Gavin E. Greenoak

Role of endogenous retroviruses as mutagens: The hairless mutation of mice

We have developed an experimental approach to distinguish the 40-60 endogenous C-type proviruses of mice and to determine their association with well characterized developmental and physiological mutations. The hairless (hr) mutation causes a variety of pleiotropic effects. Using oligonucleotide probes specific for different classes of murine leukemia virus, we have identified and cloned a provirus present in HRS/J hr/hr animals but absent in HRS/J +/+. Genetic analyses showed perfect concordance between the hr phenotype and the presence of the provirus in a number of inbred and congenic strains of mice. Molecular analysis of a haired revertant established the causal relationship since it revealed the excision of most of the proviral genome leaving behind one long terminal repeat. These findings show that virus integration caused the hairless mutation and point to the utility of naturally occurring retroviral integrations for accessing the genome of the mouse.

TOPICAL UROCANIC ACID ENHANCES UV‐INDUCED TUMOUR YIELD AND MALIGNANCY IN THE HAIRLESS MOUSE

Abstract— Epidermal urocanic acid has been postulated to be the mediator of the specific state of immunosuppression induced by UV irradiation, by which UV‐initiated tumour cells are able to evade normal recognition and can survive to grow progressively into malignant tumours. These experiments demonstrate that topical application of UV‐irradiated urocanic acid systemically suppresses the contact type hypersensitivity response to oxazolone in hairless mice. In addition, topically applied urocanic acid markedly increases the overt tumour yield and the degree of malignancy in hairless mice exposed chronically to daily minimally erythema] doses of simulated solar UV light. Topical urocanic acid also increases the number of latent UV‐initiated tumours, detectable by croton oil promotion. Therefore UV photoproducts of urocanic acid can both systemically suppress contact hypersensitivitv in the epidermis, and also enhance early survival of UV‐initiated tumour cells resulting in augmentation of UV photocarcinogenesis.

Comparison of dermal absorption of zinc from different sunscreen formulations and differing UV exposure based on stable isotope tracing.

In a pilot study to determine if zinc (Zn) from zinc oxide nanoparticles in sunscreen can penetrate human skin in vivo, nanoparticles (~30nm) of a stable isotope (52% (68)Zn enrichment) were incorporated into an essentially phytochemical-based formulation and applied to the backs of 3 human subjects twice daily for 5 days during the Southern Hemisphere winter. Blood and urine were collected prior to application and at regular intervals and up to 50 days. As observed in a larger outdoor trial following this pilot study but with a different formulation and with UV exposure: values of (68)Zn in blood continued to increase beyond the 5 day application phase with the highest measurement at 14 days after the first application; variable amounts of the (68)Zn tracer were observed in urine; and the amounts of extra Zn added to blood were small and indicate very low levels of absorption (minimal estimate <0.01% of the applied dose) through the skin. Reasons for differences in absorption detected in the stable isotope trials and previous investigations include: the sensitivity of the stable isotope method; the duration of the investigations; the number of applications of sunscreen formulation; in vitro methods with excised skin; lack of measurement of blood and urine; no skin flexing; and lack of UV exposure.

Effect of dietary lipid on UV light carcinogenesis in the hairless mouse.

Abstract— Isocaloric feeding of diets varying in lipid content to albino hairless mice has shown that their susceptibility to skin tumorigenesis induced by simulated solar UV light was not affected by the level of polyunsaturated fat, 5% or 20%. However a qualitative effect of dietary lipid was demonstrated. Mice fed 20% saturated fat were almost completely protected from UV tumorigenesis when compared with mice fed 20% polyunsaturated fat. Multiple latent tumours were detected in the saturated fat‐fed mice by subsequent dietary replenishment, suggesting that a requirement for dietary unsaturated fat exists for the promotion stage of UV‐induced skin carcinogenesis.

