Cultured cells as a model system for the study of UV-induced cytotoxicity.

In vivo, UV radiation induces a series of morphological and ultrastructural alterations in human epidermis. These and other changes eventually lead to well described pathological modifications including erythema and cancer. Morphological alterations are easier to detect in cultured cells, such as human keratinocytes or other epithelial cells. One can use different intensities of different radiation types (UV-A, -B and -C) and expose cell monolayers to different doses. In these experimental conditions it is possible to evaluate radiation risks and to provide additional information thanks to the reproducibility and the enormous amplification of the phenomena normally occurring in vivo. Alterations observed in structural studies can be summarized as the succession of the following events: (i) cell retraction with loss of cell-cell interactions; (ii) surface blebbing; and eventually (iii) cell death. Cytoskeletal components play a key role in this cascade. Morphogenesis of these changes can be ascribed to oxidative modifications due to reactive oxygen species formation following radiation that can modify both cell membrane and cytoskeleton. The use of in vitro systems can be of great relevance in the understanding of the pathogenetic mechanisms of UV radiation changes and to determine possible drugs capable of counteracting UV-mediated subcellular pathology.

Individual variations in the correlation between erythemal threshold, UV-induced DNA damage and sun-burn cell formation.

A linear correlation between erythema intensity and DNA damage upon exposure to UV has not been firmly established. Many of the deleterious effects of UV exposure do occur after exposure to suberythemal doses. After DNA damage, cells undergo DNA repair. It is commonly accepted that when the burden of damage is beyond the repair capacities, the cell undergoes programmed cell death or apoptosis. The aim of this study is to quantify the amount of UV-induced DNA damage (estimated via the measurement of DNA repair or unscheduled DNA synthesis or UDS) and cellular damage (estimated via the determination of the density of sunburn cells or SBC). If DNA damage and erythema are correlated, similar intensity of UDS and similar density of SBC should be found in volunteers irradiated with a UV dose equal to two minimal erythema doses (MED). Our results show that in 15 different individuals the same relative dose (2 MEDs) provokes UDS values, which vary within a factor of 4. An even larger variability affects SBC counts after the same relative dose. When DNA damage or SBC are plotted versus the absolute dose (i.e. the dose expressed in J/m(2)), there is a rough correlation (with several exceptions) between dose and extent of UDS and SBC counts. It seems possible to divide the volunteers into two subpopulations with different susceptibilities to UV damage. It is well known that UDS and SBC measurements are often affected by large experimental indeterminacy, yet, the analysis of our results makes it plausible to suggest that for the triggering of erythema, a common threshold value for DNA damage or for SBC count are not to be found. In conclusion, the erythema response seems to be loosely correlated with DNA damage. This suggests that the protection offered by the sunscreens against DNA damage, the molecular basis of UV-induced mutagenesis, might not be related to the sun protection factor (SPF) indicated on the label of sunscreens, which is evaluated using the erythema as an endpoint.

Optimizing the energy status of skin cells during solar radiation.

Ionizing- and ultraviolet-radiation cause cell damage or death by directly altering DNA and protein structures and by production of reactive oxygen species (ROS) and reactive carbonyl species (RCS). These processes disrupt cellular energy metabolism at multiple levels. The formation of DNA strand breaks activates signaling pathways that consume NAD, which can lead to the depletion of cellular ATP. Poly(ADP)-ribose polymerase (PARP-1) is the enzyme responsible for much of the NAD degradation following DNA damage, although numerous other PARPs have been discovered recently that await functional characterization. Studies on mouse epidermis in vivo and on human cells in culture have shown that UV-B radiation provokes the transient degradation of NAD and the synthesis of ADP-ribose polymers by PARP-1. This enzyme functions as a component of a DNA damage surveillance network in eukaryotic cells to determine the fate of cells following genotoxic stress. Additionally, the activation of PARP-1 results in the activation of a nuclear proteasome that degrades damaged nuclear proteins including histones. Identifying approaches to optimize these responses while maintaining the energy status of cells is likely to be very important in minimizing the deleterious effects of solar radiation on skin.

Factors of skin ageing share common mechanisms.

