Environment and health: 3. Ozone depletion and ultraviolet radiation.
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Animals↗
Biomedical subjects
J C van der Leun
Publications and source records attributed to J C van der Leun.
Finsen Medal Lecture 1996. Photobiology and the ozone layer.
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Animals↗
Health risks.
The health risks associated with ozone depletion will principally be those due to increased ultraviolet B (UV-B) radiation in the environment, i.e., increased damage to the eyes, the immune system, and the skin. Some new risks may also be introduced with the increased use of alternatives to the ozone-depleting substances (ODSs). Quantitative risk estimates are available for some of the UV-B-associated effects, e.g., cataract and skin cancer; however, the data are insufficient to develop similar estimates for effects such as immunosuppression and the toxicity of alternatives. Ocular damage from UV exposures includes effects on the cornea, lens, iris, and associated epithelial and conjunctival tissues. The most common acute ocular effect of environmental ultraviolet radiation (UVR) is photokeratitis. Also known as snowblindness in skiers, this condition also occurs in other outdoor recreationists. Chronic eye conditions likely to increase with ozone depletion include cataract, squamous cell carcinoma, ocular melanoma, and a variety of corneal/conjunctival effects, e.g., pterygium and pinguecula. Suppression of local (at the site of UV exposure) and systemic (at a distant, unexposed site) immune responses to a variety of antigens has been demonstrated in both humans and animals exposed to UV-B. In experiments with animals these effects have been shown to worsen the course/outcome of some infectious diseases and cancers. There is reasonably good evidence that such immunosuppression plays a role in human carcinogenesis; however, the implications of such immunosuppression for human infectious diseases are still unknown. In light-skinned populations, exposure to solar UVR appears to be the most important environmental risk factor for basal and squamous cell carcinomas and cutaneous melanoma. Originally it was believed that total accumulated exposure to UVR was the most important environmental factor in determining risk for these tumors. Recent information now suggests that only squamous cell carcinoma risk is related to total exposure. In the cases of both basal cell carcinoma and melanoma, new information suggests that increases in risk are tied to early exposures (before about age 15), particularly those leading to severe sunburns. Testing of a number of the chlorofluorocarbon (CFC) alternatives indicates that most of these chemicals have low acute toxicity, and low to moderate chronic toxicity. Some chemicals that were originally proposed as alternatives have been dropped from consideration because these tests raised concerns about toxicity and/or manufacturing difficulties. In one instance, high accidental occupational exposure was associated with liver damage, underlining the need for care in the use of these substitutes. Recent quantitative risk estimates have been developed for cataract, melanoma, and all skin cancers combined. These estimates indicate that under the Montreal Adjustments, cataract and skin-cancer incidence will peak mid-century at additional incidences of just under 3 per 100,000 and about 7 per 100,000, respectively.
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Carcinogenesis induced by UVA (365-nm) radiation: the dose-time dependence of tumor formation in hairless mice.
Although ultraviolet B (UVB wavelengths 280-315 nm) dominates the carcinogenic effect of sunlight, ultraviolet A (UVA 315-400 nm) is estimated to contribute 10-20% to the carcinogenic dose; a substantial background that is not affected by a depletion of the ozone layer. Furthermore, certain high-power modern tanning lamps emit mainly long wave UVA (UVA1; 340-400 nm). For a proper risk estimate of UVA exposure its carcinogenicity relative to that of UVB exposure needs to be determined more accurately. To this end we determined the dose-time relationship for skin tumor induction in hairless mice that were irradiated daily with custom-made Philips 365-nm sources. Irradiation of the group exposed to the highest of the four daily doses (430, 240, 140 and 75 kJ/m2) had to be discontinued because severe scratching set in after 3 months (no tumors). In the lower dose-groups the prevalence curves for skin carcinomas (percentage of tumor-bearing mice versus logarithm of time) ran virtually parallel, and were similar to those found with daily UVB exposure. However, the relationship between the daily dose (D) and the median tumor induction time (t50) appeared to differ: with UVB we found that t50 D(r) = constant, with r = 0.6, whereas with UVA1 we found r approximately 0.4. This would imply that 365-nm carcinogenesis shows less of a dose-dependency than UVB carcinogenesis, and that 365-nm radiation becomes more carcinogenic, relative to UVB, as the daily doses are lowered. This relative shift at low doses complicates extrapolation of UVB to UVA risks in humans. Based on the t50 from the lowest dose-group we found that the carcinogenicity at 365 nm (per J/m2) is 0.9 x 10(-4) times that at 293 nm, the wavelength of maximum carcinogenicity in hairless mice. This result for 365-nm carcinogenicity falls well within the margins of error of the wavelength dependency that was estimated earlier from experiments with broadband UV sources.
