During the past several years there has been debate about the origins of nonexponential intensity decays of intrinsic tryptophan (trp) fluorescence of proteins, especially for single tryptophan proteins (STP). In this review we summarize the data from diverse sources suggesting that time-dependent spectral relaxation is a ubiquitous feature of protein fluorescence. For most proteins, the observations from numerous laboratories have shown that for trp residues in proteins (1) the mean decay times increase with increasing observation wavelength; (2) decay associated spectra generally show longer decay times for the longer wavelength components; and (3) collisional quenching of proteins usually results in emission spectral shifts to shorter wavelengths. Additional evidence for spectral relaxation comes from the time-resolved emission spectra that usually shows time-dependent shifts to longer wavelengths. These overall observations are consistent with spectral relaxation in proteins occurring on a subnanosecond timescale. These results suggest that spectral relaxation is a significant if not dominant source of nonexponential decay in STP, and should be considered in any interpretation of nonexponential decay of intrinsic protein fluorescence.

Rotational reorientation times of a polar molecule neutral red (NR) have been measured in n-alkanes using steady-state fluorescence depolarization technique. The rotational dynamics of NR in alkanes is described by the Stokes–Einstein–Debye hydrodynamic theory with slip boundary condition. However, we have observed that as the size of the solvent molecule becomes bigger than the size of the solute molecule, the probe molecule experiences reduced friction and the experimentally measured reorientation times are shorter than those predicted by the hydrodynamic theory. These size effects have been analyzed using quasihydrodynamic theories.

The spectroscopic properties of α-chymotrypsin (α-Chym), l-tryptophan (Trp) and N-acetyl-l-tryptophan (NAT) solubilized in hydrated reverse micelles of sodium bis(2-ethylhexyl) sulfosuccinate in iso-octane were followed by fluorescence as a function of the amount of intramicellar water and initial pH. The lack of pH dependence observed for Trp in these systems, as opposed to what occurs in bulk water, and the similarities found for the protein in both media foresee different locations of these probes. In reverse micelles, fluorescence quenching studies using acrylamide emphasize the existence of structural alterations within the protein when its global charge changes from positive (pH = 7) to negative (pH = 10). The ensemble of the data points to an interfacial location of the zwitterionic Trp, an intermediate region of less tightly bound water for the location of the anionic Trp and NAT and an almost bulk water environment for α-Chym.

The 355 nm laser flash photolysis of argon-saturated pH 8 phosphate buffer solutions of the fluoroquinolone antibiotic flumequine produces a transient triplet state with a maximum absorbance at 575 nm where the molar absorptivity is 14 000 M⁻¹ cm⁻¹. The quantum yield of triplet formation is 0.9. The transient triplet state is quenched by various Type-1 photodynamic substrates such as tryptophan (TrpH), tyrosine, N-acetylcysteine and 2-deoxyguanosine leading to the formation of the semireduced flumequine species. This semireduced form has been readily identified by pulse radiolysis of argon-saturated pH 8 buffered aqueous solutions by reaction of the hydrated electrons and the CO₂^·− radicals with flumequine. The absorption maximum of the transient semireduced species is found at 570 nm with a molar absorptivity of 2500 M⁻¹ cm⁻¹. In argon-saturated buffered solutions, the semireduced flumequine species formed by the reaction of the flumequine triplet with TrpH stoichiometrically reduces ferricytochrome C (Cyt Fe³ ) under steady state irradiation with ultraviolet-A light. In the presence of oxygen, O₂^·− is formed but the photoreduction of Cyt Fe³ by O₂^·− competes with an oxidizing pathway which involves photo-oxidation products of TrpH.

The endocytotic pathway is profoundly altered by the UVA-induced photosensitization of HS 68 fibroblasts by the fluoroquinolone (FQ) antibiotics lomefloxacin, BAYy 3118, norfloxacin and ciprofloxacin, which preferentially localize in lysosomes. The endocytosis of low-density lipoproteins (LDL) loaded with two carbocyanine dyes compatible for effective Forster-type resonance energy transfer (FRET), namely 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) as the donor and 1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) as the acceptor, has been used as a model system. Binding of LDL to their cell surface receptors is impaired by irradiation with 10 J cm⁻² of UVA and/or treatment with 250 μM BAYy 3118 during 2 h. Perturbation of the plasma membrane by the FQ is revealed by the change in the rate of exchange of DiO from the LDL to the cell membrane as compared to untreated cells. The lysosomal degradation of LDL, demonstrated by the disappearance of FRET between DiO and DiI, is partly inhibited by the FQ. The actin filament network, involved in the fusion of mature endosomes with lysosomes, is readily destroyed upon photosensitization with the four FQ. However, actin depolymerization can be avoided by incubation of the cells with trans-epoxysuccinyl-1-leucylamido-(4-guanidino)butane, an inhibitor of lysosomal cathepsins prior to FQ photosensitization. All these data suggest that several components of the endocytotic pathway are impaired by photosensitization with these FQ.