Ultraviolet radiation dosimetry with radiochromic film

Radiochromic film is tested for its broad-band response to ultraviolet (UV) B (290-320 nm) and A (320 nm400 nm), visible and infrared radiation produced by a solar simulator and examined for dosimetry in ultraviolet radiation. Results show that MD-55-2 radiochromic film in solar and fluorescent light sources responds almost exclusively to broad-band UVA radiation with negligible colouration from UVB, visible and low level infrared radiation. A second order polynomial function approximates the change in optical density at 660 nm wavelength for film colouration with exposure to UVA from white light fluorescent and solar UV with exposures measured with a dedicated UVA dosimeter. Using a double exposure technique as used in radiation dosimetry where the film is firstly irradiated to a known UV dose, radiochromic film can be used as a quantitative measure of UVA exposure.

Skin phototyping in Asian Australians

Skin phototype was assessed in 257 Asian Australians by self‐reporting questionnaire. Minimal erythema dose, minimal melanogenic dose and minimal immediate pigment darkening dose were measured in a subgroup of 30 subjects. About 15% of Asian Australians in this study report that they have skin type I or II. Phototesting confirms that there is a UV‐sensitive group and a wide spectrum of UV‐sensitivity in this population. Whether Fitzpatricks skin typing system adequately identifies this UV‐sensitive group needs assessment by a larger study. The relationship between burning tendency and tanning capacity in Asians may differ from Caucasians.

Effect on topical 5-methoxypsoralen on tumorigenesis induced in albino and pigmented hairless mouse skin by UV irradiation

A new line of the Skh:HRII hairless pigmented mouse (black juvenile coat) is described which has been selectively bred for the capacity to respond consistently to simulated solar UV radiation with a continuous and strong tan. This mouse demonstrates a degree of protection from chronic UV-induced tumorigenesis when compared with the Skh:HRI hairless albino mouse, and has been used here to study the effect of induced melanogenesis on phototumorigenesis. Mice were irradiated for 10 weeks with incremental doses of simulated solar UV radiation (UVA + B) from a fluorescent tube source which induced tumours in 100% of albino mice and 93% of black mice by 200 days (minimally oedemal), or with 60% of this dose (sub-oedemal) which induced tumours in 85% of albino mice and 65% of black mice. Mice were also exposed to the UVA component of these radiation sources, obtained by window glass filtration. The effect of topical 5-methoxypsoralen (5-MOP) was examined, at either 0.003% with minimally oedemal UVA + B or its UVA component alone, or at 0.01% with sub-oedemal UVA + B or its UVA component alone, in both albino and black mice. The 5-MOP concentrations were selected as the maximum concentration which did not increase the erythema and oedema responses after a single exposure to minimally oedemal or sub-oedemal UVA + B. At 200 days, the tumorigenic response to sub-oedemal UVA + B was significantly increased by topical 5-MOP, to 100% in albinos and 93% in black mice. In contrast, tumorigenesis in response to minimally oedemal UVA + B was unaffected by topical 5-MOP. The UVA component alone of either irradiation regime was not tumorigenic under these conditions. When combined with topical 5-MOP, the UVA of minimally oedemal UVA + B became moderately tumorigenic, and resulted in a tumour incidence of 23% in albinos and 14.5% in black mice. However, the UVA component of sub-oedemal UVA + B, when combined with topical 5-MOP, was highly tumorigenic specifically in albino mice, inducing tumours in 93% of albino mice but in only 27% of black mice. Tan intensity resulting from minimally oedemal UVA + B was not enhanced by topical 5-MOP, and its UVA component combined with 5-MOP resulted in only a minimal tan. However, the tan intensity resulting from sub-oedemal UVA + B with topical 5-MOP was strongly increased, although its UVA component combined with 5-MOP did not produce a perceptible tan.(ABSTRACT TRUNCATED AT 400 WORDS)

The characterization of squamous cell carcinoma induced by ultraviolet irradiation in hairless mice.

