POSSIBLE ROLE OF METALLOTHIONEIN IN THE CELLULAR DEFENSE MECHANISM AGAINST UVB IRRADIATION IN NEONATAL HUMAN SKIN FIBROBLASTS

Abstract: The role of metallothionein (MT) in protecting skin cells against UVB irradiation was investigated. Fibroblast strains from normal adult (HS-K) and neonatal (NB1RGB) human skins as well as keratinocyte strains from human skin (SV40-HSK) and newborn Balb/c mouse skin (Pam 212) were exposed to UVB irradiation.
The sensitivity of HS-K and NB1RGB cells to UVB irradiation was similar; those of SV40-HSK and Pam 212 cells were two- and six-fold as sensitive to UVB irradiation as HS-K cells, respectively. The HS-K cells contained the greatest cellular reduced form of glutathione (GSH) levels compared to the three other skin cells: the levels were 13-, 7- and 6-fold of those in NB1RGB, SV40-HSK and Pam 212 cells, respectively. These results indicated that the sensitivity of skin cells to UVB irradiation was not always associated with their endogenous GSH levels. In particular, despite the fact that NB1RGB cells contained a relatively small amount of GSH, they were less sensitive to UVB irradiation.
NB1RGB cells contained 4–30 times more MT than those in other skin cells examined. The sulfhydryl residues of MT molecules in the NB1RGB cells were estimated to be mostly unoccupied by metals, suggesting they act in a similar way to those of GSH. Moreover, NB1RGB cells in which the MT content was elevated by dexamethasone (1 μ M ) or Zn²⁺ (7 μg/mL) treatment were more resistant to UVB irradiation than nontreated ones.
These results suggest that, at least in neonatal human skin fibroblasts, MT may play a role in protection against UVB irradiation.     

ENDOGENOUS GLUTATHIONE PROTECTS HUMAN SKIN FIBROBLASTS AGAINST THE CYTOTOXIC ACTION OF UVB, UVA AND NEAR-VISIBLE RADIATIONS

Both the UVB (290-320 nm) and UVA (320-380 nm) regions of sunlight damage human skin cells but, particularly at the longer wavelengths, information is scant concerning the mechanism(s) of damage induction and the roles of cellular defense mechanisms. Following extensive glutathione depletion of cultured human skin fibroblasts, the cells become strongly sensitized to the cytotoxic action of near-visible (405 nm), UVA (334 nm, 365 nm) and UVB (313 nm) but not UVC (254 nm) radiations. In the critical UVB region, the magnitude of the protection afforded by endogenous glutathione approaches that of the protection provided by excision repair. The results suggest that a significant fraction of even UVB damage can be mediated by free radical attack and that a major role of glutathione in human skin cells is to protect them from the cytotoxic action of sunlight.     

MEMBRANE DAMAGE INDUCED IN CULTURED HUMAN SKIN FIBROBLASTS BY UVA IRRADIATION

Irradiation of cultured human skin fibroblasts with ultraviolet light from 320 to 400 nm (UVA) leads to a decrease in the membrane fluidity exemplified by an enhanced fluorescence anisotropy of the lipophilic fluorescent probe 1-[4-trimethylamino)-phenyl]-6-phenylhexa-1,3,5-triene. This UVA-induced decrease in fluidity is associated with lactate dehydrogenase leakage in the supernatant. Vitamin E, an inhibitor of lipid peroxidation, exerts a protective effect on both phenomena. Therefore, this UVA-induced damage in membrane properties may be related to lipid peroxidation processes. Moreover, exponentially growing cells are more sensitive to these UVA-induced alterations than confluent cells.     

A POSSIBLE MECHANISM FOR THE ROLE OF N-RETINYLIDENE PHOSPHATIDYL ETHANOLAMINE IN THE REGENERATION OF RHODOPSIN

Abstract— A series of molecular orbital calculations on a model Schiff base comparable to protonated N -retinylidene phosphatidyl ethanolamine isomers has been made. The effect of the charged oxygen atoms of the phosphate moiety on the distribution of positive charge along the polyene chain of these isomers has been calculated. The stabilizing coulombic energy of interaction of these opposite charges and the possibility of free rotation around carbonxarbon double bonds in the electronically excited state has led to the conclusion that an 11- cis Schiff base isomer is the most probable product of the photoisomerization of an all-trans Schiff base.
The formation of a stable unprotonated all-trans Schiff base in aqueous detergent dispersion and its subsequent conversion to the protonated form, both with absorption spectra in conformity with the literature, is demonstrated.     