Cedrelone, a tetranortriterpenoid on photolysis by UV light yields a true photooxidation product 3 [14 β,15β,22β,23β-diepoxy-6-hydroxy-1,5,20(22)-meliatriene-2,7,21-trione] whose structure is well established by NMR studies and confirmed by X-ray crystallography, along with product 4 [14 β,15β-epoxy-6,23-dihydroxy-1,5,20(22)-meliatriene-2,7,21-trione]. Addition of rose bengal increases the rate of photooxidation whereas DABCO decreases rate of photolysis proving the involvement of singlet oxygen in the photooxygenation. Both the photoproducts exhibited antifeedant activity.

The tryptophan metabolite xanthurenic acid (Xan) has been isolated from aged human cataractous lenses. The photophysical properties of Xan were examined to determine if it is a potential chromophore for age-related cataractogenesis. We found that Xan produces singlet oxygen (ϕ_Δ = 0.17 in CD₃OD) with the same efficiency as the lenticular chromophore N-formyl kynurenine and quenches singlet oxygen at a rate similar (2.1 × 10⁷; CD₃OD) to other tryptophan metabolites found in the eye. As the mechanisms of induction of cataracts may also involve redox reactions, the interactions of hydrated electrons (e_aq⁻), the azide radical (N₃^·) and hydroxyl radical (OH^·) with Xan were studied using the technique of pulse radiolysis. The reaction rate constants of e_aq⁻, N₃^· and OH^· with Xan were found to be of the same order of magnitude as other tryptophan metabolites. The rate constant for reaction of Xan with e_aq⁻ solvated electrons was found to be diffusion controlled (k = 1.43 × 10¹⁰ M⁻¹ s⁻¹); the reaction with N₃^· was very fast (k = 4.0 × 10⁹ M⁻¹ s⁻¹); and with OH^· was also near diffusion controlled (k = 1.0 × 10¹⁰ M⁻¹ s⁻¹). Superoxide O₂^·− production by irradiated Xan in methanol was detected by electron paramagnetic resonance and substantiated by determining that the enhanced rate of oxygen consumption of Xan irradiated in the presence of furfuryl alcohol was lowered by superoxide dismutase.

Bispsoralen derivatives possessing two psoralens and one piperazine molecule, 1,4-bis[n′-(8-psoralenoxy) alkyl] piperazine (Bis[PsC_n]PIP, n = 4, 6, 8), show high water solubility, efficient intercalation into DNA and good photocrosslinking efficiency of DNA. Bis(PsC₄)PIP shows high lethality on bacteriophage T7 and can effectively inhibit the amplification of DNA by stopping the polymerase chain reactions in a short period of irradiation time.

Fluorometric analysis of DNA unwinding (FADU assay) was originally designed to detect X-ray–induced DNA damage in repair-proficient and repair-deficient mammalian cell lines. The method was modified and applied to detect DNA strand breaks in Chinese hamster ovary (CHO) cells exposed to ionizing radiation as well as to UV light. Exposed cells were allowed to repair damaged DNA by incubation for up to 1 h after exposure under standard growth conditions in the presence and in the absence of the DNA synthesis inhibitor aphidicolin. Thereafter, cell lysates were mixed with 0.15 M sodium hydroxide, and DNA unwinding took place at pH 12.1 for 30 min at 20°C. The amount of DNA remaining double-stranded after alkaline reaction was detected by binding to the Hoechst 33258 dye (bisbenzimide) and measuring the fluorescence. After exposure to X-rays DNA strand breaks were observed in all cell lines immediately after exposure with subsequent restitution of high molecular weight DNA during postexposure incubation. In contrast, after UV exposure delayed production of DNA strand break was observed only in cell lines proficient for nucleotide excision repair of DNA photoproducts. Here strand break production was enhanced when the polymerization step was inhibited by adding the repair inhibitor aphidicolin during repair incubation. These results demonstrate that the FADU approach is suitable to distinguish between different DNA lesions (strand breaks versus base alterations) preferentially induced by different environmental radiations (X-rays versus UV) and to distinguish between the different biochemical processes during damage repair (incision versus polymerization and ligation).

