Ronald D. Ley

ULTRAVIOLET RADIATION - INDUCED MALIGNANT MELANOMA IN Monodelphis domestica

Several lines of evidence support the hypothesis that ultraviolet radiation (UVR) is involved in the etiology of cutaneous melanoma in humans. However, progress in understanding the mechanisms involved in induction of melanotic tumors by UVR has been hindered by lack of a suitable animal model. During the course of multiple exposures (3 times/wk for 70 wk) of the South American opossum, Monodelphis domestica, to UVR, we first observed the appearance of areas of dermal melanocytic hyperplasia (MH) on the exposed skin. Post‐UVR exposure to photoreactivating light (320–500 nm) suppressed the occurrence of MH. We also observed at 100 weeks from first exposure that 10 of 46 surviving animals had developed melanotic tumors which arose, presumably, from areas of MH. Tumors on three of the 10 animals have been classified as malignant melanomas based on metastasis to lymph nodes. We conclude from these results that UVR can act as a complete carcinogen for melanoma induction and, based on the photoreactivation of MH induction, that DNA damage is involved in melanoma formation.

Sunscreen Protection Against Ultraviolet Radiation‐induced Pyrimidine Dimers in Mouse Epidermal DNA

Pyrimidine dimers were measured in epidermal DNA of SKH:HRI mice following exposure to solar‐simulated UV radiation (SSUV, 290–400 nm) or to UVA (320–400 nm). Mice were exposed to SSUV or UVA after topical application (2 mg/cm2) of vehicle, a UVB absorber (5% 2‐ethylhexyl p‐methoxycinnamate [2‐EHMC]), or a broad‐spectrum UVA absorber (5% Mexoryl®SX). The rates of induction of pyrimidine dimers in untreated animals were 5.4 ± 0.57 times 10‐4 (mean ± SEM) and 7.6 ± 0.95 times 10‐6 dimers per 108 Da of epidermal DNA per J/m2 of SSUV and UVA, respectively. Topical application of Mexoryl®SX reduced the rate of induction of pyrimidine dimers in SSUV‐exposed animals to 4.7 ± 0.44 times 10‐5 dimers per 108 Da per J/m2 for a dimer induction protection factor (PF) of 11.5 (5.4 times 10 4/4.7 times 10‐5). The rate of dimer induction in Mexoryl®SX‐treated, UVA‐ex‐posed mice was 0.95 ± 0.2 times 10‐6 dimers per 108 Da per J/m2 (PF = 8.0). The 2‐EHMC at a concentration of 5% (wt/wt) was significantly less effective than Mexoryl®SX in preventing the induction of pyrimidine dimers in animals exposed to either SSUV or UVA. The rates of dimer induction in 2‐EHMC‐treated mice were 8.2 ± 1.1 times 10‐5 and 3.8 ± 0.33 times 10‐6 dimers per Da per J/m2 of SSUV (PF = 6.6) and UVA (PF = 2.0), respectively. Upon normalizing to the efficacy for edema induction, UVA induced approximately one‐fourth the number of pyrimidine dimers per equivalent edematous response when compared to SSUV.

Ultraviolet radiation-induced lethality and repair of pyrimidine dimers in fish embryos

Pimephales promelas (fathead minnow) embryos were used to show a correlation between induction of pyrimidine dimers in DNA and embryo death. Embryo killing was measured by a lack of heart-beat and blood circulation at 48 h post-ultraviolet radiation (UVR). When the embryos were exposed to various doses of UVR from a FS-40 sunlamp followed by exposure to photoreactivating light (PRL) (320-400 nm), the number of pyrimidine dimers decreased significantly. The photorepair of dimers was accompanied by a substantial increase in embryo survival. When embryo killing was examined as a function of the number of dimers present, dimers were identified as a major lesion involved in UVR-induced killing in these fish embryos. This in vivo study on photoreactivation treatment of fish embryos shows a direct association between UVR-induced pyrimidine dimers and embryo killing. In addition, when embryos were held in the dark for 9 h after UVR, 50% of the dimers were removed by excision repair.