ACTIVATION OF NF-KB IN HUMAN SKIN FIBROBLASTS BY THE OXIDATIVE STRESS GENERATED BY UVA RADIATION

We have examined the role of the nucleus and the membrane in the activation of nuclear factor (NF)-KB by oxidant stress generated via the UVA (320–380nm) component of solar radiation. Nuclear extracts from human skin fibroblasts that had been irradiated with UVA at doses that caused little DNA damage contained activated NF-KB that bound to its recognition sequence in DNA. The UVA radiation-dependent activation of NF-KB in enucleated cells confirmed that the nucleus was not involved. On the other hand, UVA radiation-dependent activation of NF-KB appeared to be correlated with membrane damage, and activation could be prevented by a-tocopherol and butylated hydroxytol-uene, agents that inhibited UVA radiation-dependent peroxidation of cell membrane lipids. The activation of NF-KB by the DNA damaging agents UVC (200–290nm) and UVB (290–320nm) radiation also only occurred at doses where significant membrane damage was induced, and, overall, activation was not correlated with the relative levels of DNA damage induced by UVC/UVB and UVA radiations. We conclude that the oxidative modification of membrane components may be an important factor to consider in the UV radiation-dependent activation of NF-KB over all wavelength ranges examined.     

PROTECTIVE EFFECT OF SELENIUM AND ZINC ON UV-A DAMAGE IN HUMAN SKIN FIBROBLASTS

Ultraviolet A radiation participates in cytotoxicity and carcinogenesis of the skin by a mechanism involving the generation of reactive oxygen species. Endogenous antiradical defense systems utilize metalloenzymes including Se-dependent glutathione peroxidase and Cu and Zn superoxide dismutase. The aim of the present work was to determine the protective effect of two trace elements, Se and Zn, on cultured human diploid fibroblasts exposed to UV-A radiation (broad-spectrum source with a maximum intensity at 375 nm). Selenium in the culture medium (0.1 mg/L) in the form of sodium selenite increased the synthesis and activity of glutathione peroxidase by 60.5% in the absence of exposure to UV-A radiation and by 35% after irradiation with 5 J/cm² ( P = 0.043). The presence of this element significantly increased the survival of UV-A-irradiated fibroblasts ( P < 0.0001). This confirms the essential role of Se in the detoxifying activity of the enzyme. In addition, thiobarbituric acid-reacting substances (TBAR), which are lipid peroxidation markers, decreased in the presence of exogenous Se:—19% and -22% without irradiation and after irradiation with 5 J/cm² ( P = 0.056). When Zn was added at the dose of 6.5 mg/L as ZnCl₂, fibroblasts subjected to oxidizing stress induced by UV-A were protected from cytotoxicity ( P <0.0001). The TBAR production decreased significantly: -33% without irradiation and -34% after irradiation with 5 J/cm² ( P = 0.008). Superoxide dismutase activity, however, decreased after supplementing with Zn: - 26% without irradiation and - 20% after UV-A irradiation ( P = 0.017). The antioxidant properties of Zn are thus apparently independent of superoxide dismutase activity.     

CORRELATION OF UVC AND UVB CYTOTOXICITY WITH THE INDUCTION OF SPECIFIC PHOTOPRODUCTS IN T-LYMPHOCYTES AND FIBROBLASTS FROM NORMAL HUMAN DONORS

Abstract— By using specific monoclonal antibodies in situ and a computer-assisted image analysis system we have determined the relative induction of cyclobutane dimers, (6–4) photoproducts and Dewar isomers in human mononuclear cells and fibroblasts following irradiation with UVC, broad-spectrum UVB and narrow-spectrum UVB. The lamps produced these lesions in different proportions, with broad-spectrum UVB inducing a greater combined yield of (6–4) photoproducts and Dewar isomers per cyclobutane dimer than UVC or narrow-spectrum UVB. The relative induction ratios of (6–4) photoproducts compared to cyclobutane dimers were 0.15, 0.21 and 0.10 following irradiation with UVC, broad- or narrow-spectrum UVB, respectively. Although Dewar isomers were induced by UVC, their relative rate of formation compared to cyclobutane dimers was significantly greater after irradiation with either broad-spectrum or narrow-spectrum UVB. These values were 0.001, 0.07 and 0.07, respectively. With each lamp source, we have determined the survival of normal human T-lymphocytes and fibroblasts at fiuences, which induce equivalent yields of cyclobutane dimers, (6–4) photoproducts or (6–4) photoproducts plus Dewar isomers. Killing of fibroblasts appears to be associated with (6–4) photoproduct formation, whereas killing of T-lymphocytes seems to be mediated by combined (6–4) plus Dewar yields. These results emphasize the need to study the biological effects of UVB because cellular responses may be different from those following UVC irradiation.     