The induction of edema and pyrimidine dimers in epidermal DNA was determined in the skin of SKH:HR1 mice exposed to graded doses of ultraviolet radiation AI (UVAI; 340–400 nm). Exposure to UVAI induced 1.6 ± 0.08 × 10⁻⁶ (mean ± standard error of mean) pyrimidine dimers per 10⁸ Da of DNA per J/m². Edema in irradiated animals was determined as an increase in skinfold thickness. A dose of 1.8 × 10⁶ J/m² of UVAI that resulted in a 50% increase in skinfold thickness (SFT_50%) would have induced 1.0 × 10⁵ dimers per basal cell genome. A similar increase in SFT induced by full spectrum solar ultraviolet radiation (290–400 nm) would accompany the induction of 11.0 × 10⁵ pyrimidine dimers per basal cell genome. These results support a hypothesis that UVAI-induced pathological changes of the skin are mediated through the formation of nondimer photoproducts.

This research examines the behavior of ground-level solar UV radiation as measured by eight broadband meters in the continental United States during the period from late 1994 to late 1998. The goal is to define the variability that occurs in UV irradiance over time scales ranging from one to several years. The monthly integrated irradiances, from latitude 32°N to 47°N, contain large annual cycles and latitudinal gradients which depend on season. Seven of the eight sites show a maximum in July, a behavior related to proximity to the summer solstice, with modifications associated with the annual cycle in column ozone. A large interannual variability in monthly integrated irradiance appears over the 4 year period studied. A comparison of corresponding months during different years shows differences in irradiance of 20% or more in one-third of the cases analyzed. When the solar zenith angle (SZA) is held fixed in the range 60–65°, a substantial annual cycle in UV irradiance remains where the maximum monthly mean irradiance is 1.4–1.9 times the minimum, depending on location. Furthermore, the annual cycle at fixed SZA is not in phase with the normal seasonal cycle. Maximum irradiances at fixed SZA tend to occur in the October to December period, while minima cluster in April through July. The annual cycle in ozone, with maximum column values in spring and minima in autumn, explains the general character of the fixed-SZA data, although changes in cloudiness are significant contributors to interannual variability.

Ultraviolet radiation (UVR) causes systemic immune suppression, decreasing the delayed type and contact hypersensitivity responses in animals and humans and enhancing certain mycobacterial, parasitic and viral infections in mice. This study tests the hypothesis that prior exposure to UVR enhances influenza infections in mice. BALB/c female mice were exposed to 0–8.2 kJ/m² of UVR. Exposed and unexposed mice were infected intranasally three days later with 150–300 plaque-forming units/mouse (lethal dose (LD)₂₀–LD₄₀) of mouse-adapted Hong Kong Influenza A/68 (H₃N₂) virus or sham infected with 50 μL Hanks' balanced salt solution/mouse. Mortality from viral infection ranged from 25–50%. UVR exposure increased virus-associated mortality in a dose-dependent manner (up to a two-fold increase at 8.2 kJ/m²). The increased mortality was not associated with bacterial pneumonia. The highest dose of UVR also accelerated the body weight loss and increased the severity and incidence of thymic atrophy associated with influenza infection. However, UVR treatment had little effect on the increase in lung wet weight seen with viral infection, and, to our surprise, did not cause an increase in virus titers in the lung or dissemination of virus. The mice died 5–6 days after infection, too early for adaptive immune responses to have much impact. Also, UVR did not interfere with the development of protective immunity to influenza, as measured by reinfection with a lethal challenge of virus. Also, cells adoptively transferred from UVR or untreated mice were equally protective of recipient mice challenged with a lethal dose of virus. The mice resemble mice succumbing to endotoxin, and influenza infection increased the levels of tumor necrosis factor α (TNF-α) in bronchoalveolar lavage fluid and serum cortisol levels; however, UVR preexposure did not increase either of these responses to the virus. The results show that UVR increased the morbidity, mortality and pathogenesis of influenza virus in mice without affecting protective immunity to the virus, as measured by resistance to reinfection. The mechanism of enhanced mortality is uncertain, but the data raises concerns that UVR may exacerbate early responses that contribute to the pathogenesis of a primary viral infection.

