Santamarine Shows Anti-Photoaging Properties via Inhibition of MAPK/AP-1 and Stimulation of TGF-β/Smad Signaling in UVA-Irradiated HDFs

Chronic UVA exposure results in elevated reactive oxygen species in skin which leads to photoaging characterized as upregulated matrix metalloproteinase (MMP)-1 and loss of collagen. Therefore, natural antioxidants are hailed as promising agents to be utilized against photoaging. In the current study, reynosin and santamarine, two known sesquiterpene lactones isolated from Artemisia scoparia, were analyzed for their anti-photoaging properties in UVA-irradiated human dermal fibroblasts (HDFs). Results showed that UVA irradiation (8 J/cm²) upregulated the MMP-1 secretion and expression, and suppressed collagen production, which were significantly reverted by santamarine treatment (10 µM). Although both reynosin and santamarine exhibited ROS scavenging abilities, reynosin failed to significantly diminish UVA-stimulated MMP-1 release. UVA-irradiated HDFs showed increased collagen production when treated with santamarine. As a mechanism to suppress MMP-1, santamarine significantly suppressed the UVA-induced phosphorylation of p38 and JNK and nuclear translocation of p-c-Fos and p-c-Jun. Santamarine promoted collagen I production via relieving the UVA-induced suppression on TGF-β and its downstream activator Smad2/3 complex. Antioxidant properties of santamarine were also shown to arise from stimulating Nrf2-dependent expression of antioxidant enzymes SOD-1 and HO-1 in UVA-irradiated HDFs. In conclusion, santamarine was found to be a promising natural antioxidant with anti-photoaging properties against UVA-induced damages in HDFs.     

LONG-WAVE ULTRAVIOLET RADIATION INDUCES PROTEIN KINASE C IN NORMAL HUMAN KERATINOCYTES

Abstract Skin tumor promotion by phorbol ester is believed to be mediated by the phospholipid-dependent ser/ thr kinase, protein kinase C (PKC). Long-wave ultraviolet radiation (320-400 nm, UVA), which has also been shown to promote skin tumors, induces elevated levels of PKC in murine fibroblasts, suggesting that UVA may promote the development of basal and squamous cell skin cancers by a mechanism involving PKC. To examine UVA effects on PKC in a model relevant to skin, we maintained normal human epidermal keratinocytes (NHEK) in serum-free medium and exposed the cultured cells to various doses of UVA or to the phorbol ester, 12- O -tetradecanoylphorbol-13-acetate (TPA). Fifty minutes after exposure to UVA (5-20 J/cm²), PKC activity was elevated up to three-fold in NHEK cytosolic fractions, and membrane-associated PKC activity was elevated up to two-fold by UVA. The TPA treatment induced a 10-fold increase in membrane-associate PKC activity only. Immunoblot analysis suggested that a UVA-induced increase in PKC protein occurred. Both UVA and TPA reduced the cell number by 50-75% in the first 24-48 h; however, irradiated cultures began to recover at 72 h post-UVA due to an increased proliferative rate beginning after 48 h. Treatment with TPA induced a high level of differentiation as measured by cornified envelope formation. Ultraviolet A irradiation exposure was not followed by increased differentiation. These findings suggest that acute UVA exposure elevates PKC activity in human keratinocytes and may act through PKC to promote actinic skin cancer. The molecular mechanism is like to differ from that of the phorbol esters, however.     