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Time and dose dependence of acceptance of UV-induced syngeneic tumor implants in chronically UV-exposed hairless mice.
The kinetics of skin cancer induction by UV radiation has been extensively studied in hairless mice and described by Weibull statistics in which the time till 63% of the mice bear tumors is a primary parameter. However, the kinetics of the associated immunosuppression remained to be determined. To this end, we implanted a syngeneic UV-induced skin carcinoma cell line (T51/6.53) in the ventral skin of HRA/SKH hairless mice after various periods of daily dorsal UV exposure, either 150 or 75 mJ/cm2 per day UV from F40 sunlamps (regimens that when continued yield 63% of the mice with 1 mm tumors in 11.5 or 16.2 weeks, respectively). Both exposure regimens achieved a 100% acceptance (after 7 and 16 weeks, respectively). The implants failed to grow in all unirradiated control mice, but the percentage of mice in which the implants grew increased with the UV treatment time and dose. The estimated times to 63% implant acceptance were 4.3 +/- 0.8 and 8.2 +/- 0.8 weeks for the high and low daily doses, respectively. As reported earlier for shaved haired mice, there appears to be a straight reciprocity between daily UV dose and the time to tumor acceptance, i.e. the latter fully depends on the cumulative UV dose, whereas the tumor induction does not. The latter probably also depends on a pure elapse of time, i.e. UV-independent processes. A further analysis of the Weibull description indicates that immunosuppression toward the tumor requires fewer UV-driven steps that tumor induction.
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Estimates of ozone depletion and skin cancer incidence to examine the Vienna Convention achievements.
Depletion of the ozone layer has been observed on a global scale, and is probably related to halocarbon emissions. Ozone depletion increases the biologically harmful solar ultraviolet radiation reaching the surface of the Earth, which leads to a variety of adverse effects, including an increase in the incidence of skin cancer. The 1985 Vienna Convention provided the framework for international restrictions on the production of ozone-depleting substances. The consequences of such restrictions have not yet been assessed in terms of effects avoided. Here we present a new method of estimating future excess skin cancer risks which is used to compare effects of a 'no restrictions' scenario with two restrictive scenarios specified under the Vienna Convention: the Montreal Protocol, and the much stricter Copenhagen Amendments. The no-restrictions and Montreal Protocol scenarios produce a runaway increase in skin cancer incidence, up to a quadrupling and doubling, respectively, by the year 2100. The Copenhagen Amendments scenario leads to an ozone minimum around the year 2000, and a peak relative increase in incidence of skin cancer of almost 10% occurring 60 years later. These results demonstrate the importance of the international measures agreed upon under the Vienna Convention.
Atmosphere↗
Early p53 alterations in mouse skin carcinogenesis by UVB radiation: immunohistochemical detection of mutant p53 protein in clusters of preneoplastic epidermal cells.
High levels of the p53 protein are immunohistochemically detectable in a majority of human nonmelanoma skin cancers and UVB-induced murine skin tumors. These increased protein levels are often associated with mutations in the conserved domains of the p53 gene. To investigate the timing of the p53 alterations in the process of UVB carcinogenesis, we used a well defined murine model (SKH:HR1 hairless mice) in which the time that tumors appear is predictable from the UVB exposures. The mice were subjected to a series of daily UVB exposures, either for 17 days or for 30 days, which would cause skin tumors to appear around 80 or 30 weeks, respectively. In the epidermis of these mice, we detected clusters of cells showing a strong immunostaining of the p53 protein, as measured with the CM-5 polyclonal antiserum. This cannot be explained by transient accumulation of the normal p53 protein as a physiological response to UVB-induced DNA damage. In single exposure experiments the observed transient CM-5 immunoreactivity lasted for only 3 days and was not clustered, whereas these clusters were still detectable as long as 56 days after 17 days of UVB exposure. In addition, approximately 70% of these patches reacted with the mutant-specific monoclonal antibody PAb240, whereas transiently induced p53-positive cells did not. In line with indicative human data, these experimental results in the hairless mouse model unambiguously demonstrate that constitutive p53 alterations are causally related to chronic UVB exposure and that they are a very early event in the induction of skin cancer by UVB radiation.
