Exposure of the nonsteroidal anti-inflammatory drug suprofen (SUP) to UV-radiation results in the formation of radicals, reactive oxygen species (ROS), photodecarboxylated products and photoadducts with biomacromolecules. Using an ex vivo pigskin explant model, we investigated whether topical coapplication of the water-soluble antioxidants vitamin C (l-ascorbic acid, ASC), N-acetyl-l-cysteine (NAC) or l-cysteine ethylester (CYSET) with SUP reduced ultraviolet A (UVA)-induced decomposition of SUP. UVA-induced changes in antioxidant bioavailability in the stratum corneum and epidermis were also studied. Epidermal bioavailability of SUP in sham-irradiated pigskin increased 2.2- to 4.1-fold after the lowest antioxidant doses (P < 0.05). As compared with no applied antioxidant, increasing doses of all tested antioxidants resulted in increased levels of SUP and decreased levels of photoproducts (P < 0.05). A maximal protection against SUP photodegradation of 70% was found after an ASC dose of 1 μmol/cm²; these values were 60% for a NAC dose of 10 μmol/cm² and 50% for a CYSET dose of 5 μmol/cm². Skin antioxidant levels increased with increasing applied dose (P < 0.05); the bioavailability of CYSET was approximately three-fold lower than that of ASC and NAC. UVA exposure resulted in 30–50% consumption of the topically applied ASC or NAC in the stratum corneum, whereas CYSET was not consumed. In conclusion, the topically applied water-soluble antioxidants ASC, NAC and CYSET protect against UVA-induced decomposition of SUP by scavenging radicals and ROS. Coapplication of these antioxidants may therefore be an effective way to reduce or prevent the phototoxic effects of SUP in vivo.

UVA contributes to skin cancer by solar UV light. Photosensitizers are believed to play an important role in UVA carcinogenesis. We investigated the mechanism of DNA damage induced by photoexcited xanthone (XAN) analogues (XAN, thioxanthone [TXAN] and acridone [ACR]), exogenous photosensitizers, and the relationship between the DNA-damaging abilities and their highest occupied molecular orbital (HOMO) energies. DNA damage by these photosensitizers was examined using ³²P-labeled DNA fragments obtained from the p53 tumor suppressor gene. Photoexcited XAN caused DNA cleavage specifically at 5′-G of the GG sequence in the double-stranded DNA only when the DNA fragments were treated with piperidine, suggesting that DNA cleavage is due to base modification with little or no strand breakage. With denatured single-stranded DNA, the extent of XAN-sensitized photodamage was decreased. An oxidative product of G, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo), was formed by photoexcited XAN, and the 8-oxo-dGuo formation was decreased in single-stranded DNA. TXAN and ACR induced DNA photodamage as did XAN, although the order of DNA-damaging ability was XAN > TXAN > ACR. These findings suggest that photoexcited XAN analogues induce nucleobase oxidation at 5′-G of GG sequence in double-stranded DNA through electron transfer. The HOMO energies of these photosensitizers, estimated from ab initio molecular orbital (MO) calculation, decreased in the following order: XAN > TXAN > ACR. Extents of DNA damage increased exponentially with the HOMO energies of XAN analogues. This study suggests that DNA-damaging abilities of photosensitizers can be estimated from their HOMO energies.

Administration of the second-generation antihistamine, terfenadine, is sometimes associated with photosensitivity and other skin reactions. To obtain information on its photoreactivity, we used a stepwise experimental approach involving tests for photostability, phototoxicity (PT) (mouse fibroblast cell line [3T3] neutral red uptake [NRU] test) and photomutagenicity (with standard Ames salmonella tester strains TA98, TA100 and TA102). Terfenadine was not phototoxic to cultured mammalian cells under the conditions used (i.e. 5000/161 mJ cm⁻² UVA–UVB). Natural sunlight and UV radiations caused considerable drug decomposition and formation of several photoproducts. Addition of the irradiated terfenadine solution (i.e. a mixture of photoproducts) to the tester did not significantly increase background mutation frequency. Irradiation of terfenadine coplated with the TA102 strain induced a clear-cut photomutagenic response, the magnitude of which was dependent upon the precursor compound concentration and the UV dose (212/7 to 339/11 mJ cm⁻² UVA–UVB). These findings demonstrate that in vitro terfenadine is photomutagenic in absence of PT. Further in vitro and in vivo studies are therefore needed to provide an adequate safety assessment of the photochemical genotoxicity–carcinogenicity potential of terfenadine. In the meantime, patients should be advised to avoid excessive exposure to sunlight.