Ageing has been defined as the accumulation of molecular modifications which manifest as macroscopic clinical changes. Human skin, unique among mammalians insofar as it is deprived of fur, is particularly sensitive to environmental stress. Major environmental factors have been recognized to induce modifications of the morphological and biophysical properties of the skin. Metabolites from ingested or inhaled substances do affect skin, which is also sensitive to endogenous hormone levels. Factors as diverse as ultraviolet radiation, atmospheric pollution, wounds, infections, traumatisms, anoxya, cigarette smoke, and hormonal status have a role in increasing the rate of accumulation of molecular modifications and have thus been termed 'factors of ageing'. All these factors share as a common feature, the capability to directly or indirectly induce one of the steps of the micro-inflammatory cycle, which includes the expression of ICAM-1 in endothelial cells. This triggers a process leading to the accumulation of damages in the skin resulting in skin ageing since ICAM-1 expression provokes recruitment and diapedesis of circulating immune cells, which digest the extracellular matrix (ECM) by secreting collagenases, myeloperoxidases and reactive oxygen species. The activation of these lytic processes provokes random damage to resident cells, which in turn secrete prostaglandines and leukotrienes. These signaling molecules induce the degranulation of resident mast cells which release the autacoid histamine and the cytokine TNF-alpha thus activating endothelial cells lining adjacent capillaries which release P-selectin and synthesize ICAM-1. This closes a self-maintained micro-inflammatory cycle, which results in the accumulation of ECM damage, i.e. skin aging. In this paper we review the evidence that two factors able to induce macroscopical and molecular modifications in the skin, protein glycation and stretch, activate the micro-inflammatory cycle. We further present evidence that three additional factors, two external factors (electromagnetic fields and psychological stressors) and one internal factor (neuropeptides) also activate the micro-inflammatory cycles and may therefore be considered as factors of skin ageing.

Aging of human skin: review of a mechanistic model and first experimental data.

The physical, chemical, and biochemical factors that accelerate skin aging have been proposed to activate a self-maintained microinflammatory process, one of the expected end results of which is an imbalance in the turnover of macromolecules in the dermis. Surface peroxides are recognized as controllable factors of skin aging, and their accumulation is attributed to environmentally induced impairment of defense enzymes. Topical application of antioxidants decreases the rate at which skin elasticity and skin thickness are modified.

Vitamin E prevents UVB-induced cell blebbing and cell death in A431 epidermoid cells.

Cultured A431 epidermoid cells exposed to UVB (120-2400 J/m2) develop numerous blebs on their surface, detach from the plastic dish, and undergo injury and death. Numerous detached cells display fragmented nuclei, typical of apoptotic cells. Since bleb formation also occurs after oxidative stress it was assumed that the morphological variations observed are the consequence of free radical-mediated insult. In order to test this hypothesis, the antioxidant alpha-tocopherol (vitamin E) was added to cell cultures at different times, before or after irradiation. The results indicate that vitamin E inhibits UVB-induced surface blebbing as well as cell detachment from the substrate. Moreover, vitamin E is most effective in stimulating cell recovery when it is added after the end of UVB irradiation. Finally, vitamin E treatment also seems to reduce the fraction of cells undergoing death (probably those which will undergo apoptosis) after exposure to UVB radiation.

Both UVA and UVB induce cytoskeleton-dependent surface blebbing in epidermoid cells.

Data on the morphological changes induced by UVA or UVB irradiation of A431 epidermoid cells in culture are presented. After irradiation with different doses of UVB (120-2400 J m-2) or UVA (10(4)-10(5) J m-2), the membrane and cytoskeleton of these cells were analysed by immunofluorescence and scanning electron microscopy at different times after exposure (0-48 h). Both UVA and UVB alter microtubules and microfilaments and surface blebs are formed after UV irradiation. In particular, UVB induces multiple small blebs on the cells, while UVA induces one single large bleb on each cell. Since cytoskeletal damage and surface blebbing of this type are also induced by oxidative stress, these results add to the body of evidence indicating that UV radiation is capable of pro-oxidant behaviour. Specifically, the morphological changes described in this paper are reminiscent of the modifications which accompany epidermal keratinocytes during their transformation to sunburn cells after UV irradiation. The physiological implications of these findings are discussed.