Animals↗
UV radiation from sunlight: summary, conclusions and recommendations.
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Animals↗
Cell cycle kinetics following UVA irradiation in comparison to UVB and UVC irradiation.
There is limited information about the carcinogenic effect of longwave ultraviolet radiation (UVA: 315-400 nm). In particular very little is known about the relevant genotoxic damage caused by physiological doses of UVA radiation. A general response of cells to DNA damage is a delay or arrest of the cell cycle. Conversely, such cellular responses after UVA irradiation would indicate significant genotoxic damage. The aim of this study is to compare cell cycle kinetics of human fibroblasts after UVC (190-280 nm radiation), UVB (280-315 nm radiation) and UVA irradiation. Changes in the cell cycle kinetics were assessed by bivariate flow cytometric analysis of DNA synthesis and of DNA content. After UVC, UVB or UVA irradiation of human fibroblasts a suppression was seen of bromodeoxyuridine (BrdU) incorporation at all stages of S phase. The magnitude of this suppression appeared dose dependent. Maximum suppression was reached at 5-7 h after UVB exposure and directly after UVA exposure, and normal levels were reached 25 h after UVB and 7 h after UVA exposure. The lowered BrdU uptake corresponded with a lengthening of the S phase. No dramatic changes in percentages of cells in G1, S and G2/M were seen after the various UV irradiations. Apparently, UVA irradiation, like UVB and UVC irradiation, can temporarily inhibit DNA synthesis, which is indicative of genotoxic damage.
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Molecular dosimetry by flow cytometric detection of thymine dimers in mononuclear cells from extracorporally UV-irradiated blood.
UV-induced DNA damage in mononuclear leucocytes can be quantified by flow cytometry of fluorescence from a labelled monoclonal antibody that specifically binds to thymine dimers (T<-->T): specific fluorescence is already detectable after exposures of 1-2 J m-2 of 254 nm radiation and shows a linear relationship with dose. The distribution of UV fluences over an irradiated volume can thus be ascertained by measuring the UV-induced T<-->T loads of the individual cells from that volume. After irradiation of mononuclear cells in a phosphate buffer solution in a Petri dish, most cells showed a similar intensity of specific T<-->T fluorescence, forming a single sharp peak in the fluorescence histogram. This signifies an even distribution of fluences over the cells. It was noticed, however, that a variable minor fraction of mononuclear cells (usually less than 10%) could be resistant to immunostaining; this fraction was rejected from the calculation of the specific fluorescence. The flow cytometric technique was also applied to blood cells exposed in an ISOLDA device, which is in use in Russian clinics for UV irradiation of whole blood for therapeutical purposes. Only a small fraction of mononuclear cells in a sample of whole blood treated in ISOLDA acquired a detectable T<-->T load after exposure to lamps which emit predominantly either UVC or UVB light ((3.6 +/- 1.0)% and (1.8 +/- 0.4)% of all analysed cells respectively). This small fraction had received a large variation in fluences, resulting in differences in nuclear T<-->T loads by a factor of 200.(ABSTRACT TRUNCATED AT 250 WORDS)
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Substitution of equally carcinogenic UV-A for UV-B irradiations lowers epidermal thymine dimer levels during skin cancer induction in hairless mice.
Cyclobutane pyrimidine dimers (CPD) are the predominant DNA lesions induced by UV-B radiation, among these lesions thymine dimers are most frequent. Although UV-A radiation may also induce CPD, it has been found that equally cytotoxic or equally mutagenic UV-A and UV-B doses do not induce equal amounts of CPD, indicating that other DNA adducts contribute to the UV-A effects. Thus far it has not been established whether this finding can be extrapolated and also holds true for the more complex biological endpoint of skin cancer. Therefore, we compared thymine dimer levels during skin cancer induction by combined UV-A and UV-B daily exposures with the levels from equally carcinogenic daily UV-B exposures. From control experiments it was known that both groups would react similarly regarding the occurrences of carcinomas, with a median latency time of 170 +/- 10 days. After 50, 106 and 151 days of irradiation eight hairless mice (SKH:HR1) from both groups were euthanized and thymine dimers in epidermal cell suspensions were quantified by flow cytometry. Staining on DNA content enabled us to quantify thymine dimers in G0/G1-phase, in S-phase and in G2M-phase subpopulations. Both in total epidermal cell populations and in subpopulations of replicating epidermal cells thymine dimer levels were significantly lower in the UV-A/B combination group than in the UV-B group (0.010 < P < 0.025 and P < 0.005 respectively). This indicates that the carcinogenicity of UV-A relative to that of UV-B is not properly measured by thymine dimers and that other DNA lesions than CPD, for example, from reactive oxygen species, are likely to contribute to UV-A carcinogenicity.