Exposure of isolated photosystem I (PSI) complexes to illumination (2300 μE m⁻² s⁻¹) for various periods of time resulted in striking changes in their absorption spectra. A 6 nm blueshift of the absorption maximum in the red was detected after 100 min illumination. The fourth derivative of the absorption spectra verifies that the main change of the red peak was attributed to the 682 nm absorption band. Further, it was also shown that a shoulder in the absorption spectra located around 470 nm decreased after the first 5 min of illumination and almost disappeared after 40 min illumination, suggesting that chlorophyll b bound to light-harvesting complex I (LHCI) is also sensitive to excess light. A maximum inhibitory effect on the oxygen uptake rates and a strong stimulation were observed when the PSI complexes were exposed to illumination for about 20 and 40 min, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that LHCI-680 started to degrade during the first 5 min of illumination and almost completely disappeared after 40 min of illumination. These observations demonstrated that LHCI was more sensitive to illumination than the PsaA/B subunits which also presented some degradation signs after 40 min illumination. In addition, insoluble–cohesive-denatured proteins also appeared between the stacking and resolving gel after prolonged illumination (100 min). A photoprotective function of LHCI for the PSI reaction center is proposed.

We have studied the inhibition of photosynthetic electron transport by UV-A (320–400 nm) radiation in isolated spinach thylakoids. Measurements of Photosystem II (PSII) and Photosystem I activity by Clark-type oxygen electrode demonstrated that electron flow is impaired primarily in PSII. The site and mechanism of UV-A induced damage within PSII was assessed by flash-induced oxygen and thermoluminescence (TL) measurements. The flash pattern of oxygen evolution showed an increased amount of the S₀ state in the dark, which indicate a direct effect of UV-A in the water-oxidizing complex. TL measurements revealed the UV-A induced loss of PSII centers in which charge recombination between the S₂ state of the water oxidizing complex and the semireduced Q_A⁻ and Q_B⁻ quinone electron acceptors occur. Flash-induced oscillation of the B TL band, originating from the S₂Q_B⁻ recombination, showed a decreased amplitude after the second flash relative to that after the first one, which is consistent with a decrease in the amount of Q_B⁻ relative to Q_B in dark adapted samples. The efficiency of UV-A light in inhibiting PSII electron transport exceeds that of visible light 45-fold on the basis of equal energy and 60-fold on the basis of equal photon number, respectively. In conclusion, our data show that UV-A radiation is highly damaging for PSII, whose electron transport is affected both at the water oxidizing complex, and the binding site of the Q_B quinone electron acceptor in a similar way to that caused by UV-B radiation.

In 75% of cases, ovarian carcinoma has already metastasized in the abdominal cavity at the time of diagnosis. For determination of the necessity for a supplementary therapy, in addition to surgical resection, it is important to localize and stage microscopical intraperitoneal metastases of the tumor. Intraperitoneal photodetection of tumor metastases is based on preferential tumor distribution of a fluorescent tumor marker. The time-dependent differences in drug concentration between tumor and normal (T/N) tissues can be used to visualize small tumors. We performed fluorescence measurements on abdominal organs and tumor in the peritoneal cavity of rats. 5-Aminolevulinic acid (ALA)–induced protoporphyrin IX (PpIX) was used as the fluorescent marker. Three different drug doses (100, 25 and 5 mg/kg) were used and PpIX fluorescence profiles were followed up to 24 h after intravenous administration. Maximum T/N ratios were found 2–3 h after administration of ALA with all drug doses. A significant T/N tissue contrast was obtained for all abdominal organs tested after administration of 5 mg/kg.