Red Light Interferes in UVA‐Induced Photoaging of Human Skin Fibroblast Cells

The possible regulation mechanism of red light was determined to discover how to retard UVA‐induced skin photoaging. Human skin fibroblasts were cultured and irradiated with different doses of UVA, thus creating a photoaging model. Fibroblasts were also exposed to a subtoxic dose of UVA combined with a red light‐emitting diode (LED) for five continuous days. Three groups were examined: control, UVA and UVA plus red light. Cumulative exposure doses of UVA were 25 J cm^?2, and the total doses of red light were 0.18 J cm^?2. Various indicators were measured before and after irradiation, including cell morphology, viability, β‐galactosidase staining, apoptosis, cycle phase, the length of telomeres and the protein levels of photoaging‐related genes. Red light irradiation retarded the cumulative low‐dose UVA irradiation‐induced skin photoaging, decreased the expression of senescence‐associated β‐galactosidase, upregulated SIRT1 expression, decreased matrix metalloproteinase MMP‐1 and the acetylation of p53 expression, reduced the horizon of cell apoptosis and enhanced cell viability. Furthermore, the telomeres in UVA‐treated cells were shortened compared to those of cells in the red light groups. These results suggest that red light plays a key role in the antiphotoaging of human skin fibroblasts by acting on different signaling transduction pathways.     

Measurement of lens protein aggregation in vivo using dynamic light scattering in a guinea pig/UVA model for nuclear cataract

The role of UVA radiation in the formation of human nuclear cataract is not well understood. We have previously shown that exposing guinea pigs for 5 months to a chronic low level of UVA light produces increased lens nuclear light scattering and elevated levels of protein disulfide. Here we have used the technique of dynamic light scattering (DLS) to investigate lens protein aggregation in vivo in the guinea pig/UVA model. DLS size distribution analysis conducted at the same location in the lens nucleus of control and UVA-irradiated animals showed a 28% reduction in intensity of small diameter proteins in experimental lenses compared with controls (P < 0.05). In addition, large diameter proteins in UVA-exposed lens nuclei increased five-fold in intensity compared to controls (P < 0.05). The UVA-induced increase in apparent size of lens nuclear small diameter proteins was three-fold (P < 0.01), and the size of large diameter aggregates was more than four-fold in experimental lenses compared with controls. The diameter of crystallin aggregates in the UVA-irradiated lens nucleus was estimated to be 350 nm, a size able to scatter light. No significant changes in protein size were detected in the anterior cortex of UVA-irradiated lenses. It is presumed that the presence of a UVA chromophore in the guinea pig lens (NADPH bound to zeta crystallin), as well as traces of oxygen, contributed to UVA-induced crystallin aggregation. The results indicate a potentially harmful role for UVA light in the lens nucleus. A similar process of UVA-irradiated protein aggregation may take place in the older human lens nucleus, accelerating the formation of human nuclear cataract.     

PSORALEN-PHOTOSENSITIZED DAMAGE OF RAT PERITONEAL EXUDATE CELLS

Psoralen-sensitized photodamage (PUVA) of rat peritoneal exudate cells was investigated. Quartz-activated luminol-dependent chemiluminescence (ChL) was registered and the amount of trypan-positive cells was determined. Irradiation of peritoneal exudate cells in the presence of psoralen resulted in a dose-dependent monotonous inhibition of ChL. The reciprocity law of irradiation intensity and duration of irradiation was not valid for the observed inhibition of ChL: the inhibition increased with higher intensity. When psoralen previously photooxidized in ethanol (POP) was added to peritoneal exudate cell suspension, a double-phase response depending on psoralen irradiation dose was obtained: ChL activation was observed at low doses of UVA, ChL inhibition at high doses. Chemiluminescence inhibition correlated well with the increase in the number of trypan-positive cells. It may be supposed that the observed effects of PUVA or POP treatment are caused by cell cytoplasmic membrane damage.     

Suppression of an established immune response by UVA--a critical role for mast cells

Exposing experimental animals or human volunteers to UVA II (320-340 nm) radiation after immunization suppresses immunologic memory and the elicitation of delayed-in-time hypersensitivity reactions. Previous studies indicated that the mechanisms underlying UVA-induced immune suppression are similar to those described for UVB-induced immune suppression, i.e. transferred by T regulatory cells, overcome by repairing DNA damage, neutralizing interleukin (IL)-10 activity, or injecting recombinant IL-12. Here we continued our examination of the mechanisms involved in UVA II-induced suppression. Antibodies to cis-urocanic acid blocked UVA-induced immune suppression. Treating UVA-irradiated mice with histamine receptor antagonists, calcitonin gene-related peptide (CGRP) receptor antagonists or platelet activating factor receptor antagonists blocked immune suppression in UVA-irradiated mice. In light of the fact that cis-urocanic acid and CGRP target mast cells, which can then release platelet activating factor and histamine, we measured UVA-induced immune suppression in mast cell-deficient mice. No immune suppression was noted in UVA-irradiated mast cell-deficient mice. These findings indicate that exposure to UVA II activates many of the same immune regulatory factors activated by UVB to induce immune suppression. Moreover, they indicate that mast cells play a critical role in UVA-induced suppression of secondary immune reactions.     

