BILIRUBIN PHOTOTOXICITY TO HUMAN CELLS BY GREEN LIGHT PHOTOTHERAPY in vitro

Phototherapy of newborn infants with blue or green light is the most common treatment of neonatal hyperbilirubinemia. Using bilirubin bound to human lymphoid and basal skin cells we obtained the green light dose dependency of the bilirubin phototoxicity to these cell types. Cells (3–5× 10⁶/mL) were incubated with bilirubin complexed to human serum albumin (final concentrations 340 μM bilirubin, 150 μM albumin). Under these conditions all cells showed maximum binding of bilirubin. Irradiation with broadband green light (Λ_max= 512 nm) over 24 h led to a light dose-dependent population of cells, which contained no bilirubin on the cell membrane as determined by Nomarski interference microscopy. The light-induced mechanism of the disappearance of bilirubin caused lethal membrane damage to the cells (trypan blue exclusion test). The cell kill rate increased with the irradiation dose and with the fraction of cells with no bilirubin. When 90% of lymphoid cells were bilirubin free, 46% of them were dead (using 480 J cm^?1 green light). Similar results were obtained with basal skin cells. In addition, bilirubin-induced damage of cell membrane and nuclear membrane was also shown by transmission electron microscopy. Bilirubin (340 μM) in the dark led to 5% of the cells being killed. Basal skin cells bind 2.5 times more bilirubin molecules than lymphoid cells and showed a different bilirubin disappearance. Irradiation of bilirubin in carbon tetrachloride with 514.5 nm laser light showed generation of singlet oxygen via its luminescence at 1270 nm. These results demonstrate that green light phototherapy of hyperbilirubinemia may cause both skin and immune system damage.     

QUANTUM YIELDS FOR LASER PHOTOCYCLIZATION OF BILIRUBIN IN THE PRESENCE OF HUMAN SERUM ALBUMIN. DEPENDENCE OF QUANTUM YIELD ON EXCITATION WAVELENGTH

The quantum yield for laser photocyclization of bilirubin to lumirubin in the presence of human serum albumin (phi LR) was measured at five monochromatic excitation wavelengths in the range 450-530 nm. Solutions used were optically thin throughout the wavelength range and precautions were taken to exclude contributions from photocyclization of bilirubin XIII alpha impurities. The values obtained (7.2-18 x 10(-4] were lower than those previously reported and showed the following wavelength dependence: 457.9 less than 488.0 less than 501.7 less than 514.5 approximately equal to 528.7. However, the rate of lumirubin formation, normalized to constant fluence, decreased with wavelength over the same wavelength range and no evidence was found that photoisomerization of bilirubin to lumirubin is faster with green (514.5 or 528.7 nm) than with blue (457.9 or 488.0 nm) light. The stereoselectivity of the configurational isomerization of bilirubin to 4Z,15E and 4E,15Z isomers also was studied. This reaction became less regioselective for the 4Z,15E isomer with increasing wavelength. The observed wavelength dependence of phi LR and of the [4E,15Z]: [4Z,15E] ratio at photoequilibrium are consistent with an exciton coupling model in which intramolecular energy transfer can occur between the two pyrromethenone chromophores of the bilirubin molecule in the excited state. Relative rates of lumirubin formation in vivo at different excitation wavelengths and constant fluence were estimated for different optical thicknesses and for different skin thicknesses. These estimates suggest that the recently reported clinical equivalence of blue and green phototherapy lights probably reflects the marked variation of skin transmittance with wavelength more than wavelength-dependent photochemistry. The calculations also indicated that the optimal wavelength for phototherapy is probably on the long wavelength side of the bilirubin absorption maximum.     