Seven normal volunteers (six males and one female) with tanning skin types III or IV (Fitzpatrick scale) were given 10 daily subcutaneous injections of a superpotent synthetic analog of alpha-melanocyte stimulating hormone (α-MSH) over two weeks. This agent, [Nle⁴-d-Phe⁷]α-MSH, also called Melanotan-I (MT-I), was administered at a dose of 0.16 mg/kg/day (Monday–Friday), over a two week period. Tanning was measured serially using computerized light reflectance. This regimen induced tanning at 3 of 8 anatomic sites including the face, neck and forearm by comparison of baseline to (1) the end of the daily dosing period, (day 14), and (2) one week later, (day 21). Shave biopsies of the forearm taken at baseline and day 21 were analyzed by high performance liquid chromatography for eumelanin content which was measured as the permanganate oxidation product, pyrrole-2,3,5-tricarboxylic acid or PTCA. Pheomelanin content was measured as the hydroiodic acid digestion product, aminohydroxyphenylalanine (AHP). Eumelanin was also measured in the forehead skin samples of three subjects. The HPLC results show that mean (±SD) baseline eumelanin (PTCA) levels in forehead skin (n = 3) averaged 1.38 (±0.87) ng/mg of wet skin tissue weight. Higher mean baseline levels of PTCA were detected in forearm skin (2.06 ± 0.28 ng/mg wet weight, n = 7). One week after MT-I treatments ended, there was a mean (SD) 49% (±17.6%) increase in forehead skin PTCA levels compared to baseline (P = 0.019, n = 3, by paired sample T-test). The mean (SD) increase in forearm skin PTCA levels was 98% (±25.4%) over the same period (P = 0.003). In contrast, forearm pheomelanin expression following MT-I treatment did not significantly change from baseline. Overall, the MT-I regimen increased the eumelanin:pheomelanin ratio in forearm skin from 51:1 at baseline to 86:1 following MT-I (P = 0.054 by paired sample T-test). These results show that the tanning induced by MT-I in the face and forearm is associated with a significant increase in the eumelanin content of the human skin.

Covalent conjugation of a photosensitizer to a ligand that specifically recognized and internalized by a cell-surface receptor may be a way of improving the selectivity of photodynamic therapy (PDT). The class A Type-I scavenger receptor of macrophages, which among other ligands recognizes maleylated serum albumin and has a high capacity is a good candidate for testing this approach. Chlorin_e6 was covalently attached to bovine serum albumin to give conjugates with molar substitution ratios of 1:1 and 3:1 (dye to protein), and these conjugates could then be further modified by maleylation. A novel way of purifying the conjugates by acetone precipitation was developed in order to remove traces of unbound dye that could not be accomplished by size-exclusion chromatography. Conjugates were characterized by polyacrylamide gel electrophoresis and thin-layer chromatography. Photosensitizer uptake was measured by target J774 murine macrophage-like cells and nontarget OVCAR-5 human ovarian cancer cells, and phototoxicity was examined after illumination by a 660 nm diode laser by a tetrazolium assay. All of the purified conjugates were taken up by and after illumination killed J774 cells while there was only small uptake and no phototoxicity toward OVCAR-5 cells. The higher dye:protein ratio and maleylation of the conjugates both produced higher uptakes and lower survival ratios in J774 cells. The uptake and phototoxicity by J774 cells were decreased after incubation at 4°C demonstrating internalization, and confocal microscopy with organelle-specific green fluorescent probes showed largely lysosomal localization. Uptake and phototoxicity by J774 cells could both be competed by addition of the scavenger receptor ligand maleylated albumin. These data show that scavenger receptor–targeted PDT gives a high degree of specificity toward macrophages and may have applications in the treatment of tumors and atherosclerosis.

The state of aggregation of the photosensitizer meso-tetrahydroxyphenylchlorin (mTHPC) in both cell free and intracellular environment was elucidated by comparing its absorption and excitation spectra. In methanol, mTHPC existed as monomers and strongly fluoresced. In aqueous solutions such as phosphate-buffered saline (PBS), mTHPC formed nonfluorescent aggregates. Some portion of mTHPC monomerized in the presence of 10% fetal calf serum PBS. In murine myeloid leukemia M1 and WEHI-3B (JCS) cells, cytoplasmic mTHPC were monomeric. By using organelle-specific fluorescent probes, it was found that mTHPC localized preferentially at the mitochondria and the perinuclear region. Photodynamic treatment of mTHPC-sensitized leukemia cells caused rapid appearance of the apoptogenic protein cytochrome c in the cytosol. Results from flow cytometric analysis showed that the release of cytochrome c was especially pronounced in JCS cells, and well correlated with the extent of apoptotic cell death as reported earlier. Electron microscopy revealed the loss of integrity of the mitochondrial membrane and the appearance of chromatin condensation as early as 1 h after light irradiation. We conclude that rapid release of cytochrome c from photodamaged mitochondria is responsible for the mTHPC-induced apoptosis in the myeloid leukemia JCS and M1 cells.

