Photophysical characteristics of N-substituted C5–C5′-linked dihydrothymine dimers (1a,b[meso], meso compounds of [5R,5′S]-bi-5,6-dihydrothymines; 1a,b[rac], racemic compounds of [5R,5′R]-bi-5,6-dihydrothymines and [5S,5′S]-bi-5,6-dihydrothymines) in aqueous solution with varying contents of less-polar aprotic solvent such as tetrahydrofuran or dioxane have been investigated by UV-absorption, and steady-state and time-resolved fluorescence spectroscopies. Among the C5–C5′-linked dimers, (5R,5′S)-bi-5,6-dihydro-1-methylthymine (1a[meso]) showed a redshifted weak UV-absorption band at 270–350 nm and excimer fluorescence emission at λ_max = 370 nm with a quantum yield (Φ_F) of ∼0.1 in phosphate buffer (pH < 10) at 293 K. Racemic compound of 5,6-dihydro-1-methylthymine dimer (1a[rac]), meso and racemic compounds of 5,6-dihydro-1,3-dimethylthymine dimers (1b[meso] and 1b[rac]) in phosphate buffer were nonfluorescent under similar conditions. The UV-absorption and fluorescence spectral characteristics of 1a[meso] in aqueous solution were interpreted in terms of intramolecular stacking interactions between the dihydropyrimidine chromophores leading to a preferential “closed-shell” conformation both in the ground state and the excited singlet state. In basic solutions at pH > pK_a = 11.7, the fluorescence quantum yield of 1a[meso] decreased due to a dominant “open-shell” conformation resulting from the electrostatic repulsion between the deprotonated dihydrothymine chromophores of 1a[meso] in a dianion form.

Azoaldolase is obtained from rabbit muscle aldolase by adding an azo chromophore to a cysteine side chain in each of the four enzyme subunits. The enzyme becomes photosensitive whereas both its catalytic activity and the michaelian kinetics are retained. Chromophore excitation causes E to Z isomerization of the azo bond, and mutually influences the protein–substrate equilibria. The various isomerization and substrate binding equilibria have been investigated under the hypothesis of a cyclic process described by four linked equilibrium constants. The mechanism of the light effect is a continuous adaptation of the specific parameters of the active protein, that is substrate recognition and rate of the catalyzed process. Absorbed light allows the rapid modification of the concentrations of various related molecules, depending on the used frequencies. At present such a mechanism has not been described in photobiology; so azoaldolase can be taken as a model for a possible new mechanism of light regulation of a biological system, based on changes in the molecular recognition by an active protein against its substrate.

The octahedral rhodium complex, cis-dichloro bis(1,10 phenanthroline)rhodium(III) chloride (BISPHEN), is known to form covalent linkages with DNA involving the attachment of the metal to a base. In order to determine the sequence selectivity of this chemistry, solutions of the complex containing one of the double-stranded DNA plasmids, pBSSK.c-raf (eco) or pBSKS .XE.LTR-F (a construct that contains sequences derived from the long terminal repeat [LTR] region of the human immunodeficiency virus) have been irradiated using UVA light. The DNA samples were denatured after irradiation, a primer was annealed to one of the strands, and a complementary strand was constructed using a polymerase enzyme. Polyacrylamide gel sequencing analysis was used to reveal stops created in the complementary strand caused by the polymerase encountering a metal-bound base. The data indicate that “hot spots” primarily occur at, or adjacent to, guanines (G), with a particularly strong preference for strings of G. In the latter case, the hottest spot is at the 5′G. These results are consistent with our previously postulated mechanism for the covalent binding chemistry which involves photooxidation of deoxyguanosine by the excited state of the metal complex as the primary photochemical step.

In order to develop monitoring and assessment systems of biologically effective doses of solar-UV radiation, concurrent measurements of spectral photometry and spore dosimetry were conducted in summer months at four sites in Japan and Europe. Effectiveness spectra were derived by multiplying spectral irradiance in 0.5 nm steps between 290 and 400 nm with the inactivation efficiency of the spores determined using monochromatic radiation of fine wavelength resolution. Shapes of the effectiveness spectra were very similar at the four sites exhibiting major peaks at 303.5, 305.0, 307.5 and 311.0 nm. The dose rates for spore inactivation from direct survival measurements and from calculations by the integration of the effectiveness spectra were compared for 174 data points. The ratios (observed/calculated) of the two values were concordant with a mean of 1.26 (±0.24 standard deviation [SD]). The possible causes for the variations and slightly larger observed values are discussed.