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Cells with UV-specific DNA damage are present in murine lymph nodes after in vivo UV irradiation.
Ultraviolet radiation is absorbed in the skin, especially in the epidermis. After ultraviolet irradiation the number of major histocompatibility complex class II+, adenosine triphosphatase+ Langerhans cells and Thy-1+ dendritic epidermal cells in the epidermis decreases. Whether this decrease is due to migration of these cells or to loss of membrane markers is not clear. To address this question we have used the monoclonal antibody H3 directed against cyclobutyl thymine dimers-a form of DNA damage that is specifically induced by ultraviolet radiation-to investigate whether H3+ cells are present in the draining lymph nodes of the skin after ultraviolet irradiation of hairless, inbred mice (HRA/Skh). After a single dose of ultraviolet radiation (Westinghouse FS40, 1.5 kJ/m2), H3+ cells were present in the paracortex of the draining lymph nodes. No positive cells were found in the blood of irradiated mice. These results suggest that the H3+ cells in the lymph nodes originate from the skin. The number of H3+ cells in the draining lymph nodes increased the first 24 h after irradiation and then stabilized. Immunohistochemical double staining revealed that all H3+ cells were major histocompatibility complex II+, and that only a fraction of the cells were NLDC-145 positive. No V gamma 3 T-cell receptor bearing cells could be found in the lymph nodes after UV irradiation of the skin.
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Induction and disappearance of thymine dimers in human skin exposed to UVB radiation: flow cytometric measurements in replicating and nonreplicating epidermal cells.
We have earlier reported on determining UV-induced DNA damage in murine epidermal cell suspensions by flow cytometric analysis of the fluorescence from a fluorescein isothiocyanate-labeled antibody (H3) directed against thymine dimers (T < > T). Here we present an optimization of the technique for analysis of epidermal cell suspensions from 4 mm biopsies from human skin. Cells with different DNA contents can easily be distinguished in flow cytometry by the intensity of DNA-specific 7-amino-actinomycin D fluorescence. Genuine G2-M-phase cells can further be distinguished from cell doublets by pulse-shape discrimination. Thus, T < > T levels in individual cells with different DNA contents (i.e. G0-G1, S or G2-M phases) can be determined after in vivo exposure of human skin to environmentally relevant UVB (280-315 nm) doses. The method was applied to measure the decrease of T < > T in nonreplicating cells (G0-G1 phase) and replicating cells (S phase or G2-M phase) from seven volunteers exposed to twice their minimal erythema dose. The reduction in the average T < > T-specific fluorescence at 24 h after exposure was 46% (ranging between 16% and 66%) for the G0-G1 cells and 70% (ranging between 37% and 100%) for the S + G2-M cells. The difference was statistically highly significant. Determination of individual DNA repair capacities with this method can become a convenient diagnostic tool for patients with DNA repair disorders, or it may even be used to identify individuals with low repair proficiencies and increased risk of developing skin cancers.
Cell Division↗
Ultraviolet radiation--induced impairment of the early initiating and the late effector phases of contact hypersensitivity to picrylchloride: regulation by different mechanisms.
Two types of antigen-specific T cells are needed for the elicitation of contact hypersensitivity reactions. They act in an obligate sequence to mediate the early initiating and late effector phases of contact hypersensitivity, which are accompanied by skin-swelling responses at 2 and 24 h after challenge, respectively. The magnitude of the late ear swelling depends on that of the early swelling. We studied the influence of ultraviolet radiation on both phases of contact hypersensitivity to picrylchloride. Mice were exposed to subedemal doses of ultraviolet radiation on the shaved backs for four consecutive days. Four days later mice were sensitized on non-irradiated skin. Four days after sensitization mice were challenged on the ears, and swelling was measured 2, 4, and 24 h after challenge. The early and late phases of contact hypersensitivity were largely suppressed in ultraviolet-irradiated, actively sensitized mice. Transfer of immune lymphoid cells from donor mice that were sensitized 4 d earlier induced early and late components of contact hypersensitivity in naive recipients after challenge. Transfer of immune lymphoid cells from donors that were sensitized 1 d earlier only induced the early component of contact hypersensitivity. Ultraviolet irradiation of donor mice significantly reduced the capacity of the immune lymphoid cells to induce contact hypersensitivity. We show that lymphoid cells responsible for the early and late components of contact hypersensitivity are both affected.
