wavelengths in the solar ultraviolet range. Photochem. Photobiol., 47, 405412.[ISI][Medline]
- D'Agostini,F., Balansky,R.M., Camoirano,A. and De Flora,S. (2000) Interactions between N-acetylcysteine and ascorbic acid in modulating mutagenesis and carcinogenesis. Int. J. Cancer, 88, 702707.[CrossRef][ISI][Medline]
- Gallagher,C.H., Path,F.R.C., Canfield,P.J., Greenoak,G.E. and Reeve,V.E. (1984) Characterization and histogenesis of tumors in the hairless mouse produced by low-dosage incremental ultraviolet radiation. J. Invest. Dermatol., 83, 169174.[ISI][Medline]
- Izzotti,A., Balansky,R.M., Cartiglia,C., Camoirano,A., Longobardi,M. and De Flora,S. (2003) Genomic and transcriptional alterations in mouse fetus liver after transplacental exposure to cigarette smoke. FASEB J., 17, 11271129.[Abstract/Free Full Text]
- Martin,K.R., Trempus,C., Saulnier,M., Kari,F.W., Barrett,J.C. and French,J.E. (2001) Dietary N-acetyl-L-cysteine modulates benzo[a]pyrene-induced skin tumors in cancer-prone p53 haploinsufficient Tg.AC (v-Ha-ras) mice. Carcinogenesis, 22, 13731378.[Abstract/Free Full Text]
- Steenvoorden,D.P., Hasselbaink,D.M. and Beijersbergen van Henegouwen,G.M. (1998) Protection against UV-induced reactive intermediates in human cells and mouse skin by glutathione precursors: a comparison of N-acetylcysteine and glutathione ethylester. Photochem. Photobiol., 67, 651656.[ISI][Medline]
- Kobayashi,S., Takehana,M. and Tohyama,C. (1996) Glutathione isopropyl ester reduces UVB-induced skin damage in hairless mice. Photochem. Photobiol., 63, 106110.[ISI][Medline]
- Aruoma,O.I., Halliwell,B., Hoey,B.M. and Butler,J. (1989) The antioxidant action of N-acetylcysteine. Free Radic. Biol. Med., 6, 593597.[CrossRef][ISI][Medline]
- Izzotti,A., Orlando,M., Gasparini,L., Scatolini,L., Cartiglia,C., Tulimiero,L. and De Flora,S. (1998) In vitro inhibition by N-acetylcysteine of oxidative DNA modifications detected by 32P postlabeling. Free Radic. Res., 28, 165178.[ISI][Medline]
- Ashida,M., Bito,T., Budiyanto,A., Ichihashi,M. and Ueda,M. (2003) Involvement of EGF receptor activation in the induction of cyclooxygenase-2 in HaCaT keratinocytes after UVB. Exp. Dermatol., 12, 445452.[CrossRef][ISI][Medline]
- Wan,Y., Wang,Z., Shao,Y., Xu,Y., Voorhees,J. and Fisher,G. (2000) UV-induced expression of GADD45 is mediated by an oxidant sensitive pathway in cultured human keratinocytes and human skin in vivo. Int. J. Mol. Med., 6, 683688.[ISI][Medline]
- Bowden,G.T. (2004) Prevention of non-melanoma skin cancer by targeting ultraviolet-B-light signalling. Nature Rev. Cancer, 4, 2335.[CrossRef][ISI][Medline]
- Rieber,M. and Rieber,M.S. (2003) N-acetylcysteine enhances UV-mediated caspase-3 activation, fragmentation of E2F-4 and apoptosis in human C8161 melanoma: inhibition by ectopic Bcl-2 expression. Biochem. Pharmacol., 65, 15931601.[CrossRef][ISI][Medline]
- Steenvoorden,D.P. and Beijersbergen van Henegouwen,G.M. (1998) Glutathione synthesis is not involved in protection by N-acetylcysteine against UVB-induced immunosuppression in mice. Photochem. Photobiol., 68, 97100.[CrossRef][ISI][Medline]