The ultraviolet-A (UVA) component of sunlight produces in cutaneous cells a highly toxic oxidative stress mediated by redox cycling reactions of Fe ions. A tight regulation of cell iron uptake and storage by iron regulatory proteins (IRP) of keratinocytes and fibroblasts avoids these damaging reactions. We report here that about 40 J/cm² of UVA are required to inactivate half of the binding capacity of apo-IRP-1 to iron responsive elements (IRE) of RNA whereas 15 J/cm² already inhibit half of the holo-IRP-1 aconitase activity. No increase in the holo-IRP-1 activity is observed during the apo-IRP-1 photoinactivation suggesting that UVA does not trigger a shift between these two forms. As opposed to holo-IRP-1, which contains a 4Fe–4S cluster, apo-IRP-1 has no UVA chromophore. Thus it should be inactivated indirectly by reactive oxygen species generated by the UVA-induced endogenous photo-oxidative stress. The apo-IRP-1 photoinactivation is weakly prevented by the lipophilic oxyradical scavenger vitamin E but not by the hydrophilic azide anion, a singlet oxygen quencher or by diethyldithiocarbamate, a superoxide dismutase inhibitor. However, full protection against photoinactivation of the apo form is observed after incubation with N-acetylcysteine but the latter only partially protects the aconitase function of the holo-IRP-1 from photoinactivation. The marked difference in the kinetics of photoinactivation of the apo and holo forms, the light dose–independent effect of the sulfhydril group reagent, 2-mercaptoethanol and the partial protection brought by the ferric ion complexing agent desferrioxamine suggest that the photochemistry of the 4Fe–4S cluster of the holo form plays little, if any, role in the photoinactivation of the apo-IRP-1/IRE interaction. It is concluded that the apo/holo equilibrium is irreversibly destroyed by UVA irradiation.

Solar irradiance measurements from Ushuaia (Argentina) and Palmer and McMurdo Stations in Antarctica covering four seasons from mid-1993 through early 1997 have been analyzed and their variations compared with column ozone changes. UV irradiances were weighted for biological effectiveness using a published biological weighting function for dose-dependent inhibition of photosynthesis by phytoplankton from the Weddell Sea. All calculations involved integrated daily UV doses and visible exposures (weighted UV and unweighted visible irradiances, respectively). The results show that daily biologically effective total UV doses underwent large short-term variations at all three sites, with day-to-day increases up to 236% at Ushuaia, 285% at Palmer and 99% at McMurdo. Parallel changes in visible exposure indicated that the total UV changes were preponderantly due to variations in cloudiness. On a 12-month basis, daily biologically effective UV doses correlated strongly with visible exposures (R ≥ 0.99). Anticorrelations of total UV with ozone, on the other hand, were poor (R > −0.11). The largest daily biologically effective UV doses, and their day-to-day increases, occurred as part of the normal variability related to cloud cover and were seldom associated with significant ozone depletion. UV dose/visible exposure ratios tended to reflect ozone depletion events somewhat more consistently than UV doses alone. With the Weddell Sea phytoplankton weighting function used in this study, antarctic ozone hole events were seldom readily discernible in the biologically effective UV record. The results suggest that, where the UV sensitivity of organisms was similar to that of the Weddell Sea phytoplankton, seasonal ozone depletion had no appreciable effect on annual primary productivity during the 1993–1997 period. Additional data on the geographical and seasonal variation of biological weighting functions are desirable for more comprehensive assessments of ozone depletion effects at high southern latitudes.

