Skin Of Control Animals Research Articles

58P - Melanoma affects clock gene machinery of several organs in tumor-bearing mice

Background: The accumulation of soluble factors in the tumor microenvironment and their release into the bloodstream lead to systemic effects, resulting in cancer-associated syndromes such as cachexia. Another association observed in the latter scenario is chrono-disruption, which has been related with tumor onset and development. In fact, a positive relationship between master clock integrity and healthiness has already been disclosed; however, it is unclear whether the effects of chronodisruption in cancer are locally or systemically exerted. It is known that lung adenocarcinoma reprograms liver metabolism via a pro-inflammatory response without affecting liver clock genes. Our study evaluated whether a non-metastatic skin tumor can affect clock gene machinery of other organs. Methods: Eight to sixteen-week old C57BL/6J male mice were inoculated subcutaneously with B16-F10 cells (or PBS, control animals), single housed at 22 ± 2 °C, kept under 12/12h light/dark cycle, and received food and water ad libitum. Two weeks after inoculation mice were euthanized 2 hours after lights on (ZT2) or 2 hours after lights off (ZT14). Skin of control animals and samples of non-tumoral adjacent skin, tumor, liver, lung, and brown adipose tissue (BAT) were collected to evaluate the expression of clock genes (Per1, Per2, Bmal1, and Nr1d1) by qPCR. Results: No oscillatory profile of clock genes was detected in skin of control animals, tumor adjacent skin, and tumor itself of inoculated animals; Bmal1 expression was reduced in adjacent skin and tumor as compared to skin of control animals. In liver and lung tissue, Per1 and Per2 oscillated in control animals, and tumor inoculation did not affect this oscillatory profile. Temporal oscillation of Bmal1 and Nr1d1 in the liver was lost in tumor-bearing mice. In BAT, Per1 and Bmal1 oscillatory expression was also lost in tumor-bearing mice. In all organs analyzed. Bmal1 transcript was reduced in tumor-bearing mice when compared to control animals. Conclusions: The presence of a non-metastatic tumor in the skin alters clock machinery in adjacent skin, liver, lungs, and BAT. These data bring new knowledge of how tumor macro-environment affects clock machinery, resulting in a likely chrono-disruption of the organism as a result of a localized tumor in the skin. Legal entity responsible for the study: University of Sao Paulo (USP) and Sao Paulo Research Foundation (FAPESP) Funding: Sao Paulo Research Foundation (FAPESP) and National Council of Technological and Scientific Development (CNPq). Disclosure: All authors have declared no conflicts of interest.

Increased expression of GAP-43 in small sensory neurons after stimulation by NGF indicative of neuroregeneration in capsaicin-treated rats

Intraplantar injections of human recombinant nerve growth factor (rhNGF-β) into the hind paw of capsaicin-treated adult rats are known to lead to a recovery of depleted peptide transmitter substances, to the immunohistochemical reappearance of peptidergic innervation in the skin and in the dorsal horn of the spinal cord, as well as to a recovery of the function of capsaicin-lesioned neurons. In the present study a marker peptide for neuronal regeneration and outgrowth, growth associated protein 43 (GAP-43), was investigated in lumbar dorsal root ganglia (DRGs) and in the hindpaw skin, in order to differentiate which population of the sensory neurons responds with a neuroregenerative behaviour. In situ hybridization histochemistry (ISH) revealed that at day 8 after the capsaicin treatment GAP-43 expression was significantly increased in small DRG cells as compared to control animals, and treatment with NGF in capsaicinized rats lead to an even more pronounced increase of GAP-43 expression in the small-sized cell population. Intraepidermal labelling of GAP-43 peptide was observed in the skin of control animals, but was markedly reduced in the animals that were treated with capsaicin alone. However, intraepidermal GAP-43 immunoreactive (GAP-43-IR) fibres nearly fully recovered in the capsaicin+NGF-treated group. These results indicate that the population of small DRG cells shows spontaneous regenerative activity after a capsaicin lesion which does not lead to a successful recovery of nerve terminals in the skin. Only after an additional NGF treatment small DRG cells show an even stronger regenerative response which now also involves structural reorganization of neuron membranes and axogenesis in the periphery.

