Ch Gallagher

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.

D-Amino acid residue in the C-type natriuretic peptide from the venom of the mammal, Ornithorhynchus anatinus, the Australian platypus.

The C‐type natriuretic peptide from the platypus venom (OvCNP) exists in two forms, OvCNPa and OvCNPb, whose amino acid sequences are identical. Through the use of nuclear magnetic resonance, mass spectrometry, and peptidase digestion studies, we discovered that OvCNPb incorporates a D‐amino acid at position 2 in the primary structure. Peptides containing a D‐amino acid have been found in lower forms of organism, but this report is the first for a D‐amino acid in a biologically active peptide from a mammal. The result implies the existence of a specific isomerase in the platypus that converts an L‐amino acid residue in the protein to the D‐configuration.

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.

NMR studies of diffusional water permeability of red blood cells from macropodid marsupials (kangaroos and wallabies)

1. The water permeabilities of the red blood cell (RBC) membranes of five species of macropodid marsupials were monitored by using a Mn(2+)-doping 1H nuclear magnetic resonance (NMR) technique. 2. Since this appears to be the first time that this type of measurement at 400 MHz for 1H has been reported, an analysis of instrumental parameters influencing the estimated value of the water exchange time (Te), and of the diffusional water permeability (Pd), was performed on samples of human RBC. 3. It was found that a short interpulse delay in the Carr-Purcell-Meiboom-Gill pulse sequence had to be used (around 100 microseconds) to avoid an underestimation of the relaxation times, that occurred as the result of molecular diffusion through non-uniform local magnetic fields in and around the cells. 4. There were no significant differences, in the water permeabilities of the RBC membranes, between the five species (Macropus rufogriseus, M. parma, M. eugenii, M. parryi and Wallabia bicolor). 5. There were also no significant differences between various colonies of M. eugenii living in different habitats. 6. The average values of Pd were 9.1 x 10(-3) cm/sec at 24.6 degrees C, 10.4 x 10(-3) cm/sec at 30 degrees C, 12.6 x 10(-3) cm/sec at 37 degrees C, and 14.7 x 10(-3) cm/sec at 42.1 degrees C; these were more than twice as high as those for human RBC. 7. In agreement with the high water permeability the RBC of macropodids displayed a relatively low activation energy of water diffusion across their membranes, approximately 21 kJ/mol, compared with 25 kJ/mol for human RBC.

Conformations of platypus venom C-type natriuretic peptide in aqueous solution and sodium dodecyl sulfate micelles

Nuclear magnetic resonance spectroscopy was used to investigate the conformations of the platypus venom C-type natriuretic peptide A (OvCNPa) in aqueous solutions and in solutions containing sodium dodecyl sulfate (SDS) micelles. The chemically synthesized OvCNPa showed a substantial decrease in flexibility in aqueous solution at 10 degrees C, allowing the observation of medium- and long-range nuclear Overhauser enhancement (NOE) connectivities. Three-dimensional structures calculated using these data showed flexible and reasonably well-defined regions, the locations of which were similar in the two solvents. In aqueous solution, the linear part that spans residues 3-14 was basically an extended conformation while the cyclic portion, defined by residues 23-39, contained a series of beta-turns. The overall shape of the cyclic portion was similar to that observed for an atrial natriuretic peptide (ANP) variant in aqueous solution. OvCNPa adopted a different conformation in SDS micelles wherein the N-terminal region, defined by residues 2-10, was more compact, characterised by turns and a helix, while the cyclic region had turns and an overall shape that was fundamentally different from those structures observed in aqueous solution. The hydrophobic cluster, situated at the centre of the ring of the structure in aqueous solution, was absent in the structure in the presence of SDS micelles. Thus, OvCNPa interacts with SDS micelles and can possibly form ion-channels in cell membranes.

Nmr studies of diffusional water permeability of erythrocytes from eight species of marsupial

Abstract 1. 1.The diffusional water permeability (Pd) of the red blood cell (RBC) membranes of eight species of marsupial were monitored by using a Mn2+-doping 1H nuclear magnetic resonance (NMR) technique at 400 MHz (9.4T). 2. 2.There were differences in the water permeability of the RBC membranes among the species investigated. 3. 3.On the basis of parameters characterizing the RBC water permeability the animals could be divided in two groups. 4. 4.For the first group, including the large macropodid marsupials, eastern grey kangaroo (Macropus giganteus) and red kangaroo (M. rufus), the values of Pd were 5.4–6.1 × 10−3 cm/sec at 24.6°C; 6.6–7.2 × 10−3 cm/sec at 30°C; 7.8–9.1 × 10−3cm/sec at 37°C and 9.3°-11.1 × 10-3 cm/sec at 42.1°C. 5. 5.The values of the activation energy for the diffusion process (Ea,d) ranged from 24–27 kJ/mol. 6. 6.For the second group, including bandicoot (Isoodon macrourus), bilby (Macrotis lagotis sagitta), Tasmanian devil (Sarcophilus harrisii), koala (Phascolarctis cinerens), brush tailed possum (Trichosurus vulpecula), and Goodfellows tree kangaroo (Dendrolagus goodfellowi), the values of Pd were higher, ranging from 8.1–10.4 × 10−3 cm/sec at 24.6°C; 8.9–11.8 × 10−3 cm/sec at 30°C; 10.3–13.7 × 10−3cm/sec at 37°C and 11.9–15.3 × 10−3cm/sec at 42.1°C. 7. 7.In parallel with the high water permeability the RBC of the marsupials in the second group exhibited lower values of Ea,D ranging from 15–21 kJ/mol, except for Goodfellows tree kangaroo, for which the Ea,D was ~ 25 kJ/mol.

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)

Metabolism of [14C]benzo[a]pyrene in vivo in the rat

[14C]Benzo[a]pyrene ([14C]B[a]P) injected intraperitoneally into rats appeared rapidly in liver, lung and kidney, and remained detectable in these tissues for at least 7 days. A large proportion (7--13%) of the 14 C became covalently bound to tissue macromolecules, probably primarily proteins. Subcellular organelles of the liver were all found to bind the carcinogen, the microsomes most rapidly and the light mitochondrial fraction taking up 14C later. Nuclear bound 14C was detected in both liver and lung. Purification of the cytosolic 14C from liver revealed specific binding to the same cytosolic proteins purified from the in vitro reaction of [14C] B[a]P.

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.

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.