Fluorescence lifetime and anisotropy measurements were made on the red fluorescent protein (DsRed) from tropical coral of the Discosoma genus, both at single-molecule and bulk concentrations. As expected from previous work, the fluorescence lifetime of DsRed in solution is dependent on laser power, decreasing from an average fluorescence lifetime in the beam of about 3.3 ns at low power (3.5 ns if one extrapolates to zero power) to about 2.1 ns at 28 kW/cm². At the single-molecule level, exciting with 532 nm, 10 ps laser pulses at 80 MHz repetition rate, DsRed particles entering the laser beam initially have a lifetime of about 3.6 ns and convert to a form having a lifetime of about 3.0 ns with a quantum yield of photoconversion on the order of 10⁻³ (calculated in terms of photons per DsRed tetramer). The particles then undergo additional photoconversion with a quantum yield of roughly 10⁻⁵, generating a form with an average lifetime of 1.6 ns. These results may be explained by rapid photoconversion of one DsRed monomer in a tetramer, which acts as an energy transfer sink, resulting in a lower quantum yield for photoconversion of subsequent monomers. Multiparameter correlation and selective averaging can be used to identify DsRed in a mixture of fluorophores, in part exploiting the fact that fluorescent lifetime of DsRed changes as a function of excitation intensity.

Mass spectrometric and ultraviolet absorption spectral evidence are presented for the assignment of structures to three products detected in the reaction mixtures formed upon the photolysis of aqueous solutions of the nucleotide 2′-deoxyguanosine 5′-monophosphate (dGMP) with light of wavelengths >270 nm. The products for which structures are assigned are spiroiminodihydantoin 2′-deoxyribonucleotide (1), 2,2-diamino-4-([2-deoxy-5-monophosphate-β-d-erythro-pentofuranosyl]amino)-5-(2H)-oxazolone (oxazolone 2′-deoxyribonucleotide, 2) and 2-amino-5-([2-deoxy-5-monophosphate-β-d-erythro-pentofuranosyl]amino)-4H-imidazol-4-one (imidazolone 2′-deoxyribonucleotide, 3). These results, when combined with mechanistic data presented in an earlier communication, provide support for the proposal that the irradiation of dGMP with UVB light leads to the formation of singlet oxygen. The UV absorption spectral properties of the imidazolone make this product a reasonable candidate to rationalize the autosensitization of dGMP degradation reported in the earlier communication.

The fluorescence lifetime of ethidium bromide (EB), a widely used fluorescent dye, has been monitored in water solution versus trehalose concentration in order to learn how the presence of the disaccharide modifies the hydration of EB, which is used in this study as a model probe. The interest in trehalose, a naturally occurring bioprotector (osmolyte), stems from its ability to stabilize biomolecules under stress situations. The observed effects of trehalose on EB fluorescence properties have been compared with those induced by sucrose and glucose. Lifetime measurements have been performed by frequency domain fluorometry (2–40 MHz), and from the analysis of their changes versus sugar concentration, the EB–sugar interaction parameters have been obtained. The effect of trehalose on the EB decay in glasses has also been studied both by exploring the heterogeneity of lifetime decay and by single-molecule imaging. Trehalose appears to be more efficient in changing the EB fluorescence parameters, such as the emission lifetime, and it leads to a degree of heterogeneity higher than that induced by the other sugars. When EB is embedded in trehalose glasses, the heterogeneity of the emission and of the bleaching time is further enhanced.