- Nijmeijer,B.A., Steenvoorden,D.P.T., Beijersbergen van Henegouwen,G.M.J., Roza,L. and Vink,A.A. (1998) The mechanism of N-acetylcysteine photoprotection is not related to dipyrimidine photoproducts. J. Photochem. Photobiol. B Biol., 44, 225230.[CrossRef][ISI][Medline]
- Bissett,D.L., Chatterjee,R. and Hannon,D.P. (1990) Photoprotective effect of superoxide-scavenging antioxidants against ultraviolet radiation-induced chronic skin damage in the hairless mouse. Photodermatol. Photoimmunol. Photomed., 7, 5662.[ISI][Medline]
- Darr,D., Combs,S., Dunston,S., Manning,T. and Pinnell,S. (1992) Topical vitamin C protects porcine skin from ultraviolet radiation-induced damage. Br. J. Dermatol., 127, 247253.[ISI][Medline]
- Catani,M.V., Rossi,A., Costanzo,A., Sabatini,S., Levrero,M., Melino,G. and Avigliano,L. (2001) Induction of gene expression via activator protein-1 in the ascorbate protection against UV-induced damage. Biochem. J., 15, 7785.
- Shamberger,R.J. (1984) Genetic toxicology of ascorbic acid. Mutat. Res., 133, 135159.[CrossRef][ISI][Medline]
- Lee,K.W., Lee,H.J., Suhr,Y.-J. and Lee,C.Y. (2003) Vitamin C and cancer chemoprevention: reappraisal. Am. J. Clin. Nutr., 78, 10741078.[Abstract/Free Full Text]
- Benatti,U., Morelli,A., Guida,L. and De Flora,A. (1983) The production of activated oxygen species by an interaction of methemoglobin with ascorbate. Biochem. Biophys. Res. Commun., 11, 980987.
- Reelfs,O., Tyrrell,R.M. and Pourzand,C. (2004) Ultraviolet A radiation-induced immediate iron release is a key modulator of the activation of NF-kappa B in human skin fibroblasts. J. Invest. Dermatol., 122, 14401447.[CrossRef][ISI][Medline]
- Cooke,M.S., Mistry,N., Ahmad,J. et al. (2003) Deoxycytidine glyoxal: lesion induction and evidence of repair following vitamin C supplementation in vivo. Free Radic. Biol. Med., 34, 218225.[CrossRef][ISI][Medline]
- Meves,A., Stock,S.N., Beyerle,A., Pittelkow,M.R. and Peus,D. (2002) Vitamin C derivative ascorbyl palmitate promotes ultraviolet-B-induced lipid peroxidation and cytotoxicity in keratocytes. J. Invest. Dermatol., 119, 11031108.[CrossRef][ISI][Medline]
- Kitazawa,M., Podda,M., Thiele,J., Traber,M.G., Iwasaki,K., Sakamoto,K. and Packer,L. (1997) Interactions between vitamin E homologues and ascorbate free radicals in murine skin homogenates irradiated with ultraviolet light. Photochem. Photobiol., 65, 355365.[ISI][Medline]
- Broekmans,W.M., Vink,A.A., Boelsma,E., Klopping-Ketelaars,W.A., Tijburg,L.B., van't Veer,P., van Poppel,G. and Kardinaal,A.F. (2003) Determinants of skin sensitivity to solar irradiation. Eur. J. Clin. Nutr., 57, 12221229.[CrossRef][ISI][Medline]
- Dunham,W.B., Zuckerkandl,E., Reynolds,R., Willoughby,R., Marcuson,R., Barth,R. and Pauling,L. (1982) Effects of intake of L-ascorbic acid on the incidence of dermal neoplasms induced in mice by ultraviolet light. Proc. Natl Acad. Sci. USA, 79, 75327536.[Abstract]