GAP-43 immunoreactivity is enhanced after UV irradiation in the peripheral nervous system of the rat

UV irradiation evoked thermal and mechanical hypersensitivity in the glabrous skin of the rat hind paw. During the period of hyperalgesia skin samples were taken from irradiated areas of the skin and the corresponding segments of the spinal cord, and the expression of growth-associated protein 43 (GAP-43) was quantitatively measured. Although GAP-43 was present in a small number of fibers in the skin of control animals, the total length of GAP-43 positive fibers per unit area was significantly increased within 3 days after irradiation in both ipsi- and contralateral hind limbs. The time course of GAP-43 expression corresponded to that of mechanical hyperalgesia. GAP-43 immunopositivity was at its maximum on day 3 after UV irradiation and declined to control level at day 14. Increased GAP-43 immunoreactivity was similar in both hind paws except on day 3 when it was significantly more on the ipsilateral side. Most GAP-43-positive fibers were arborizing branches in the skin, but strong positivity was found around blood vessels and around sweat glands. Glyoxylic acid-induced fluorescent-catecholamine marking was associated with blood vessels without extravascular sprouting in control or UV-irradiated skin. At 3 and 5 days after UV irradiation GAP-43-positive profiles were present in the spinal L4-5 segments. GAP-43 immunoreactivity was detected on both sides in the dorsal horn with greater intensity on the ipsilateral side. We conclude that the increased expression of GAP-43 in neuronal processes of the ipsi- and contralateral paw after UV irradiation is most unlikely to be directly associated with nerve sprouting. GAP-43 expression occurred mainly in sensory fibers.

Influence of Inorganic and Organic Dietary Zinc on Its Concentration in Blood Serum, Bones and Hair, and Catalytical Activity of Selected Serum Enzymes in Pigs

Rupic. V .. L. Ivandija. S. Luterotti. M. Dominis-Kramaric: lnfiuence of Inorganic and Organic Dietary Zinc on its Concentration in Blood Serum, Bones and Hair, and on Catalytical Activity of SOllie Sen/III En~Yllles in Pigs. Acta vet. Bmo 1997.66: 75-85. The influence of dietary zinc originating from either inorganic or organic source on zincdepleted pigs was investigated. Within the 30 d period preceding the trial. all the animals (German Landrace x Pietrain x Large White x Swedish Landrace weaned crossbreds) were depleted of zinc and divided into three groups. 14 animals in each. During the experimental fattening period (105 d). the control group received no extra zinc. whereas the two trial groups were fed a diet with zinc supplement either as inorganic salt (ZnS04) or metalo-organic chelate (zinc methionate). On average. the respective supplements contained 84.3 mg Zn.kg· l diet and 40.9 mg Zn.kgl diet. Evaluation of the nutritional effects of zinc was based on its concentration in the blood serum, bone and hair. Moreover. the effect of zinc on catalytical activity of alkaline phosphatase. aspartate aminotransferase. alanine aminotransferase and gamma-glutamyltransferase in serum was studied. A decrease of catalytical activity of both aspartate aminotransferase and alanine aminotransferase was found along with signs of parakeratosis on the skin of control animals but no correlation with serum zinc was established. In contrast to this finding. a linear correlation between catalytical activity of alkaline phosphatase and of gamma-glutamyltransferase and serum zinc level was found with the respective correlation coefficients of 0.90 and -0.72. Nutritional efficiency of diet zinc was evidenced by both higher (P < 0.05) zinc level in blood serum. bone and hair and by the state of hepatobiliary tract and bones. and favourable performance results of trial animals compared to controls. None of trial animals suffered from any health disturbances. Progressive parakeratosis appeared. however. in control animals and lasted during the second month of the trial. It was accompanied by deterioration of performance data of the affected animals. Our data suggest that bioavailability of zinc from the chelate was better than of that derived from inorganic salt. particularly in the period of rapid growth. This presumbaly resulted in reduced elimination of zinc. Based on better nutritional efficiency in pigs and on ecological benefits. zinc methionate should be preferred to ZnS04 as a zinc containing diet additive. Growth, pigs. ~illc sulphate. ~illc methionate. bioavailability, nutrition The improvement of body mass gain and carcass quality are among major goals of all pork producers. One of the ways to achieve this is to provide the animals with necessary minerals from the sources of favourable bioavailability. Circulation of zinc and its biological importance in animals (cellular replication. lung tissue CO, release, sexual maturation, wound healing. fertility and reproduction) have been extensively investigated. Zinc is an essential trace element, and it is an integral part of some 300 enzymes (carbonic anhydrase, alkaline phosphatase, alcohol dehydrogenase, RNA and DNA polymerases, 5-nucleotidase, etc.) which take part in protein synthesis, metabolism of carbohydrates and nucleic acids (Mi I ne 1994). It is assumed that Zn derived from compounds with amino acids stimulates