COMPARISON OF EFFECTS OF UVA, UVB AND UVC ON GROWTH AND VIABILITY OF MITOGEN-STIMULATED HUMAN PERIPHERAL BLOOD CELLS

Abstract— Peripheral blood mononuclear cells were irradiated with UVA, UVB or UVC. The highest exposure dose used in each waveband reduced the number of viable cells to one-third the control cell population after 3 days in culture. Exposure of these cells to half as much UV from each waveband resulted in an equivalent or greater degree of inhibition of their proliferative response to mitogen as measured by lymphoblast transformation, [³H]-thymidine uptake and viable cell number on day 3 in culture. The pattern of inhibition was distinct for each waveband. UVA interfered with blastogenesis on the first 2 days of culture at doses which had considerably less effect on viable cell number. UVA also depressed the first round of DNA synthesis, which was detectable on the second day of culture. By day 3 in culture, however, the UVA-induced reduction in both the number of lymphoblasts and the uptake of [³H]-thymidine was a direct reflection of reduced numbers of viable cells. UVB did not interfere with blastogenesis in mitogen-stimulated cultures to the same degree as did UVA. Only the highest dose of UVB depressed blast transformation more than viable cell number on day 1; by day 2 lower doses were also inhibitory. In contrast UVC had little effect on blastogenesis at any time; a reduced number of lymphoblasts observed on days 2 and 3 in culture was a direct reflection of a reduced number of viable cells rather than a reduced percent of these cells undergoing blast transformation. As with UVA-irradiated, mitogen-stimulated cells, [³H]-thymidine uptake was also depressed in both UVB and UVC irradiated, mitogen-stimulated cells on day 2. However, only UVB continued to depress DNA synthesis more than viable cell number after 3 days of culture. These results suggest that UVA, UVB and UVC may interfere with any one or more of the signals involved in the response to mitogen, be they the recognition of mitogen by T cells or accessory cells, the transformation of lymphocytes into lymphoblasts or the activation of lymphoblasts to synthesize DNA.     

UVA‐Induced DNA Damage and Apoptosis in Red Blood Cells of the African Catfish Clarias gariepinus

Ultraviolet‐A light (UVA)‐induced DNA damage and repair in red blood cells to investigate the sensitivity of African catfish to UVA exposure is reported. Fishes were irradiated with various doses of UVA light (15, 30, and 60 min day⁻¹ for 3 days). Morphological and nuclear abnormalities in red blood cells were observed in the fish exposed to UVA compared with controls. Morphological alterations such as acanthocytes, crenated cells, swollen cells, teardrop‐like cells, hemolyzed cells, and sickle cells were observed. Those alterations were increased after 24 h exposure to UVA light and decreased at 14 days after exposure. The percentage of apoptosis was higher in red blood cells exposed to higher doses of UVA light. No micronuclei were detected, but small nuclear abnormalities such as deformed and eccentric nuclei were observed in some groups. We concluded that exposure to UVA light induced DNA damage, apoptosis, and morphological alterations in red blood cells in catfish; however, catfish were found to be less sensitive to UVA light than wild‐type medaka.     