DNA-PROTEIN CROSSLINKING IN NORMAL HUMAN SKIN FIBROBLASTS EXPOSED TO SOLAR ULTRAVIOLET WAVELENGTHS

Three normal human skin fibroblast cell lines were exposed to the simulated solar UV radiation produced by a fluorescent sunlamp under conditions in which the wavelength components shorter than either 295, 305 or 315 nm were excluded. The level of DNA-protein crosslinks (DPC) was then measured in those cells using the alkaline elution technique either immediately after irradiation or following a 24 h incubation. In each case, cells were exposed to fluences that induce similar levels of DPC. For cells exposed to 10 kJ m(-2) of sunlamp UV > 295 nm, the level of DPC exhibited a 2-5-fold increase following incubation. In contrast, 40-100% of the DPC were removed upon incubation of cells irradiated with either 100 kJ m(-2) of sunlamp UV > 305 nm or 150 kJ m(-2) of sunlamp UV > 315 nm. A major difference between the effects induced by these wavelength regions is that, in addition to DPC, a very high level of pyrimidine dimers is also produced by sunlamp UV > 295 nm, whereas much lower dimer yields result from treatment with either sunlamp UV > 305 nm or sunlamp UV > 315 nm. A potential role for type II DNA topoisomerase in the formation of these DPC resulting from either the change in conformational structure caused by the presence of a high level of dimers or an involvement of this enzyme in dimer excision repair is discussed.     

INVESTIGATIONS ON THE MECHANISM OF CHLORPROMAZINE PHOTOTOXICITY: EFFECTS ON LYSOSOMES OF CULTURED HUMAN FIBROBLASTS

Abstract The effect of chlorpromazine (CPZ) and UVA on lysosomes of cultured normal human fibroblasts has been investigated. Acid phosphatase (ACPase) activity in 12 000 g pellet of cells treated with CPZ (10 μg/m l ) and UVA (6 × 10⁴ J/m²) was found to be decreased as compared with non-treated, CPZ or UVA treated control cells. This decrease, however, was not accompanied by a concomitant increase in ACPase activity in the 12 000 g supernatant. The addition of Triton X-100 to cells pretreated with CPZ + UVA resulted in only a moderate increase in ACPase activity of the 12 000 g supernatant. ACPase activity of the cells incubated in media containing preirradiated CPZ was also found to he decreased. These results indicate that CPZ + UVA directly inactivate lysosomal enzymes, possibly without affecting the membrane.     

SUNLIGHT-INDUCED PYRIMIDINE DIMERS IN HUMAN SKIN FIBROBLASTS IN COMPARISON WITH DIMERIZATION AFTER ARTIFICIAL UV-IRRADIATION

Abstract— We compared artificial UV-sources such as germicidal- or sun-lamps with summer noon sunlight in Switzerland for selective efficiency in the induction of pyrimidine dimers in the DNA of human cells. In our studies we determined cytosine-thymine (C-T) as well as thymine-thymine dimer densities (T-T) by high pressure liquid chromatography in cultures of xeroderma pigmentosum cells of group A. Using far-UV light from a germicidal lamp, we found a rate of formation per Jirr² for C-T and T-T of 0.0019% and 0.0024%, respectively, of the total thymine radioactivity in hydrolysates of [³H]thymidine labeled cells. After irradiation with an unfiltered sunlamp we measured a rate of formation of 0.0005% per Jm-² both for C-T and T-T, based on the sunlamp emission of 297 ±4 nm wavelength. Utilization of Kodacel- or Mylar-filters lowered the rate of dimerization by a factor of 2 and 60, respectively. One hour of irradiation with noon summer sunlight induced 0.038 ±0.012% C-T and 0.036 ±0.011% T-T. This extent of dimer production is equivalent to 15 Jm^-2 of far-UV exposure at 254 nm.     

EARLY ALTERATIONS OF ACTIN IN CULTURED HUMAN KERATINOCYTES AND FIBROBLASTS EXPOSED TO LONG-WAVELENGTH RADIATIONS. POSSIBLE INVOLVEMENT IN THE UVA-INDUCED PERTURBATIONS OF ENDOCYTOTIC PROCESSES

Abstract Exposure of cultured MRC5 human fibroblasts or NCTC 2544 human keratinocytes to mild doses of ultraviolet A (UVA: 320-400 nm) radiations markedly decreased the actin reactivity with fluorescein-labeled phalloidin. This indicates a change in the degree of poly merization of actin and thus in the organization of actin filaments. Such a phenomenon might be involved in the previously reported UVA-induced inhibition of specific and nonspecific endocytotic processes.     