Modulation by L-histidine of H2O2-mediated damage of cellular and isolated DNA.

The mechanism by which L-histidine modulates H2O2-induced damage to DNA has been investigated by alkaline and neutral gel electrophoresis of cellular DNA, by measuring the conversion of purified supercoiled DNA to its relaxed and linear forms and by the ESR spin-trapping technique. L-Histidine greatly increased the amount of H2O2-mediated DNA single-strand breaks. DNA double-strand breaks were produced only in cells exposed to H2O2 and L-histidine. The addition of a cell permeable chelator such as o-phenanthroline (unlike EDTA, DTPA and desferrioxamine) prevented both DNA single- and double-strand breakage induced by H2O2 plus L-histidine. In vitro, the profile of the dose-response curve for the ferrous iron-mediated, H2O2-dependent DNA nicking was modified by the addition of L-histidine. At low H2O2 concentrations, corresponding to the maximum extent of DNA cleavage, L-histidine was protective. At higher H2O2 concentrations L-histidine enhanced the formation of DNA single-stand breaks and produced DNA double-strand breaks. The increase in H2O2-mediated DNA nicking by L-histidine depended on the L-histidine:Fe(II) ratio, the maximal rate occurring at a molar ratio of 10(3):1 and being independent of the concentration of DNA. Thus, it appeared that intracellular iron mediated both DNA single- and double-strand breaks induced by H2O2 plus L-histidine. Results of ESR experiments seemed to rule out the involvement of the hydroxyl radical by itself in DNA cleavage mediated by the L-histidine:Fe(II):H2O2 system.

L-histidine-mediated enhancement of hydrogen peroxide-induced cytotoxicity: relationships between DNA single/double strand breakage and cell killing.

Results presented in this study demonstrate an association between the L-Histidine-mediated enhancement of H2O2-induced cytotoxicity and the formation of DNA double strand breakage (DSB), whereas no relationship exists between the increased cytotoxic response and DNA single strand breakage (SSB). Indeed, the higher lethality and the production of DNA DSB occurred in oxidatively-injured cells regardless of whether the exposure to L-Histidine was performed before or during challenge with the oxidant. In fact, the increased level of DNA SSB detected in cells simultaneously exposed to the oxidant and the amino acid was not observed in cells pre-treated with L-Histidine and then challenged with hydrogen peroxide. Further experiments have demonstrated an association between the kinetics of DNA DSB formation and the enhancement of the cytotoxic response. In conclusion, intracellular L-Histidine seems to mediate the formation of DNA DSB and the increased growth-inhibitory response elicited by the oxidant. In addition, these results suggest that the enhancement of DNA SSB is produced by the extracellular/plasma membrane fraction of the amino acid and not causally related to the L-Histidine-mediated increase of the growth-inhibitory response to H2O2-treated cells.

Protonation of the imidazole ring prevents the modulation by histidine of oxidative DNA degradation.

When supercoiled DNA is incubated with Fe(II) at pH 7 in the presence of hydrogen peroxide, the rate of nicking first increases with increasing H2O2 concentration to reach a maximum, then decreases and eventually increases again. When 0.1 mM histidine is added at neutral pH at low H2O2 concentration (< 3 mM), it hinders the nicking of DNA; when it is added at high H2O2 concentrations (> 10 mM), it enhances the rate of nicking. When similar experiments are performed at slightly acidic pH (4.5) the biphasic behavior is maintained, independent of the presence of histidine. One can conclude that the protonation of imidazole (pK = 5.9) abolishes the capability of histidine to modulate the oxidative degradation of DNA. Results of electron spin resonance experiments suggest that at low H2O2 concentration, the protective effect of histidine could be the consequence of its capability to bind OH. radicals.

Differential effects of histidine on hydrogen peroxide-induced bacterial killing and DNA nicking in vitro.