Exposure to ultraviolet radiation results in increased levels of intradermal cis-urocanic acid (cUCA) and alters cutaneous immunity by interfering with processing and presentation of antigen by Langerhans cells. Reports on effects of systemic immunotoxicity with 30 day cUCA exposure in laboratory rodents include thymic atrophy, thymic hypocellularity and decreased T-cell–mediated immunity; however, immune effects of single exposure or 5 day cUCA administration, which may better mimic human exposures, are poorly defined. The present study initially evaluated immune effects of single, 5 day, and 4 week cUCA exposure in C57BL/6N mice. Single administration of intradermal cUCA resulted in decreased splenocyte phagocytosis that persisted for 30 days after cUCA exposure. Five day consecutive cUCA exposure decreased numbers of phenotypically mature CD4 CD8⁻ and CD4⁻CD8 (single positive) thymocytes, increased CD4 CD8 (double positive) immature thymocytes and increased splenocyte proliferation. Prolonged cUCA exposure (4 weeks) caused profound thymic hypocellularity and splenic hypercellularity and increased splenic macrophage chemiluminescence. Because of this apparent sensitivity of C57BL/6N mice to cUCA, thymic hypocellularity was compared between C57BL/6N and C3H/HeN mice dosed with cUCA, and was found to be more pronounced in the C57BL/6N strain. These results are an extension of previous conclusions on immune modulation caused by cUCA in the spleen and thymus. Further, the observed variation in sensitivity between the mouse strains is consistent with known genetic susceptibility of these strains to the immunomodulatory effects of exposure to sunlight.

Nonsteroidal anti-inflammatory drug (NSAID)–photoinduced DNA damage in human peripheral blood mononuclear cells measured using the alkaline comet assay is presented. Whereas Tiaprofenic Acid–photoinduced DNA damage was promptly induced (i.e. observed at relatively low radiation doses), Ketoprofen-photoinduced DNA damage was delayed. This prompt and delayed effect is observed with UVA (320–400 nm), UVB (290–320 nm) and solar-simulated radiation and is attributed to the different photochemical properties of NSAID. The results from these experiments, carried out in living cells, confirm the speculations of NSAID-photoinduced DNA damage brought up by the many experiments conducted in solution.

In previous work, we evaluated the effects of ultraviolet (UV = 280–400 nm) radiation on the early life stages of a planktonic Calanoid copepod (Calanus finmarchicus Gunnerus) and of Atlantic cod (Gadus morhua). Both are key species in North Atlantic food webs. To further describe the potential impacts of UV exposure on the early life stages of these two species, we measured the wavelength-specific DNA damage (cyclobutane pyrimidine dimer [CPD] formation per megabase of DNA) induced under controlled experimental exposure to UV radiation. UV-induced DNA damage in C. finmarchicus and cod eggs was highest in the UV-B exposure treatments. Under the same spectral exposures, CPD loads in C. finmarchicus eggs were higher than those in cod eggs, and for both C. finmarchicus and cod embryos, CPD loads were generally lower in eggs than in larvae. Biological weighting functions (BWF) and exposure response curves that explain most of the variability in CPD production were derived from these data. Comparison of the BWF revealed significant differences in sensitivity to UV-B: C. finmarchicus is more sensitive than cod, and larvae are more sensitive than eggs. This is consistent with the raw CPD values. Shapes of the BWF were similar to each other and to a quantitative action spectrum for damage to T7 bacteriophage DNA that is unshielded by cellular material. The strong similarities in the shapes of the weighting functions are not consistent with photoprotection by UV-absorbing compounds, which would generate features in BWF corresponding to absorption bands. The BWF reported in this study were applied to assess the mortality that would result from accumulation of a given CPD load: for both C. finmarchicus and cod eggs, an increased load of 10 CPD Mb⁻¹ of DNA due to UV exposure would result in approximately 10% mortality.

We tested the hypothesis that cyanobacterial cells have sufficient acclimation potential to tolerate UVB when it is applied in a natural quantum ratio to growth photosynthetically active radiation (PAR). We grew Synechococcus under 50 (Low) or 300 (High) μmol PAR m⁻²·s⁻¹ and then exposed the cells to 0.125 (Low) or 0.75 (High) μmol UVB m⁻²·s⁻¹. The PAR:UVB quantum ratios were near natural for both the Low-PAR:Low-UVB and the High-PAR:High-UVB treatments, but UVB was in excess of typical aquatic PAR:UVB for Low-PAR:High-UVB treatments. The cellular light history determined the UVB responses of Synechococcus. High-PAR cells initially had fewer cpc transcripts encoding phycocyanin, lower phycocyanin content, and more psbAII/AIII transcripts encoding the D1:2 photosystem II (PSII) protein isoform. Higher PAR potentiated them to tolerate an appropriate UVB level without short-term inhibition of PSII or growth. Low-PAR cells rapidly altered psbAII/AIII and cpc gene expression and tolerated appropriate Low UVB. Low-PAR:High-UVB cells, in contrast, suffered short-term inhibition of PSII and growth. In all treatments UVB induced transient loss of cpc transcripts, possibly to free resources for psbAII/AIII expression, which is important for UVB resistance. The drop in cpc transcripts was not part of a general shock response because rbcL transcript pools were stable upon UVB exposure.

Polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BaP), are ubiquitous environmental carcinogens. BaP is metabolized in vivo to reactive intermediates that become covalently bound to DNA and form BaP–DNA adducts, an initial event in carcinogenesis. Ultraviolet A (UVA) synergizes with BaP to significantly enhance genetic damage and accelerate carcinogenic processes. This study was initiated to investigate in vivo cellular changes related to carcinogenesis induced by repeated exposures to BaP plus UVA. Simulated chronic exposure to an environmental carcinogen and sunlight was conducted through biweekly topical application of BaP followed 2 h later by UVA exposure over a 10 week period. BaP diol epoxide (BPDE)–DNA adducts were measured in vivo by immunohistochemistry using an anti-BPDE–DNA monoclonal antibody. Oxidative DNA damage was measured by the detection of 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation using high-performance liquid chromatography. Alterations in the cell cycle that were relevant to carcinogenesis were revealed by changes in p53, as identified in vivo using a polyclonal anti-p53 antibody. We found that cells containing BPDE–DNA adducts and nuclear p53 expression significantly increased between 2 and 10 weeks of BaP–UVA treatment, whereas neither BPDE–DNA adducts nor significant changes in p53 were observed in untreated skin. Using regression analysis, oxidative 8-OHdG damage also showed a parallel increase over 2–10 weeks (r = 0.80). These results indicate that genetic damage caused by exposures to BaP plus UVA accumulates with time and increases the potential for inductive events leading to carcinogenesis and tumor formation.

The development of near-infrared fluorescent contrast agents and imaging techniques depends on the deep penetration of excitation light through several centimeters of tissue and the sensitive collection of the re-emitted fluorescence. In this contribution, the sensitivity and depth penetration of various fluorescence-enhanced imaging studies is surveyed and compared with current studies using continuous wave (CW) and frequency-domain photon migration (FDPM) measurements with planar wave illumination of modulated excitation light at 100 MHz and area collection of reemitted fluorescent light using a previously developed modulated intensified charge-coupled device camera system. Fluorescence was generated from nanomolar to micromolar solutions of indocyanine green (ICG) in a 100 μL volume submerged at 1–4 cm depths in a 1% Liposyn® solution to mimic tissue scattering properties. Enhanced depth penetration and sensitivity are achieved with optimal filter rejection of excitation light, and FDPM rejection of background light is not achieved using CW methods. We show the ability to detect as few as 100 fmol of ICG from area illumination of 785 nm light (5.5 mW/cm²) and FDPM area collection of 830 nm fluorescent light generated from 3 cm below the phantom surface. The lowered noise floor of FDPM measurements enables greater sensitivity and penetration depth than comparable CW measurements.

Photoimmunotherapy was introduced two decades ago but has been studied infrequently in vivo and is virtually untested clinically. Progress has been limited because high-quality, well-characterized photosensitizer immunoconjugates (PICs) have been difficult to make. Here, we describe the development of an innovative conjugation method for producing water-soluble PICs that are free of insoluble aggregates and free of unacceptable amounts of noncovalently associated photosensitizer impurities. The method exploits two procedures previously untried in this research area. First, a small number of antibody lysines (<3 per antibody) are polyethylene glycolated (PEGylated) using a 10 kDa branched polyethylene glycol (PEG), which dramatically enhances PIC solubility and reduces PIC aggregation. Second, a 50% dimethyl sulfoxide–50% aqueous two-solvent system is used to prevent photosensitizer aggregation and noncovalent interactions. These measures allow efficient covalent linkage of the photosensitizer BPD Verteporfin (BPD) to antibody lysines, thorough purification of the resulting PICs (verified by sodium dodecyl sulfate–polyacrylamide gel electrophoresis), maintenance of PIC antigen-binding activity (verified by cellular binding–uptake assays) and reduction of nonspecific cellular uptake (e.g. macrophage capture) of the PICs. Loading levels could be varied controllably in the range ≤11 BPD/antibody. PICs of the C225 anti–epidermal growth factor receptor (EGFR) chimeric monoclonal antibody killed EGFR-overexpressing A-431 cells photodynamically but did not significantly affect EGFR-negative NR6 cells. Although fluorescence measurements demonstrated that the PICs were quenched by as much as an order of magnitude compared with free BPD, an impressive 90% reduction in A-431 cell viability was achieved using 20 J/cm² of 690 nm light after a 40 h incubation with the C225 PICs. The results suggest that PEGylated BPD-C225 PICs merit further investigation in animal models of EGFR-overexpressing cancers.