Differential Activation of Signaling Pathways by UVA and UVB Radiation in Normal Human Epidermal Keratinocytes(†)

Ultraviolet (UV) radiation from the solar spectrum is a major etiological factor for many cutaneous pathologies including cancer. By understanding changes in cell signaling pathways induced by UVA and UVB, novel strategies for prevention and treatment of UV‐related pathologies could be developed. However, much of the information in the literature from various laboratories cannot cross talk because of difficulties associated with the use of ill‐defined light sources and physiologically irrelevant light dosimetry. Herein, we have assessed the effect of exposure of normal human epidermal keratinocytes (NHEK) to UVA (2 and 4 J cm^?2) or UVB (20 and 40 mJ cm^?2) radiation. Employing western blot analysis, we found that exposure of NHEK to UVB, but not UVA, phosphorylates JNK1/2 at Th¹⁸³/Tyr¹⁸⁵, STAT3 at Ser⁷²⁷, AKT at Ser⁴⁷³ and increases c‐Fos expression, whereas exposure to UVA, but not UVB, phosphorylates AKT at Thr³⁰⁸. UVB as well as UVA exposure leads to increased phosphorylation of (1) ERK1/2 at Th²⁰²/Tyr²⁰⁴; (2) p38 at Th¹⁸⁰/Tyr²⁰⁴; (3) STAT3 at Tyr⁷⁰⁵; (4) mTOR at Thr²⁴⁴⁸; and (v) p70S6k at Thr⁴²¹/Ser⁴²⁴; enhanced expression of PI3K (p85) and c‐jun; and nuclear translocation of NFκB proteins. These findings could be considered as a beginning for understanding the differential effects of UVA and UVB in the human skin and may have implications both with respect to risk assessment from exposure to solar UV radiation, and to target interventions against signaling events mediated by UVA and UVB.     

Extracorporeal Photochemotherapy (Photopheresis) Induces Apoptosis in Lymphocytes: A Possible Mechanism of Action of PUVA Therapy

Abstract— The mechanism of action of psoralen plus UVA (PUVA) and photopheresis is not entirely understood. These therapies are assumed to be immunomodulating partly by gradually decreasing leukocyte viability. We investigated whether this delayed form of cell death was due to apoptosis. Untreated and treated (PUVA exposed) leukocytes obtained from six patients with systemic sclerosis and (untreated) leukocytes from healthy control individuals were studied. Qualitative gel electrophoresis and quantitative in situ nick translation analysis of DNA fragmentation was performed. Apoptosis of the treated cells did occur (gel electrophoresis) after 24 h. At t = 0 h, immediately after exposure to PUVA, there was no evidence of DNA fragmentation in the treated cells. The percentage of treated cells undergoing apoptosis was 20–55% at t = 24 h ( in situ nick translation). The untreated leukocytes of the patients and the healthy individuals showed no distinctive rise in apoptotic cells. Apoptosis of the leukocytes after PUVA or photopheresis treatment might be a mechanism of action and might explain the therapeutic response.     

COMPARATIVE EFFECT OF UVA AND UVB ON CULTURED RABBIT LENS

Abstract Effects on lens physiology of UVB and UVA used separately and sequentially were investigated using 4 week old rabbit lenses in organ culture. Narrowband UVB at 0.3 J/cm²= joules/lens (1 h exposure) has little effect on sodium and calcium concentrations in the lens interior or transparency of lenses subsequently cultured for 20 h after a 1 h exposure. With an incident energy of 3 J/cm² of broadband UVB (295–330 nm), lenses become opaque and slightly swollen with significant ion imbalances during culture over a 1 day period. In contrast, lenses exposed to approximately 6–24 J/cm² of UVA (330–400 nm) remain transparent after 1 day of culture. Extended culture up to 4 days reveals no signs of opacification. Ion homeostasis and normal lens hydration are also maintained in UVA-irradiated lenses. The presence of 95% oxygen during UVA irradiation is also without effect. Broadband UVA irradiation is damaging, however, if lenses are first exposed to subthreshold doses of narrowband UVB (307 ± 5 nm) irradiation, viz . 0.3 J/cm². Thus, sequential UVB/UVA irradiation at subthreshold doses causes impaired active cation transport and accumulation of sodium and calcium accompanying lens opacification.     

The expression and role of protein kinase C in neonatal cardiac myocyte attachment, cell volume, and myofibril formation is dependent on the composition of the extracellular matrix.