Bilirubin- and light induced cell death in a murine lymphoma cell line

Cells from the mouse lymphoma cell line L5178Y-R were exposed to blue light from phototherapy lamps in the presence of solutions of 160 microM bilirubin supplemented with serum albumin. HPLC analysis showed that the bilirubin solution was photooxidised as a function of increasing light dose. The cells were stained with trypan blue to score necrosis, and apoptosis was assayed by the terminal deoxynucleotide transferase assay (TdT) or by studying the nuclear structure in cells stained with propidium iodide. A rapidly developing apoptosis was observed after light doses killing 60-80% of the cells as judged from the trypan blue exclusion test. The fraction of apoptotic cells was smaller than the fraction of necrotic cells. Exposure of the cells to fractions of light at a high dose rate was compared to the effect of the same total dose at a lower dose rate given as a single fraction. No large differences were found, however, there was a tendency of a higher degree of necrosis as well as apoptosis in the cells receiving the light in fractions at a high dose rate.     

CONFIGURATIONAL PHOTOISOMERIZATION OF BILIRUBIN IN VITRO–II. A COMPARATIVE STUDY OF PHOTOTHERAPY FLUORESCENT LAMPS AND LASERS

Abstract— A comparative investigation of configurational photoisomerization of bilirubin (Z.Z-BR) bound to human serum albumin (HSA) in vitro produced by fluorescent lamps currently employed in phototherapy of neonatal jaundice has been carried out by using a fast absorbance technique. Photoequilibrium Z, E-BR concentrations and rise-times are reported. Similar measurements effected with monochromatic laser lines in the blue-green spectral region are presented for comparison purposes.
Narrow-spectrum fluorescent lamps (violet, special-blue, filtered-green) produce Z,E-BR concentrations at photoequilibrium almost equal to those obtained with monochromatic lines. The photoequilibrium rise-times, however, strongly depend on the spectral bandwidth of the excitation light. Special-blue, daylight and green lamps produce Z,E-BR concentrations = 34,31, and 12%, respectively. For green lamps, the intense UV-blue-Hg lines are responsible for more than 50% of Z,E-BR concentration. Green light is found to be quite ineffective in forming Z,E isomers and very efficient in causing Z,E-BR to revert to native Z.Z-BR. Moreover, simultaneous irradiation of BR solution with special-blue and green fluorescent lamps produces almost complete inhibition of the Z→E reaction at suitable green/blue intensity ratio.
On the basis of these results a possible mechanism is presented to explain the clinical success of fluorescent green lamp phototherapy and the greater production of structural BR isomers recently reported for green than that for white lamps.     

Photochemistry of nucleic acids in cells.

A survey of the recent aspects of the main photoreactions induced by far-UV radiation in cellular DNA is reported. This mostly includes the formation of cyclobutadipyrimidines, pyrimidine(6-4)pyrimidone photoadducts and related Dewar valence isomers in various eukaryotic and prokaryotic cells, as monitored by using either specific or more general assays. Information is also provided on mechanistic aspects regarding the formation of 5,6-dihydro-5-(alpha-thyminyl) thymine, the so-called "spore photoproduct" within far-UV-irradiated bacterial spores. The second major topic of the review deals with the effects of near-UV radiation and visible light on cellular DNA which are mostly mediated by photosensitizers. The main photoreactions of furocoumarins with DNA, one major class of photosensitizers used in the phototherapy of skin diseases, involve a [2 + 2] cycloaddition to the thymine bases according to an oxygen-independent mechanism. In contrast a second type of photosensitized reaction which appears to play a major role in the genotoxic effects of both near-UV and visible light requires the presence of oxygen. The photodynamic effects which are mediated by either still unidentified endogenous photosensitizers or defined exogenous photosensitizers lead to the formation of a wide spectrum of DNA modifications including base damage, oligonucleotide strand breaks and DNA-protein cross-links.     