- Pauling,L., Willoughby,R., Reynolds,R., Blaisdell,B.E. and Lawson,S. (1982) Incidence of sqamous cell carcinoma in hairless mice irradiated with ultraviolet light in relation to intake of ascorbic acid (vitamin C) and of D,L-alpha-tocopheryl acetate (vitamin E). Int. J. Vitam. Nutr. Res. Suppl., 23, 5382.[Medline]
- Robinson,A.B., Hunsberger,A. and Westall,F.C. (1994) Suppression of squamous cell carcinoma in hairless mice by dietary nutrient variation. Mech. Ageing Dev., 76, 201214.[CrossRef][ISI][Medline]
- May,J.M., Cobb,C.E., Mendiratta,S., Hill,K.E. and Burk,R.F. (1998) Reduction of the ascorbyl free radical to ascorbate by thioredoxin reductase. J. Biol. Chem., 273, 2303923045.[Abstract/Free Full Text]
- Meister,A. (1992) On the antioxidant effects of ascorbic acid and glutathione (Commentary). Biochem. Pharmacol., 44, 19051915.[CrossRef][ISI][Medline]
- Meister,A. (1994) Glutathione, ascorbate and cellular protection. Cancer Res., 54 (suppl.), 1969S1975S.[Medline]
- Rose,R.C. and Bode,A.M. (1992) Tissue-mediated regeneration of ascorbic acid: is the process enzymatic? Enzyme, 46, 196203.[ISI][Medline]
- Winkler,B.S., Orselli,S.M. and Rex,T.S. (1994) The redox couple between glutathione and ascorbic acid: a chemical and physiological perspective. Free Radic. Biol. Med., 17, 339349.[CrossRef]
- Kennedy,D.O., Matsumoto,M., Kojima,A. and Matsui-Yuasa,I. (1999) Cellular thiols status and cell death in the effect of green tea polyphenols in Ehrlich ascites tumor cells. Chem. Biol. Interact., 122, 5971.[CrossRef][ISI][Medline]
- Jiao,D., Smith,T.J., Yang,C.S., Pittman,B., Deasi,D., Amin,S. and Chung,F.-L. (1997) Chemopreventive activity of thiol conjugates of isothiocyanates for lung tumorigenesis. Carcinogenesis, 18, 21432147.[Abstract]
- Berrigan,M.J., Gurtoo,H.L., Sharma,S.D., Struck,R.F. and Martinello,A.J. (1980) Protection by N-acetylcysteine of cyclophosphamide metabolism related in vivo depression of mixed function oxygenase activity and in vitro denaturation of cytochrome P-450. Biochem. Biophys. Res. Commun., 93, 797803.[ISI][Medline]
- De Flora,S., Camoirano,A., Izzotti,A., Zanacchi,P., Bagnasco,M. and Cesarone,C.F. (1991) Antimutagenic and anticarcinogenic mechanisms of aminothiols. In Nygaard,F. and Upton,A.C. (eds) Anticarcinogenesis and Radiation Protection III. Plenum Press, New York, NY, pp. 275285.
- D'Agostini,F., Bagnasco,M., Giunciuglio,D., Albini,A. and De Flora,S. (1998) Oral N-acetylcysteine inhibition of doxorubicin-induced clastogenicity and alopecia: interaction between the two drugs in preventing primary tumors and lung micrometastases in mice. Int. J. Oncol., 13, 217224.[ISI][Medline]
- Doroshow,J.H., Locker,J.Y., Ifrim,I. and Myers,C.E. (1981) Prevention of doxorubicin cardiac toxicity in the mouse by N-acetylcysteine. J. Clin. Invest., 68, 10531064.[ISI][Medline]
- Moison,R.M., Rijnkels,J.M., Podda,E., Righel,F., Tomasello,F., Caffieri,S. and Beijersbergen van Henegouwen,G.M. (2003) Topically applied vitamin C and cysteine derivatives protect against UVA-induced photodegradation of suprofen in ex vivo pig skin. Photochem. Photobiol., 77, 343348.[CrossRef][ISI][Medline]
Received October 19, 2004;
revised December 14, 2004;
accepted December 15, 2004.