Oxidizing species (OS), produced by photosensitization or derived from cytotoxic agents, activate apoptotic pathways. We investigated whether two different OS, formed at the same subcellular sites, have equivalent ability to initiate apoptosis in HL-60 cells. Our previous work showed that absorption of visible light by rose bengal (RB) produces singlet oxygen exclusively, whereas absorption of ultraviolet A produces RB-derived radicals in addition to singlet oxygen. Singlet oxygen, but not the RB-derived radicals, induced nuclear condensation and DNA fragmentation into nucleosome-size fragments in a dose dependent manner. In contrast, the RB-derived radicals caused greater lipid oxidation than singlet oxygen. These results indicate that different OS, produced at the same subcellular sites, do not have the same ability to induce apoptosis and that the ability of an OS to initiate lipid oxidation does not necessarily correlate with its ability to induce apoptosis.

UV irradiation enhances transcription of a number of cellular and viral genes. We have compared dose responses for alterations in expression from reporter constructs driven by the human and murine cytomegalovirus (CMV) immediate early (IE) promoters in cells from patients with deficiencies in nucleotide excision repair (complementation groups of xeroderma pigmentosum and Cockayne syndrome) following UV exposure, or infection with UV-damaged recombinant vectors. Results suggest that unrepaired damage in active genes triggers increased reporter activity from constructs driven by the CMV promoters in human fibroblasts. Similar to human fibroblasts, HeLa cells and cells from Li–Fraumeni syndrome patients (characterized by an inherited mutation in the p53 gene) also displayed an increase in reporter activity following UV exposure; however, this response was absent in all simian virus 40 (SV40)-transformed cell lines examined. This suggests that a pathway affected by SV40-transformation (other than p53) plays an essential role in UV-enhanced expression from the CMV IE promoter.

The contribution of DNA strand breaks accumulating in the course of nucleotide excision repair to upregulation of the p53 tumor suppressor protein was investigated in human dermal fibroblast strains after treatment with 254 nm ultraviolet (UV) light. For this purpose, fibroblast cultures were exposed to UV and incubated for 3 h in the presence or absence of 1-β-d-arabinofuranosylcytosine (araC) and/or hydroxyurea (HU), and then assayed for DNA strand breakage and p53 protein levels. As expected from previous studies, incubation of normal and ataxia telangiectasia (AT) fibroblasts with araC and HU after UV irradiation resulted in an accumulation of DNA strand breaks. Such araC/HU-accumulated strand breaks (reflecting nonligated repair-incision events) following UV irradiation were not detected in xeroderma pigmentosum (XP) fibroblast strains belonging to complementation groups A and G. Western blot analysis revealed that normal fibroblasts exhibited little upregulation of p53 (∼1.2-fold) when incubated without araC after 5 J/m² irradiation, but showed significant (three-fold) upregulation of p53 when incubated with araC after irradiation. AraC is known to inhibit nucleotide excision repair at both the damage removal and repair resynthesis steps. Therefore, the potentiation of UV-induced upregulation of p53 evoked by araC in normal cells may be a consequence of either persistent bulky DNA lesions or persistent incision-associated DNA strand breaks. To distinguish between these two possibilities, we determined p53 induction in AT fibroblasts (which do not upregulate p53 in response to DNA strand breakage) and in XP fibroblasts (which do not exhibit incision-associated breaks after UV irradiation). The p53 response after treatment with 5 J/m² UV and incubation with araC was similar in AT, XPA, XPG and normal fibroblasts. In addition, exposure of XPA and XPG fibroblasts to UV (5, 10 or 20 J/m²) followed by incubation without araC resulted in a strong upregulation of p53. We further demonstrated that HU, an inhibitor of replicative DNA synthesis (but not of nucleotide excision repair), had no significant impact on p53 protein levels in UV irradiated and unirradiated human fibroblasts. We conclude that upregulation of p53 at early times after exposure of diploid human fibroblasts to UV light is triggered by persistent bulky DNA lesions, and that incision-associated DNA strand breaks accumulating in the course of nucleotide excision repair and breaks arising as a result of inhibition of DNA replication contribute little (if anything) to upregulation of p53.