The extracellular matrix (ECM) is a dynamic component of tissues that influences cellular phenotype and behavior. We sought to determine the role of specific ECM substrates in the regulation of protein kinase C (PKC) isozyme expression and function in cardiac myocyte attachment, cell volume, and myofibril formation. PKC isozyme expression was ECM substrate specific. Increasing concentrations of the PKC delta inhibitor rottlerin attenuated myocyte attachment to randomly organized collagen (1, 5, and 10 microM), laminin (5 and 10 microM), aligned collagen (5 and 10 microM), and fibronectin (10 microM). Rottlerin significantly decreased cell volume on laminin and randomly organized collagen, and inhibited myofibril formation on laminin. The PKC alpha inhibitor G? 6976 inhibited attachment to randomly organized collagen at 6 nM but did not affect cell volume. The general PKC inhibitor Bisindolylmalemide I (10 and 30 microM) did not affect myocyte attachment; however, it significantly decreased cell volume on randomly organized collagen. Our data indicate that PKC isozymes are expressed and utilized by neonatal cardiac myocytes during attachment, cell growth, and myofibril formation. Specifically, it appears that PKC delta and/or its downstream effectors play an important role in the interaction between cardiac myocytes and laminin, providing further evidence that the ECM influences cardiac myocyte behavior.     

Effect of UVA radiation on membrane fluidity and radical decay in human fibroblasts as detected by spin labeled stearic acids

The ultraviolet A (UVA) radiation component of sunlight (320-400 nm) has been shown to be a source of oxidative stress to cells via generation of reactive oxygen species. We report here some consequences of the UVA irradiation on cell membranes detected by electron paramagnetic resonance (EPR) spectroscopy. Paramagnetic nitroxide derivatives of stearic acid bearing the monitoring group at different depths in the hydrocarbon chain were incorporated into human fibroblasts membranes to analyze two main characteristics: kinetics of the nitroxide reduction and membrane fluidity. These two characteristics were compared for control and UVA-irradiated (0-250 kJ/m(2)) cells. The term relative redox capacity (RRC) was introduced to characterize and to compare free radical reduction measured by EPR with some well-known viability/clonogenicity tests. Our results showed that UVA-irradiation produces a more rigid membrane structure, especially at higher doses. Furthermore, we found that trends agree in survival measured by neutral red (NR), trypan blue (TB), and clonogenic efficiency compared with RRC values measured by EPR for low and medium exposure doses. Above 100 kJ/m(2), differences between these tests were observed. Antioxidant effect was modeled by alpha-tocopherol-acetate treatment of the cells before UVA irradiation. While NR, TB and clonogenicity tests showed protection at the highest UVA doses (>100 kJ/m(2)), results obtained with EPR measurements, both membrane fluidity and kinetics, or using MTT test did not exhibit this protective effect.     

Comparative effects of UVA and UVB irradiation on the immune system of fish

Aquatic organisms can be harmed by the current levels of solar ultraviolet radiation. We have recently shown that exposure of fish to UVB irradiation alters the functioning of the fish immune system, but the effects of UVA radiation are unknown. The present study continues this work by characterizing UVA irradiation-induced immunological changes in fish. Roach, a cyprinid fish, were exposed to a single dose of either UVA (3.6 J/cm2) or UVB (0.5 J/cm2) irradiation. Both irradiations suppressed transiently mitogen-stimulated proliferation of blood lymphocytes. UVA, but not UVB, decreased hematocrit, plasma protein, and plasma immunoglobulin levels and increased the proportions of blood cells classified as unidentified leukocytes, possibly consisting of UVA-damaged lymphocytes. UVB, but not UVA, altered the functioning of head kidney and blood phagocytes, induced granulocytosis and lymphocytopenia in the blood and increased plasma cortisol concentration. These results imply that both UVA and UVB are potent modulators of the immune defence of fish.     