TWO GENES CONTRIBUTE TO DIFFERENT EXTENTS TO THE HEME OXYGENASE ENZYME ACTIVITY MEASURED IN CULTURED HUMAN SKIN FIBROBLASTS AND KERATINOCYTES: IMPLICATIONS FOR PROTECTION AGAINST OXIDANT STRESS

Abstract— Activation of expression of the heme oxygenase (HO) gene appears to be involved in a cellular defense system in mammalian cells. We now demonstrate that while HO-1 mRNA levels are strongly inducible in dermal fibroblasts they are barely inducible in human epidermal keratinocytes following oxidative stress (UVA radiation and hydrogen peroxide). Paralleling this result was the observation that HO-2 mRNA levels were low in dermal fibroblasts but were high in epidermal keratinocytes. In neither case was the HO-2 gene inducible. The expression of the two HO genes led to enzymatic activity in both types of skin cells with an approximately 2.5-fold higher level of enzymatic activity present in keratinocytes compared with fibroblasts derived from the same biopsy. In addition, ferritin levels, which have been found to be augmented via the HO-dependent release of iron from endogenous heme sources, were two- to three-fold higher in keratinocytes compared with matching fibroblasts. This higher ferritin pool would result in an enhancement of cellular iron sequestering capacity that may confer increased resistance to oxidative stress. Indeed, keratinocytes showed less UVA radiation-dependent cell membrane damage than fibroblasts. These results are consistent with the hypothesis that HO expression in human epidermis and dermis is related to cellular defense mechanisms that operate in human skin.     

THE EFFECT OF THE ANTIPSORIATIC DRUG METABOLITE ETRETIN (Ro 10-1670) ON UVB IRRADIATION INDUCED CHANGES IN THE METABOLISM OF ARACHIDONIC ACID IN HUMAN KERATINOCYTES IN CULTURE

[¹⁴C]Arachidonic acid was avidly incorporated into human keratinocytes in culture and following exposure to UVB irradiation of 9 mJ/cm² (erythemally effective, EE) substantial amounts of ¹⁴C-radiolabel were released from the cells. The release of radiolabel was accompanied by a decrease in the labelling of phosphatidylethanolamine whereas the labelling of triacylglycerols and cholesteryl esters was increased. Keratinocytes produced significant amounts of prostaglandin E₂ (PGE₂) and following UVB irradiation of 9 mJ/cm² (EE) the formation of prostaglandin E₂ was increased.
Etretin (Ro 10-1670), the active metabolite of the antipsoriatic drug etretinate (Ro 10-9359), affected significantly neither the total release of radiolabel induced by UVB nor the formation of prostaglandin E₂. However, in the presence of etretin the UVB irradiation induced transfer of [^l4C]arachidonic acid into triacylglycerols and cholesteryl esters was not increased as much as in the corresponding experiments without etretin. On the basis of the present study it appears that etretin does not interfere with the release of arachidonic acid in amounts which could be related to the therapeutic effects of the combination of retinoids with UVB irradiation (Re-UVB) in the treatment of psoriasis.     

UNSCHEDULED DNA SYNTHESIS: A QUANTITATIVE INDICATOR OF RESIDUAL IMMUNODETECTABLE PYRIMIDINE DIMERS IN HUMAN FIBROBLASTS AFTER ULTRAVIOLET-B IRRADIATION

We have addressed the question whether the level of UV-B induced DNA damage can be accurately assessed by the measurement of the rate of unscheduled DNA synthesis (UDS). Cultured human fibroblasts were irradiated with UV radiation at 290, 313 or 365 nm. The LD50 was 85 J/m2 at 290 nm, 4500 J/m2 at 313 nm, and 70 kJ/m2 at 365 nm. The analysis of UDS measurements indicate complete arrest of repair processes within 24 h after irradiation, irrespective of the dose (in the range 10-60 J/m2 at 290 nm, and 250-1000 J/m2 at 313 nm). Irradiation at 365 nm failed to yield detectable evidence of UDS. Incubation of irradiated cells with an antiserum directed against both 6-4 type and cyclobutane-type pyrimidine dimers shows a clear parallelism between the disappearance of the antibody-binding determinants and the variation of the rate of UDS vs time after the end of the irradiation. Thus it is concluded that in UV-B irradiated normal cultured human fibroblasts, the lack of UDS reflects the absence of immunodetectable pyrimidine dimers.     