The adverse health effects caused by increased exposure to ultraviolet radiation (UVR) due to deterioration of stratospheric ozone are of major concern. These health effects include sunburn, skin cancer, cataracts and immune suppression. Immune suppression has been associated with the release of cytokines, a defect in antigen presentation, induction of suppressor T cells and suppression of contact hypersensitivity (CH). CH is typically assessed by the mouse ear swelling test (MEST). Previous studies have demonstrated enhanced CH responses with vitamin A acetate (VAA) dietary supplementation assessed by MEST and the local lymph node assay (LLNA). To determine the effect that VAA has on UVR-induced immune suppression, we examined both the induction and elicitation phases of CH using murine models. The MEST was used to evaluate the interaction of UVR and VAA on CH elicitation. However, a positive MEST response requires that the induction phase as well as the elicitation phase of CH be functional. The LLNA was used to evaluate the interaction of UVR and VAA only on CH induction. We tested the hypothesis that mice maintained on a VAA-enriched diet are more resistant to UVR-induced immune suppression (CH) than those maintained on a control diet. Mice were maintained on a VAA-enriched or the control diet for 3 weeks and then exposed to UVR 3 days prior to sensitization with 2,4-dinitrofluorobenzene (DNFB). VAA enhanced the MEST response in both UVR-exposed and non–UVR-exposed mice. The VAA-enriched diet did not significantly alter the LLNA response in either UVR- or non–UVR-exposed mice. However, there was significant suppression in CH by UVR as measured by the LLNA. These results indicate that (1) the VAA-enriched diet does not restore the number of proliferating cells in the CH induction phase of UVR-induced immunosuppression; (2) the immunosuppressive effects of UVR affect the induction phase of CH; and (3) the LLNA should be examined as an alternative to the MEST for measurement of UVR-induced immunosuppression. The data indicate that the VAA-enriched diet enhanced the elicitation response (MEST) but not the earlier induction phase (LLNA). Further studies are necessary to define mechanisms of action, but modulation of cytokines and effects of specific lymphocyte subsets, as well as systemic effects and local modulation at the site of elicitation are possible. Additionally, future studies to evaluate the effect of the VAA-enriched diet when multiple doses of both UVR and DNFB are used would be of interest for both the LLNA and MEST endpoints.

Plectonema boryanum UTEX 485 cells were grown at 29°C and 150 μmol m⁻² s⁻¹ photosynthetically active radiation (PAR) and exposed to PAR combined with ultraviolet-A radiation (UV-A) at 15°C. This induced a time-dependent inhibition of photosystem II (PSII) photochemistry measured as a decrease of the chlorophyll a fluorescence ratio, F_v/F_m, to 50% after 2 h of UV-A treatment compared to nontreated control cells. Exposure of the same cells to PAR combined with UV-A ultraviolet-B radiation (UV-B) caused only a 30% inhibition of PSII photochemistry relative to nontreated cells. In contrast, UV-A and UV-A UV-B irradiation of cells cultured at 15°C and 150 μmol m⁻² s⁻¹ had minimal effects on the F_v/F_m values. However, cells grown at 15°C and lower PAR irradiance (6 μmol m⁻² s⁻¹) exhibited similar inhibition patterns of PSII photochemistry as control cells. The decreased sensitivity of PSII photochemistry of P. boryanum grown at 15°C and 150 μmol m⁻² s⁻¹ to subsequent exposure to UV radiation relative to either control cells or cells grown at low temperature but low irradiance was correlated with the following: (1) a reduced efficiency of energy transfer to PSII reaction centers; (2) higher levels of a carotenoid tentatively identified as myxoxanthophyll; (3) the accumulation of scytonemin and mycosporine amino acids; and (4) the accumulation of ATP-dependent caseinolytic proteases. Thus, acclimation of P. boryanum at low temperature and moderate irradiance appears to confer significant resistance to UV-induced photoinhibition of PSII. The role of excitation pressure in the induction of this resistance to UV radiation is discussed.

High-dose chemotherapy combined with autologous transplantation using bone marrow or peripheral blood-derived stem cells (PBSC) is now widely used in the treatment of hematologic malignancies as well as some solid tumors like breast cancer (BC). However, some controversial results were recently obtained in the latter case. The presence of malignant cells in the autograft has been associated with the recurrence of the disease, and purging procedures are needed to eliminate this risk. The aim of this study was to evaluate the potential of the photosensitizer 4,5-dibromorhodamine methyl ester (TH9402), a dibrominated rhodamine derivative, to eradicate multiple myeloma (MM) and BC cell lines, while sparing more than 50% of normal pluripotential blood stem cells from healthy volunteers. The human BC MCF-7 and T-47D and MM RPMI 8226 and NCI-H929 cell lines were used to optimize the photodynamic purging process. Cell concentration and the cell suspension thickness as well as the dye and light doses were varied in order to eventually treat 1–2 L of apheresis. The light source consisted of two fluorescent scanning tubes emitting green light centered about 515 nm. The cellular uptake of TH9402 was measured during the incubation and washout periods and after photodynamic treatment (PDT) using spectrofluorometric analysis. The limiting dilution assay showed that an eradication rate of more than 5 logs is obtained when using a 40 min incubation with 5–10 μM dye followed by a 90 min washout period and a light dose of 5–10 J/cm² (2.8 mW/cm²) in all cell lines. Agitating the 2 cm thick cell suspension containing 20 × 10⁶ cells/mL during PDT was essential for maximal photoinactivation. Experiments on mobilized PBSC obtained from healthy volunteers showed that even more drastic purging conditions than those found optimal for maximal eradication of the malignant cell lines were compatible with a good recovery of hematopoietic progenitors cells. The absence of significant toxicity towards normal hematopoietic stem cells, combined with the 5 logs eradication of cancer cell lines induced by this procedure suggests that TH9402 offers an excellent potential as an ex vivo photodynamic purging agent for autologous transplantation in MM and BC treatment.