Ferritin is a multimeric protein consisting of heavy and light chains assembled in different tissue-specific ratios, which can protect cells from oxidative stress by storing reactive iron (Fe). Because the lens is constantly exposed to UV irradiation, we studied its effects on ferritin synthesis and Fe metabolism in cultured lens epithelial cells with and without ascorbic acid (Asc). UVB caused a large increase in accumulation of newly synthesized ferritin chains; this increase was additive to that induced by Asc. In contrast to the Asc-induced increase in Fe storage, Fe storage in ferritin was unaltered by UVB. Although UVB increased accumulation of newly synthesized ferritin chains, total ferritin levels were unaltered. In contrast, Asc, which induced a quantitatively similar increase in accumulation of newly synthesized ferritin chains, doubled the total amount of ferritin. Because UVB did not change Fe storage in ferritin or the size of the labile Fe pool, it was hypothesized and then determined that these newly synthesized chains did not assemble into functional holoferritin. Numerous studies detail the effects of various treatments on de novo ferritin synthesis; however, this study provides a cautionary note regarding the conclusions of such studies in the absence of data indicating assembly of functional ferritin molecules.

Conservation of energetically “expensive” metabolites is facilitated by enzymatic intra- and intermolecular channeling mechanisms. Our previous in vitro kinetic studies indicate that Vibrio harveyi reduced nicotinamide adenine dinucleotide phosphate–flavin mononucleotide (NADPH-FMN) oxidoreductase flavin reductase P (FRP) can transfer reduced riboflavin 5′-phosphate (FMNH₂) to bacterial luciferase by direct channeling. However, no evidence has ever been reported for such an FMNH₂ channeling between these two enzymes in vivo. The formation of a donor–acceptor enzyme complex, stable or transient, is mandatory for direct metabolite channeling between two enzymes regardless of details of the transfer mechanisms. In this study, we have obtained direct evidence of in vitro and in vivo FRP–luciferase complexes that are functionally active. The approach used is a variation of a technique previously described as Bioluminescence Resonance Energy Transfer. Yellow fluorescence protein (YFP) was fused to FRP to generate an active FRP–YFP fusion enzyme, which emits fluorescence peaking at 530 nm. In comparison with the normal 490 nm bioluminescence, an additional 530 nm component was observed in both the in vitro bioluminescence from the coupled reaction of luciferase and FRP–YFP and the in vivo bioluminescence from frp gene–negative V. harveyi cells that expressed FRP–YFP. This 530 nm bioluminescence component was not detected in a control in which a much higher level of YFP was present but not fused to FRP. Such findings indicate an energy transfer from the exited emitter of luciferase to the FRP component of the luciferase–FRP–YFP complex. Hence, the formation of an active complex of luciferase and FRP–YFP was detected both in vitro and in vivo.

Since 1986, people have been informed that they get about 80% of their lifetime ultraviolet (UV) dose by the age of 18. This belief originated from the mathematical conclusion that diligent use of sunscreens (sun protection factor 15 or higher) during the first 18 years of life would reduce the lifetime incidence of nonmelanoma skin cancers by 78%. These data were misconstrued to mean that individuals also got about 80% of their lifetime dose of UV by the age of 18 (linear relationship). However, these calculations were based on the incidence of nonmelanoma skin cancers being related to the square of the UV dose. Careful analysis of UV exposure data shows that Americans actually get less than 25% of their lifetime UV dose by the age of 18. This finding also appears to be true worldwide because Australia, UK and The Netherlands report a similar UV exposure pattern. UV-initiated damage early in life can be promoted by subsequent exposures to progress into tumors later in life. For example, the nonmelanoma skin cancer, squamous cell carcinoma, is dependent on the cumulative UV dose. Thus, a better educational approach for reducing skin cancers would be to instruct fair-skinned individuals to protect themselves throughout their lives from being exposed to too much UV radiation.