Our novel approach was to compare the pharmacokinetics of 5-aminolevulinic acid (ALA), ALA-n-butyl and ALA-n-hexylester induced protoporphyrin IX (PpIX), together with the phototoxicity after photodynamic therapy (PDT) in human skin in vivo, using iontophoresis as a dose-control system. A series of four increasing doses of each compound was iontophoresed into healthy skin of 10 volunteers. The kinetics of PpIX metabolism (n = 4) and the response to PDT (n = 6) performed 5 h after iontophoresis, were assessed by surface PpIX fluorescence and post-irradiation erythema. Whilst ALA-induced PpIX peaked at 7.5 h, highest PpIX fluorescence induced by ALA-n-hexylester was observed at 3–6 h and no clear peak was seen with ALA-n-butylester. With ALA-n-hexylester, more PpIX was formed after 3 (P < 0.05) and 4.5 h, than with ALA or ALA-n-butylester. All compounds showed a linear correlation between logarithm of dose and PpIX fluorescence/phototoxicity at 5 h, with R-values ranging from 0.87 to 1. In addition, the ALA-n-hexylester showed the tendency to cause greater erythema than ALA and ALA-n-butylester. Fluorescence microscopy (n = 2) showed similar PpIX distributions and penetration depths for the three drugs, although both ALA esters led to a more homogeneous PpIX localization. Hence, ALA-n-hexylester appears to have slightly more favorable characteristics for PDT than ALA or ALA-n-butylester.

Rising ultraviolet-B (UVB, 280–320 nm) radiation has been proposed as a factor which may explain nonnormal amphibian population declines. Accordingly research has been directed toward estimating the photolyase activity of several amphibian species in order to predict a species' resilience to UV damage. Unfortunately, in spite of published research which demonstrated that the activity of one of the principal photorepair enzymes, photolyase, can be induced, these estimates did not address the potential for in vivo induction by environmental factors present in situ. We show here that wood frog (Rana sylvatica) embryos exposed to periods of ambient solar radiation (1) displayed significantly different photolyase activities from embryos exposed to equivalent periods of dark; and (2) were positively correlated with the UVB fluence received in vivo. Such results suggest that previous conclusions regarding the relationship between photorepair and population decline must be reevaluated. Estimating amphibian photorepair is a complicated process, and caution must be exercised when interpreting such data.

Recently, intravascular low-power red laser light (LPRLL) therapy has been proposed for the prevention of postangioplasty restenosis due to the observed inhibition of experimental neointimal formation. The objective of this study was to determine the impact of endoluminal LPRLL on vascular levels of inducible nitric oxide synthase (iNOS) and cyclic guanosine monophosphate (cGMP) to help define the mechanism of this effect. Eight atherosclerotic male adult New Zealand White rabbits weighing 4–6 kg were used in these studies. The iliac arteries were treated in separate zones with: (1) balloon inflation only; (2) laser illumination only; and (3) balloon inflation laser illumination. An uninjured zone of the iliac artery served as a control. Laser irradiation (630 nm) was delivered to the vessel wall via a Cold^™ laser Illuminator (Cook, Inc., Bloomington, IN), with a 3 mm–diameter balloon. Experiments demonstrated that vascular cGMP levels obtained immediately following treatment in the balloon only group was the lowest (0.29 ± 0.05 pmol/mg protein) and significantly lower compared with the uninjured controls (1.01 ± 0.07 pmol/mg protein) (P < 0.001). In the laser only treated group cGMP levels were significantly increased (2.87 ± 0.12 pmol/mg protein) compared with the uninjured control (P < 0.001) and the balloon only group (P < 0.001). Vascular cGMP levels in the balloon laser group (2.09 ± 0.07 pmol/mg protein) was also increased compared to the balloon only (P < 0.001) and control (P < 0.001) groups. Qualitative analysis of Western blot demonstrated that laser illumination induces iNOS. In contrast balloon dilatation did not induce iNOS. Balloon laser treatment, however, tended to restore the expression of iNOS. Our study demonstrated that intravascular low dose laser irradiation induces iNOS and elevates vascular cGMP in an in vivo atherosclerotic rabbit model.