It is well known that UV exposure of human skin induces DNA damage, and the cumulative effect of such repeated damage is an important contributor to the development of skin cancer. Here, we demonstrate UV dose- and time-dependent induction of DNA damage in the form of cyclobutane pyrimidine dimers (CPD) in skin cells following a single exposure of human skin to UV radiation. CPD cells were identified by an immunohistochemical technique using monoclonal antibodies to thymine dimers. The percentage of CPD cells was UV dose–dependent, even a suberythemal (0.5 minimal erythemal dose [MED]) dose resulted in detectable level of cells that contained pyrimidine dimers. Forty-eight hours after irradiation the percent of total epidermal cells positive for CPD ranged from 19 ± 8, 36 ± 10, 57 ± 12 and 80 ± 10, and total percent dermal cells positive for CPD ranged from 1 ± 1, 7 ± 3, 16 ± 3 and 20 ± 5, respectively, following 0.5, 1.0, 2.0 and 4.0 MED. CPD were also observed in deeper reticular dermis, which suggest the penetrating ability of UV radiation into the skin. The change in CPD cells from 0.5 to 240 h post-UV exposure in both epidermal and dermal compartments of the skin was also quantitated. CPD cells were observed in skin biopsies at early time points after UV exposure which remained elevated for 48 h, then declined significantly by 3 days post-UV. A close examination of the skin at and after 3 days following UV exposure indicates the significant removal of DNA damaged cells from the epidermis. Ten days after UV exposure the levels of CPD cells in both epidermis and dermis were not significantly different from that in unirradiated skin.

Light fractionation with dark periods of the order of hours has been shown to considerably increase the efficacy of 5-aminolevulinic acid-photodynamic therapy (ALA-PDT). Recent investigations have suggested that this increase may be due to the resynthesis of protoporphyrin IX (PpIX) during the dark period following the first illumination that is then utilized in the second light fraction. We have investigated the kinetics of PpIX fluorescence and PDT-induced damage during PDT in the normal skin of the SKH1 HR hairless mouse. A single illumination (514 nm), with light fluences of 5, 10 and 50 J cm⁻² was performed 4 h after the application of 20% ALA, to determine the effect of PDT on the synthesis of PpIX. Results show that the kinetics of PpIX fluorescence after illumination are dependent on the fluence delivered; the resynthesis of PpIX is progressively inhibited following fluences above 10 J cm⁻². In order to determine the influence of the PpIX fluorescence intensity at the time of the second illumination on the visual skin damage, 5 95 and 50 50 J cm⁻² (when significantly less PpIX fluorescence is present before the second illumination), were delivered with a dark interval of 2 h between light fractions. Each scheme was compared to illumination with 100 J cm⁻² in a single fraction delivered 4 or 6 h after the application of ALA. As we have shown previously greater skin damage results when an equal light fluence is delivered in two fractions. However, significantly more damage results when 5 J cm⁻² is delivered in the first light fraction. Also, delivering 5 J cm⁻² at 5 mW cm⁻² 95 J cm⁻² at 50 mW cm⁻² results in a reduction in visual skin damage from that obtained with 5 95 J cm⁻² at 50 mW cm⁻². A similar reduction in damage is observed if 5 45 J cm⁻² are delivered at 50 mW cm⁻². PpIX photoproducts are formed during illumination and subsequently photobleached. PpIX photoproducts do not dissipate in the 2 h dark interval between illuminations.