Inhibition of hair growth by subcutaneous injection of a sympathetic neurotoxin, 6-hydroxydopamine in neonatal mice

The effects of a sympathetic neurotoxin, 6-hydroxydopamine (6-OHDA), on hair growth in neonatal mice were examined. Newborn mice were injected once subcutaneously in the mid-dorsal region with 6-OHDA (0.3 mg/g body weight) and bovine serum albumin (BSA) or with BSA only (controls) on day 0. By day 10, distinct areas of hairless skin were observed surrounding the sites treated with 6-OHDA. The areas of hairless skin were smaller at 15 days of age and were covered with hair by 20 days of age. Control sites injected with BSA only were indistinguishable from the surrounding skin at all ages examined. Microscopic and morphometrical analyses of skin obtained from the neonatal mice at various ages showed that the hairless skin in 6-OHDA-treated mice was thinner than the skin of control animals. The thinning of the skin and delay in hair growth induced by 6-OHDA treatment showed a significant difference from the skin of control animals injected with BSA only. Immunohistochemical experiments demonstrated that the administration of 6-OHDA had caused the complete depletion of tyrosine hydroxylase-immunoreactive fibers (sympathetic fibers) around blood vessels and piloerector muscles and in nerve bundles throughout the dermis and subcutaneous tissue. These findings indicate that the sympathetic neurons are associated with skin thickness and hair growth in neonatal mice.

Human recombinant interleukin-1 beta up-regulates elastin gene expression in dermal fibroblasts. Evidence for transcriptional regulation in vitro and in vivo.

The effects of human recombinant interleukin (IL)-1 beta on elastin gene expression were studied in human skin fibroblast cultures by Northern hybridization and transient transfection experiments. Incubation of the cells with IL-1 beta elevated the elastin mRNA steady-state levels by approximately 3- to 4-fold. A similar increase was noted at the protein level, when estimated by indirect immunofluorescence of cultured cells. This effect was independent of the on-going protein synthesis, as tested by incubation with cycloheximide. Transient transfections of the dermal fibroblasts with a human elastin promoter/chloramphenicol acetyltransferase (CAT) reporter gene construct suggested transcriptional regulation, since the CAT activity in cells incubated with IL-1 beta was similarly increased approximately 3-fold. Enhancement of the human elastin promoter activity by IL-1 beta was also noted in fibroblast cultures established from the skin and lungs of transgenic mice which have integrated the human promoter/CAT construct into their genome and express it in a tissue-specific manner. Furthermore, subcutaneous injection of IL-1 beta to the mice resulted in a approximately 4-fold elevation of the CAT activity in the skin after a 30-h incubation, as compared to the CAT activity in the skin of control animals. Collectively, these data indicate that IL-1 beta up-regulates elastin gene expression in fibroblast cultures as well as in the skin in vivo, and the activation occurs at the transcriptional level.

Prolactin and thyroid hormone interactions in salt and water balance in the newt Notophthalmus viridescens

Hypophysectomy lowered plasma sodium in summer newts to approximately 80% of control levels. Treatment of hypophysectomized animals with corticosterone, prolactin, or corticosterone + prolactin did not correct the pituitary deficiency. Thyroxine treatment (0.1 or 0.5 μg every other day for 2 weeks) of intact summer newts depressed plasma sodium to approximately 40% of control values. Thyroxine treatment of winter animals (0.1 μg every other day for 2 weeks) depressed plasma sodium to 90% of control values. In addition, thyroxine treatment of winter animals depressed urine sodium, more than doubled the rate of molting, and increased the osmotic permeability of the skin from 71 to 103 μl water/cm 2/hr. Newts treated with both thyroxine (0.1 μg every other day) and prolactin (10 μg every other day) had levels of plasma sodium identical to controls in both summer and winter experiments. In the winter experiment, prolactin + thyroxine treatment lowered urine sodium, more than doubled molting rate, decreased osmotic permeability of the skin (from 70 to 48 μl water/cm 2/hr) and decreased short-circuit current (from 30 to 16 μA/cm 2) in isolated skin preparations. In addition, the skin of animals receiving both prolactin and thyroxine was more slippery than the skin of control animals or of animals treated with thyroxine alone, suggesting that they were secreting excess mucus. It is suggested that the effects of these hormones on the skin may at least partially account for their effects on plasma and urine sodium.