PHOTOSENSITIZED CARCINOGEN DEGRADATION AND THE POSSIBLE ROLE OF SINGLET OXYGEN IN CARCINOGEN ACTIVATION

Abstract— Dye sensitized photo-oxidation of various chemical carcinogens including 2-acetylaminofluorene, benzo(a)pyrene, 9,10-dimethyl-1,2-benzanthracene and 4-nitroquinoline- N -oxide has been studied in the presence and absence of oxygen. Visible light from a filtered 150 W Xe arc was used for photolysis. and carcinogen destruction was monitored spectrophotometrically. The carcinogens were solubilized in aqueous solution within micelles of various lipid-like surfactants. Photosensitized destruction of carcinogens by several triplet sensitizers has been observed. Enhanced damage in the presence of oxygen is also found. These results are consistent with photochemical oxidative damage to carcinogens being produced in aerated solutions via the sensitizer triplet state, possibly involving singlet oxygen. These manifold reactions are catalysed by micelles which concentrate polycyclic hydrocarbons in the non-polar environment within the micelles where the exclusion of water probably increases reactive species lifetimes. This model photochemical system mimics the aerobic metabolic activation of chemical carcinogens, which in part is believed to be mediated by various forms of activated oxygen.     

PHOTOREPAIR OF PYRIMIDINE DIMERS IN HUMAN SKIN IN VIVO

Abstract— The exposure of human skin in vivo to UV radiation emitted from a sunlamp induces the formation of pyrimidine dimers. The number of dimers, as detected by UV-endonuclease, decreases following exposure of the UV–irradiated skin to visible wavelengths of light. These results suggest that humans possess a mechanism by which pyrimidine dimers are photorepaired upon illumination of human skin in vivo with visible light.     

Dawson结构钨钒磷杂多化合物催化苯酚过氧化氢的羟化作用及其羟化机理

合成了系列Ｄａｗｓｏｎ结构钨钒磷杂多化合物（Ｃｐｙｒ）７＋ｎＰ２Ｗ１８－ｎＶｎＯ６２（ｎ＝１～３），并用ＩＲ，ＮＭＲ等手段对其结构进行了表征．考察了这类杂多化合物对苯酚过氧化氢的羟化活性，探讨了反应机理．     

THE EFFECT OF LASER IRRADIATION ON THE RELEASE OF bFGF FROM 3T3 FIBROBLASTS

Studies have shown that low-level laser irradiation increases the proliferation of fibroblasts in cell culture. The mechanism of action is unknown. Basic fibroblast growth factor (bFGF) is a multifunctional polypeptide that has been detected in most tissues and which supports cell proliferation and differentiation. The purpose of this study was to determine whether laser irradiation (660 nm) can stimulate production of bFGF from fibroblast cells in cell culture. Our study showed that fibroblasts irradiated with laser energy at 2.16 J/cm² demonstrated increased cell proliferation and enhanced production of bFGF, whereas fibroblasts irradiated with laser energy at 3.24 J/cm² neither demonstrated increased cell proliferation or an enhanced release of bFGF as compared to the control group. These results provide direct evidence that the proliferation of fibroblasts as a result of stimulation by low level laser irradiation may be associated with the autocrine production of bFGF from fibroblasts.     

MECHANISM OF OXYGEN DETOXIFICATION IN NEONATAL RAT LUNG TISSUE

Abstract— Pulmonary macrophages obtained from neonatal rats contain approximately four times the activity of cyanide resistant superoxide dismutase and catalase compared with the cells from adult animals. The activity is highest immediately after birth and diminishes with age until the minimum level is reached at approximately 3 weeks of age. Superimposed upon this change in basal activity is the capability of neonatal cells to synthesize additional cyanide resistant superoxide dismutase and catalase when either the animals or isolated pulmonary macrophages are exposed to 95–100% oxygen. The inductive effect begins at 2–3 days after birth, peaks at 10 days, and disappears at approximately 15 days after birth. In contrast to adult rats, neonatal rats are extremely resistant to the toxic effects of oxygen. If, however, the oxygen mediated increase in both enzymes is prevented or the maximum effective pulmonary macrophage number is diminished in test animals, these animals become vulnerable to toxic effects of oxygen exposure, observed by gross and histologic examination of lung tissue, in a manner similar to adult animals. These data indicate that both cyanide-resistant superoxide dismutase and catalase may be part of the endogenous defense mechanisms which provide neonatal rats with an exceptional resistance to oxygen toxicity.