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Interaction between ultraviolet A and ultraviolet B radiations in skin cancer induction in hairless mice.
The rate of tumor induction by UV-A radiation rises more slowly with time and accumulated dose than that by UV-B radiation. It has recently been shown that this difference disappears when frank papillomas are excluded from the analysis. Thus, the rate of development of "nonpapillomas" (mainly squamous cell carcinomas and precursors) can be fully characterized by a typical tumor induction time, e.g., the time until 50% of the mice bear tumors. This has opened the possibility to investigate how UV-A and UV-B exposures add up in the induction of squamous cell carcinomas, which is an important issue in risk assessments of artificial UV-A sources for cosmetic or medical purposes. We present the results of an experiment in which 6 groups of 24 albino SKH:HR1 mice were treated daily for 600 days with either effective UV-A radiation, effective UV-B radiation, or combinations of both. The observed times it took for 50% of the mice to bear tumors in the combination groups were compared with those calculated on the basis of arithmetical addition of effective UV-A and effective UV-B doses. We did not find a statistically significant (P < 0.05) deviation from additivity.
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Wavelength dependence of skin cancer induction by ultraviolet irradiation of albino hairless mice.
Information on the variation in carcinogenicity with wavelength is crucial in risk assessments for skin cancers induced by UV radiation. Until recently the wavelength (lambda) dependencies of other detrimental UV effects, such as sunburn, have been used as substitutes. Direct information on the lambda dependency can only be obtained from animal experiments. To this end we accumulated a large data set on skin tumors induced by chronic UV exposure of albino SKH:HR1 mice (14 different broadband UV sources and about 1100 mice); the data come from the Photobiology Unit of the former Skin and Cancer Hospital in Philadelphia and from the Department of Dermatology of the University of Utrecht. The lambda dependency was extracted from this data set (a statistically satisfactory description with chi 2 = 13.4, df = 7) and represented by the Skin Cancer Utrecht-Philadelphia action spectrum, i.e., a set of factors to weight the exposures at different wavelengths according to their respective effectiveness (inversely proportional to the daily exposure required for a median tumor induction time of 300 days). The fits obtained with other already available action spectra proved to be poor (chi 2 > 60, df = 11). The maximum effectiveness was found at 293 nm, and above 340 nm the effectiveness showed a shoulder at about 10(-4) of the maximum. A sensitivity analysis of the final solution for the lambda dependency showed a large margin of uncertainty above 340 nm and an information gap below 280 nm. The large variation in tumor responses in the present data set can be transformed to a coherent, common dose-response relationship by proper spectral weighting with this single action spectrum.
Animals↗
Ultraviolet carcinogenesis: environmental effects of ozone depletion.
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Atmosphere↗
Flow cytometric immunofluorescence assay for quantification of cyclobutyldithymine dimers in separate phases of the cell cycle.
Quantitative immunofluorescence assays for the measurement of cyclobutyldithymine dimers (T <> T) based on computer-assisted immunofluorescence microscopy have recently been described. Here we present a modified assay for T <> T based on flow cytometry. This method has the advantage that T <> T can be quantified in separate phases of the cell cycle by the fluorescent counterstaining of nuclear DNA and subsequent selection on DNA content. The H3 monoclonal antibody directed at T <> T binds to partially denatured DNA in situ. The antibody is labeled with fluorescein isothiocyanate (FITC) and DNA is stained with the intercalating dye 7-amino-actinomycin D. FITC fluorescence increases linearly with dose of UV-C radiation (up to 45 J/m2) of cultured human fibroblasts. A linear fluorescence-dose relationship was also found for epidermal cells of SKH:HR1 hairless mice after in vivo irradiation with UV-B (FS40 sunlamp, up to 3750 J/m2). This technique allows a quick assessment of UV damage levels in 10,000s of cells and makes immunofluorescence of DNA damage more accessible to other research groups.
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