&NA; Squamous cell carcinomas (SCCs) induced by ultraviolet irradiation in hairless mice were characterized according to their growth, gross appearance and light and transmission electron microscopic features. SCCs arose directly from irradiated skin (ab initio) or progressed from pre‐existing epidermal tumours and lesions. SCCs could be graded using guidelines established for human tumours. SCCs comprised 60.8% of the tumours examined. Of these, 35.6% were designated as grade 1,27.7% as grade 2, 7.9% as grade 3 and 28.7% as grade 4. Spindle cell tumours suspected of being SCCs were included in grade 4. Grades 1, 2 and 3 could not be distinguished on the basis of growth and gross appearance. Those arising ab initio presented as either red, ulcerated lesions or as raised, white, verrucose lesions. Grade 4 SCCs that arose ab initio presented as rapidly growing, red, spherical lesions. Those that arose from pre‐existing tumours or lesions had no characteristic appearance, and variable growth. Light microscopically, grade 4 SCCs with an obvious point of origin from epidermis or other epidermal tumours, and putative grade 4 SCCs without such a point of origin, were characterized commonly by spindle cells, pleomorphic giant or multinucleated cells and individual cell reticular fibres. Ultrastructurally, spindle cells, although poorly differentiated, were distinct from fibroblastic proliferations and had few tonofilaments or desmosomes, and were inconsistently surrounded by basal lamina‐like material. On the basis of these characteristics, and despite inconclusive positivity with immunoperoxidase staining for keratin and pre‐keratin, it was concluded that these spindle cell tumours were most probably of identical squamous cell origin. Metastases to the lungs were noted in association with a grade 1, grade 2 and a grade 3 SCC and represented a rate of 4.8%. Although it appears that spindle cell SCC represents the end stage of progression for local malignancy, it may not be so for metastatic potential.

PYRIMIDINE DIMER INDUCTION AND REMOVAL IN THE EPIDERMIS OF HAIRLESS MICE: INEFFICIENT REPAIR IN THE GENOME OVERALL AND RAPID REPAIR IN THE H‐ras SEQUENCE

Excision repair of pyrimidine dimers was examined at the genome overall in three strains of hairless (hr/hr) and congenic wild‐type mice, as well as in the expressed H‐ras gene in hairless mice. The assay used a pyrimidine dimer‐specific endonuclease from Micrococcus luteus and alkaline agarose gel electrophoresis. From 0 to 25% of endonuclease‐sensitive sites were removed at the genome level in either hairy or hairless mice but about 50% were removed in the H‐ras gene in hairless mice by 24 h after exposure to 5.4 J/cm2 UV (290‐400 nm) irradiation. No differences were observed in the repair capacity between hairy and hairless mice, thus eliminating defective DNA repair as the explanation for the greater susceptibility to UV carcinogenesis in hairless mice.

Ultrastructure of Ultraviolet Radiation-Induced Hairless Mouse Skin Carcinogenesis, With Special Reference to the Epidermal-Dermal Junction

&NA; The ultrastructure of ultraviolet (UV)‐induced skin pathology was studied in mice to complement previously reported gross and light microscopic findings, and to assess further the usefulness of the animal model for study of sunlight associated epidermal tumours in man. Hairless albino (HRA/Skh‐1) mice were exposed to a minimal erythemal dose from a filtered light source emitting both UVA and UVB, approximating solar emission. Samples of normal and hyperplastic skin, pedunculated papillomas, carcinomas in situ and invasive squamous cell carcinomas were processed for transmission electron microscopy once their identity was confirmed by light microscopic examination. Keratinocyte pleomorphism became more marked and cell to cell contact diminished as malignancy developed. For papillomas, carcinomas in situ and invasive squamous cell carcinomas, there was a progressive disruption of the epidermal junction which became marked upon frank invasion. Most of the differences between the various categories of pathological change, therefore, were not absolute but rather of degree, supporting the notion that invasive squamous cell carcinoma represents an end stage for malignancy which may arise de novo, directly from hyperplastic skin, or proceed from other tumour types. The similarity in structure of the mouse tumours to comparable tumours in man supports the usefulness of the animal model and suggests that the results have implications for sunlight associated tumours in man.