Dose Response for Ultraviolet Radiation A–induced Focal Melanocytic Hyperplasia and Nonmelanoma Skin Tumors in Monodelphis domestica¶

Four groups of 30 dorsally shaved opossums (Monodelphis domestica) were exposed to graded doses of ultraviolet radiation A (UVA) (320–400 nm) three times per week for 90 weeks. Animals were monitored for the appearance of focal melanocytic hyperplasia (FMH) and nonmelanoma skin tumors (NMST) during the course of the exposures and for an additional 20 weeks following termination of exposures. FMH is the putative precursor for melanoma in the opossum. The lowest dose of UVA (2.5 × 103 J/m2) used in this study was selected based on the action spectrum for the induction of melanoma in a fish model. The prediction was that 2.5 × 103 J/m2 would induce FMH in the opossum if the action spectra for the induction of FMH in the opossum and melanoma in the fish were the same. The highest UVA dose was 2.5 × 105 J/m2. Only the highest dose of UVA gave a statistically significant induction of FMH and NMST in the opossum. As in previous studies, the FMH appeared earlier than the NMST during the course of exposures and the final prevalence of FMH was lower than the final prevalence of NMST. Overall, the results of this study indicate that the efficacy of UVA to induce FMH in the opossum is not as great as would be predicted from the action spectrum for melanoma induction in a fish model.

DNA damage is involved in the induction of opacification and neovascularization of the cornea by ultraviolet radiation

Studies were conducted to examine ultraviolet radiation (UVR)-induced alterations of the cornea of the gray, short-tailed opossum. Monodelphis domestica, and the effect of post-UVR illumination to photoreactivation light (PRL, 320-500 nm). As photoreactivation treatment specifically monomerizes pyrimidine dimers, an amelioration of the UVR-induced biological end-point would implicate DNA as being a primary chromophore for induction of that end-point. Corneas of anesthetized, four-month-old, opossums were exposed to 250 J m-2 (0.025 J cm-2) from a Westinghouse FS20 sunlamp either one or three times a week for up to 13 exposures. The corneas of 4-5 animals received either: (a) 90 min of PRL immediately prior to UVR; (b) PRL immediately following UVR; (c) PRL alone; or (d) UVR alone. Eyes were examined with a slit lamp microscope 24 hr following each exposure and scored for the appearance of opacification and neovascularization of the cornea. In animals exposed to UVR alone, 2-5 exposures, depending on whether the exposures were given once or three times per week, were required to obtain opacification and neovascularization in 50% of the irradiated corneas. The onset of both opacification and neovascularization in 50% of the corneas required 8-11 exposures when the UVR was immediately followed by PRL. Based on the specificity of photoreactivation repair to act solely on pyrimidine dimers, these observations suggest that UVR-induced pyrimidine dimers in corneal DNA are involved in UVR-induced opacification and neovascularization of the cornea of Monodelphis domestica.

LACK OF CORRELATION BETWEEN SUPPRESSION OF CONTACT HYPERSENSITIVITY BY UV RADIATION AND PHOTOISOMERIZATION OF EPIDERMAL UROCANIC ACID IN THE HAIRLESS MOUSE

Abstract The immunological consequences of exposure to UVA (320–400 nm) radiation are unclear. This study describes the relationship between the generation of epidermal cis‐urocanic acid and the ability to respond to a contact‐sensitizing agent, in hairless mice exposed to different UV radiation sources, which incorporate successively greater short‐wavelength cutoff by filtration of the radiation from fluorescent UV tubes. Mice were exposed to these radiation sources at doses systematically varying in UVB radiation content but supplying increasing proportions of UVA radiation. All radiation sources were found to generate approximately 35%cis‐urocanic acid in the epidermis, thus normalizing the sources for cis‐urocanic acid production. However, only those sources richest in short‐wavelength UVB resulted in suppression of the systemic contact hypersensitivity response. These sources also induced the greatest erythema reaction, measured as its edema component, in the exposed skin. A strong correlation was thus demonstrated between the induction of edema and the suppression of contact hypersensitivity, but there appeared to be no correlation between the generation of epidermal cis‐urocanic acid and suppression of contact hypersensitivity. The sources richest in UVA content did not result in suppression of contact hypersensitivity: furthermore mice previously irradiated with such UVA‐rich sources were refractory to the immunosuppressive action of exogenous cis‐urocanic acid. A protective effect of the increased UVA content thus appeared to be inhibiting immunosuppression by the available endogenously generated or exogenously applied cis‐urocanic acid.