The hydrogen peroxide dose-response curves for Escherichia coli killing and DNA nicking in vitro display remarkably similar bimodal patterns. The concentrations of the oxidant resulting in maximum mode one killing, however, exceeds by two orders of magnitude those resulting in the mode one DNA nicking response. Addition of histidine differentially affects the experimental curves describing the dose-dependency for bacterial killing and DNA damage in vitro. Indeed, the lethal effect elicited by the oxidant in the presence of the amino acid is strictly concentration-dependent and thus the inactivation curve loses its bimodal character. In marked contrast, histidine abolishes DNA damage generated by low concentrations of hydrogen peroxide (< 100 microM) in the in vitro system (the mode one DNA nicking response) but greatly increases DNA damage produced by concentrations of the oxidant higher than 1 mM (the mode two DNA nicking response). Experimental results also suggest that treatment of covalently closed circular double-stranded super-coiled DNA with hydrogen peroxide, in the presence of both histidine and iron, may result in the formation of DNA double strand breakage, a type of lesion which is not efficiently produced by the oxidant in the absence of the amino acid. Taken together, the above results indicate that histidine differentially affects the in vitro DNA cleavage and E. coli lethality induced by hydrogen peroxide and suggest that different molecular events mediate mode one DNA nicking in vitro and mode one killing of bacterial cells.

DNA nicking by ultraviolet radiation is enhanced in the presence of iron and of oxygen.

Double-stranded covalently closed circular supercoiled DNA (ccc DNA) from plasmid pUK 9 was irradiated in vitro at defined wavelengths in the UV region (290, 313 and 365 nm). The nicking was monitored by electrophoresis on agarose gels, ethidium staining and densitometric quantitation of supercoiled and relaxed moieties. At the explored wavelengths, the dose required for introducing one nick per million phosphodiester bonds diminishes with increased concentration of added ferric iron, whereas the effect of cupric iron is practically negligible. Adding metal chelators or bubbling argon prior to the irradiation results in a dramatic increase in the dose required for introducing one nick per million phosphodiester bonds. Taken together, these results seem to indicate that iron and oxygen play a role as cofactors in the UV-induced nicking of ccc DNA in vitro.

Modulation of DNA breakage induced via the Fenton reaction.

The conversion of the covalently closed circular double-stranded supercoiled DNA (pBR322) to a relaxed circle was used to investigate DNA nicking induced by Fe2+ and H2O2. In our experimental conditions of ionic strength (150 mM NaCl), pH = 7 and temperature (37 degrees C), the dose-response curve for the ferrous iron mediated H2O2 dependent DNA nicking is peculiar. For a fixed concentration of ferrous iron (2 microM), the concentration of H2O2 producing a maximum extent of DNA nicking was about 10-30 microM. The DNA single-strand breakage decreased with an increase of H2O2 concentration. We have investigated the effects of several factors such as the nature of the buffer, ionic strength, temperature and pH. Buffer components leading to the autoxidation of ferrous iron to ferric iron (phosphate) or to the scavenging of reactive oxygen species (Tris) greatly alter the dose-response curve. The H2O2 concentrations required for producing the maximum extent of DNA single-strand breaks at 4 degrees C and 56 degrees C were respectively 30 microM and 3 microM. At pH = 10, the pattern of the dose-response curve was totally different. The data showed that the peculiar dose-response curve for the ferrous iron mediated H2O2 dependent DNA nicking greatly depended on the experimental conditions.

L-glutamine prevents the L-histidine-mediated enhancement of hydrogen peroxide-induced cytotoxicity.

Results presented in this study demonstrate that L-glutamine, a competitive inhibitor of L-histidine uptake, inhibits in a concentration-dependent fashion the L-histidine-mediated enhancement of H2O2-induced cytotoxicity. L-Glutamine also prevents the induction of DNA double strand breaks (DSB) but does not affect the enhancing effect of L-histidine on DNA single strand break induction by H2O2. Taken together, these data demonstrate that L-histidine, in order to allow the formation of DNA double strand breakage and increase the toxicity elicited by the oxidant, has to enter the cell. In addition, these results indicate that the enhancement of DNA single strand breakage is a consequence of the action of the amino acid at the extracellular level and/or outer surface of the plasma membrane and does not appear related to the mechanism whereby L-histidine increases the cytotoxic response to H2O2. The latter mechanism very likely involves the formation of DNA DSB.