Detection and Prevention of Ocular Phototoxicity of Ciprofloxacin and Other Fluoroquinolone Antibiotics†

Fluoroquinolone (FLQ) drugs are a potent family of antibiotics used to treat infections including ocular infections. To determine if these antibiotics may be phototoxic to the eye, we exposed human lens epithelial cells to 0.125–1 mm FLQs (ciprofloxacin [Cipro], lomefloxacin [Lome], norfloxacin [Nor] and ofloxacin [Ofl]), the precursor quinolone nalidixic acid (Nalid) and UVA radiation (2.5 J cm⁻²). Based on fluorescence confocal microscopy, FLQs are diffused throughout the cytoplasm and preferentially located in the lysosomes of lens epithelial cells. Neither FLQ exposure alone nor UVA exposure alone reduced cell viability. However, with exposure to UVA radiation the FLQs studied (Cipro, Nor, Lome and Ofl) induced a phototoxic reaction that included necrosis, apoptosis, loss of cell viability as measured by MTS, and membrane damage as determined by the lactate dehydrogenase assay. Both Nalid and all FLQs studied (Cipro, Nor, Lome and Ofl) photopolymerized the lens protein α-crystallin. Phototoxic damage to lens epithelial cells and/or α-crystallin will lead to a loss of transparency of the human lens. However, if precautions are taken to filter all UV radiation from the eye while taking these antibiotics, eye damage may be prevented.     

Photo-oxidation of 6-Thioguanine by UVA: The Formation of Addition Products with Low Molecular Weight Thiol Compounds

The thiopurine, 6-thioguanine (6-TG) is present in the DNA of patients treated with the immunosuppressant and anticancer drugs azathioprine or mercaptopurine. The skin of these patients is selectively sensitive to UVA radiation—which comprises >90% of the UV light in incident sunlight—and they suffer high rates of skin cancer. UVA irradiation of DNA 6-TG produces DNA lesions that may contribute to the development of cancer. Antioxidants can protect 6-TG against UVA but 6-TG oxidation products may undergo further reactions. We characterize some of these reactions and show that addition products are formed between UVA-irradiated 6-TG and N-acetylcysteine and other low molecular weight thiol compounds including β-mercaptoethanol, cysteine and the cysteine-containing tripeptide glutathione (GSH). GSH is also adducted to 6-TG-containing oligodeoxynucleotides in an oxygen- and UVA-dependent nucleophilic displacement reaction that involves an intermediate oxidized 6-TG, guanine sulfonate (G^SO3). These photochemical reactions of 6-TG, particularly the formation of a covalent oligodeoxynucleotide–GSH complex, suggest that crosslinking of proteins or low molecular weight thiol compounds to DNA may be a previously unrecognized hazard in sunlight-exposed cells of thiopurine-treated patients.     

Cell Cycle Effects and Concomitant p53 Expression in Hairless Murine Skin after Longwave UVA (365 nm) Irradiation: A Comparison with UVB Irradiation

Abstract— Ultraviolet A (UVA,315–400 nm) radiation is known to be a complete carcinogen, but in contrast to UVB (280-315 nm) radiation, much of the cell damage is oxygen dependent (mediated through reactive oxygen species), and the dominant premutational DNA lesion(s) remains to be identified. To investigate further the basic differences in UVA and UVB carcinogenesis, we compared in vivo cellular responses, viz. cell cycle progression and transient p53 expression in the epidermis, after UVA1 (340-400 nm) exposure with those after broadband UVB exposure of hairless mice. Using flow cytometry we found a temporary suppression of bromodeoxyuridine (BrdU) uptake in S-phase cells both after UVB and UVA1 irradiation, which only in the case of UVB is followed by an increase to well over control levels. With equally erythemogenic doses (1-2 MED), the modulation of BrdU uptake was more profound after UVB than after UVA1 irradiation. Also, a marked transient increase in the percentage of S-phase cells occurred both after UVB and after UVA1 irradiation, but this increase evolved more rapidly after UVA1 irradiation. Further, p53 expression increased both after UVB and UVA1 irradiations, with peak expression already occurring from 12 to 24 h after UVA1 exposure and around 24 h after UVB exposure. Overall, UVA1 radiation appears to have less of an impact on the cell cycle than UVB radiation, as measured by the magnitude and duration of changes in DNA synthesis and cells in S phase. These differences are likely to reflect basic differences between UVB and UVA1 in genotoxicity and carcinogenic action.