Comparison between ultraviolet-visible and near-infrared elastic scattering spectroscopy of chemically induced melanomas in an animal model

The work reported compares elastic scattering spectroscopy (ESS) for diagnosis of pigmented skin lesions in two spectral regions: UV-visible and near infrared (NIR). Given the known strong absorption by melanin in the near-UV to mid-visible range of the spectrum, such a comparison can help determine the optimum wavelength range of ESS for diagnosis of pigmented skin lesions. For this purpose, four South American opossums are treated with dimethylbenz(a)anthracene on multiple dorsal sites to induce both malignant melanomas and benign pigmented lesions. Skin lesions are examined in vivo with ESS using both UV-visible and NIR, with wavelength ranges of 330 to 900 nm and 900 to 1700 nm, respectively. Both portable systems use the same fiber optic probe geometry. ESS measurements are made on the lesions, and spectral differences are grouped by diagnosis from standard histopathological procedure. Both ESS datasets show strong spectral trends with the histopathological assignments, and the data suggest a model for the underlying basis of the spectral distinction between benign and malignant pigmented nevi.

Photobiology of Monodelphis domestica

The gray, short-tailed opossum, Monodelphis domestica, has been used for photobiologic studies since 1984. The presence of a light-activated DNA repair pathway in the tissues of Monodelphis has been used to identify pyrimidine dimers in DNA as initiating events for a number of ultraviolet radiation (UVR)-induced pathologies of the skin and cornea. Furthermore, Monodelphis, unlike common laboratory rodents, is susceptible to the induction of melanoma by UVR alone.

Monodelphis domestica: A NEW ANIMAL MODEL FOR STUDIES IN PHOTODERMATOLOGY*

The ability to photoreverse pyrimidine dimers in DNA of the South American opossum Monodelphis domestica provides a powerful tool with which to probe the role of pyrimidine dimers in ultraviolet radiation (UVR)‐induced histopathologic changes of the skin of this mammal. We have observed that post‐UVR exposure to photoreactivation light not only reversed pyrimidine dimers in epidermal DNA, but also suppressed the capacity of UVR to induce macroscopic and microscopic changes in the skin of M. domestica.

Enhanced pyrimidine dimer removal in repair-proficient murine fibroblasts transforme with the denV gene of bacteriophage T4

The denV gene of bacteriophage T4, which encodes the pyrimidine dimer-specific repair enzyme endonuclease V, was introduced into murine fibroblasts with normal rodent pyrimidine dimer repair capabilities. Endonuclease V recognizes ultraviolet radiation (UVR)-induced pyrimidine dimers and produces single-strand breaks adjacent to the dimers. These nicks may serve as substrates to initiate excision repair of pyrimidine dimers by endogenous enzymes. In the present study, murine fibroblasts stably transfected with denV were able to remove 50-80% of UVR-induced pyrimidine dimers, while control cells removed only about 20% of dimers under the same conditions of pyrimidine dimer induction and repair. For both control and denV-transfected cells, repair continued for at least 24 h after exposure. When removal of UVR-induced photoproducts was initiated by endogenous excision repair mechanisms, an average of 38 nucleotides were replaced per dimer removed, as determined by bromouracil photolysis; denV-initiated excision repair, on the other hand, resulted in removal of an average of 6 nucleotides per dimer repaired. The enhanced pyrimidine dimer repair capabilities conferred by denV gene expression did not appear to improve post-UVR survival.