We have previously shown that keratinocytes in vitro can convert biologically inactive vitamin D₃ to the hormone calcitriol. The present study was initiated to test whether ultraviolet B (UVB)-induced photolysis of provitamin D₃ (7-dehydrocholesterol, [7-DHC]) which results in the formation of vitamin D₃ also leads to the generation of calcitriol in keratinocytes. Submerged monolayers of HaCaT keratinocytes were preincubated with 7-DHC (25 μM) at 37°C and irradiated with monochromatic UVB at different wavelengths (effective UV-doses: 7.5–60 mJ/cm²), or a narrow-band fluorescent lamp Philips TL-01 (UVB-doses: 125–1500 mJ/cm²). Irradiation with both sources of UVB resulted in the generation of different amounts of previtamin D₃ in our in vitro model followed by time-dependent isomerization to vitamin D₃ and consecutive formation of calcitriol in the picomolar range. Unirradiated cultures or cultures exposed to wavelengths >315 nm generated no or only trace amounts of calcitriol. The conversion of vitamin D₃ generated after UVB irradiation to calcitriol is inhibited by ketoconazole indicating the involvement of P450 mixed function oxidases in this chemical reaction. The generation of calcitriol was wavelength- and UVB dose dependent and reached approximately 18-fold higher levels after irradiation at 297 nm than at 310 nm (effective UVB dose: 30 mJ/cm²). Hence, keratinocytes may be a potential source of biologically active calcitriol within epidermis, when irradiated with therapeutical doses of UVB.

Normal hematopoietic progenitor cells from 129S6/SvEv mice are substantially less sensitive to Merocyanine 540 (MC540)-mediated photodynamic therapy (PDT) than hematopoietic progenitors from sex- and age-matched C57BL/6 mice. When exposed to a combination of MC540 and light commonly used for the extracorporeal purging of hematopoietic stem cells, granulocyte/macrophage progenitors (CFU-GM) from C57BL/6 mice are depleted 7.9-fold whereas CFU-GM from 129S6/SvEv and (C57BL/6 × 129S6/SvEv) F1 mice are depleted 1.4- and 2-fold, respectively. The same rank order of sensitivity is also found with regard to unipotent progenitors of granulocytes and macrophages and with regard to early and late erythroid progenitors. The resistance of hematopoietic progenitors from 129S6/SvEv mice to MC540-PDT appears to be the result of reduced dye binding rather than the result of high levels of intracellular glutathione. These findings have practical implications for the design of preclinical tests of PDT in animal models. They may also provide a useful tool for future investigations into the molecular determinants of sensitivity to MC540-PDT.

Riboflavin (RF) is a normal component of the eye lens which triggers a strong photosensitizing activity when exposed to light. Upon irradiation with short wavelength radiations below 400 nm, RF-photosensitized damage may occur. However, vitamin C is present at high concentrations in the normal lens and plays an important role in inhibiting these photosensitization processes. An in vitro simple model was used with the objective of understanding better the relationships between vitamin C and oxygen concentrations on the mechanisms of RF-mediated photodegradation of tryptophan (Trp), a target particularly sensitive to photo-oxidation. Under nitrogen, the RF decomposition reached its maximal value, and vitamin C and Trp photo-oxidation was negligible. When increasing oxygen pressure, RF photodegradation dropped and vitamin C photo-oxidation strongly increased and was maximal at 100% O₂. RF-induced photodegradation of Trp first increased with oxygen concentration, up to 40 μM O₂, and then decreased. RF and Trp degradation were significantly protected by vitamin C so that no more than 20% of the substrates concentration were oxidized in the presence of vitamin C higher than 0.8 mM. From our results we conclude that in the specific conditions of the normal lens, the high vitamin C concentration (2 mM) is compatible with the UVA radiation hazard, despite the presence of RF. However, if lenticular vitamin C decreases below 0.8 mM, photodegradation of RF may occur and Trp may therefore be photo-oxidized by a Type-I mechanism.

The spectroscopy and photochemistry of protoporphyrin IX in ethanol and in Triton X-100 micelle solution have been examined using near-infrared two-photon excitation (TPE). TPE will allow photodynamic therapy with highly localized light dosage. We have determined that the photochemistry subsequent to TPE is very similar to that found for one-photon excitation. Moreover, the photoproducts observed possess very intense TPE fluorescence spectra, which allows their detection at low relative concentrations.