GENETIC TOXICOLOGY OF THE PHOTOSENSITIZATION OF CHINESE HAMSTER CELLS BY PHTHALOCYANINES

Chloroaluminum phthalocyanine (CAPC) was recently shown to photosensitize cell killing in culture and tumor destruction in vivo. Because this compound is potentially useful in the photodynamic therapy of cancer, its properties as a genotoxic agent were evaluated. Applying the technique of alkaline elution to study DNA integrity, it was found that CAPC could produce single-strand breaks in the DNA of Chinese hamster cells after exposure to white fluorescent light. At equicytotoxic doses, the number of DNA strand breaks produced by CAPC photosensitization was about three times lower than that induced by X-irradiation. During incubation in growth medium after exposure to CAPC-plus-fluorescent light, cells rejoined DNA strand breaks at a rate similar to that observed after X-irradiation. Resistance to 6-thioguanine (6-TG') or to ouabain (OUA') were used as end points of mutagenic potential. Following a treatment that caused -90% cell killing, there was a slight mutagenic effect, i.e. the frequencies were increased by -40% above the background or spontaneous mutations. However, this enhancement was not statistically significant. Taken together, the foregoing, plus an earlier observation that there is no variation in the sensitivity of cells to CAPC + light through the cell cycle, lead to the inferences that DNA damage does not play a major role in cell killing and that the mutagenic potential of this treatment is small.     

Therapeutic Effects of Full Spectrum Light on the Development of Atopic Dermatitis‐like Lesions in NC/Nga Mice

Full spectrum light (FSL) includes UVA, visible light and infrared light. Many studies have investigated the application of FSL in severe cases of atopic dermatitis (AD) in humans; however, FSL has not yet been studied in an animal model. The purpose of this study was to evaluate the therapeutic effects of FSL on AD‐like skin lesions using NC/Nga mice, with the aim of mitigating itching and attenuating the expression of adhesion molecules. We examined the effects of FSL on mite allergen‐treated NC/Nga mice by assessing skin symptom severity, ear thickness, serum IgE levels, and the cytokine expression. We examined the histology of lesions using hematoxylin–eosin, toluidine blue and immunohistochemical staining. Our findings suggest that FSL phototherapy exerts positive therapeutic effects on Dermatophagoides farinae (Df)‐induced AD‐like skin lesions in NC/Nga mice by reducing IgE levels, thus promoting recovery of the skin barrier. The mechanisms by which FSL phototherapy exerts its effects may also involve the inhibition of scratching behavior, reduction of IL‐6 levels and reductions in adhesion molecule expression. The present study indicates that FSL phototherapy inhibits the development of AD in NC/Nga mice by suppressing cytokine, chemokine and adhesion molecule expression, and thus, could potentially be useful in treating AD.     

Continuing damage to rat retinal DNA during darkness following light exposure

The damaging effects of visible light on the mammalian retina can be detected as functional, morphological or biochemical changes in the photoreceptor cells. Although previous studies have implicated short-lived reactive oxygen species in these processes, the termination of light exposure does not prevent continuing damage. To investigate the degenerative processes persisting during darkness following light treatment, rats were exposed to 24 h of intense visible light and the accumulation of DNA damage to restriction fragments containing opsin, insulin 1 or interleukin-6 genes was measured as single-strand breaks (ssb) on alkaline agarose gels. With longer dark treatments all three DNA fragments showed increasing DNA damage. Treatment of rats with the synthetic antioxidant dimethylthiourea prior to light exposure reduced the initial development of alkali-sensitive strand breaks and allowed significant repair of all three DNA fragments. The time course of double-strand DNA breaks was also examined in specific genes and repetitive DNA. Nucleosomal DNA laddering was evident immediately following the 24 h light treatment and increased during the subsequent dark period. The increase in the intensity of the DNA ladder pattern suggests a continuation of enzymatically mediated apoptotic processes triggered during light exposure. The protective effects of antioxidant suggests that the light-induced DNA degradative process includes both early oxidative reactions and enzymatic processes that continue after cessation of light exposure.     

Fluorescence Excitation Spectrum of Bilirubin in Blood: A Model for the Action Spectrum for Phototherapy of Neonatal Jaundice

A recent report (Lamola et al. 2013 Pediatric Research, 74, 54–60) presents a semiempirical model for facile calculation of an action spectrum for bilirubin photochemistry in vivo using the most current knowledge of the optics of neonatal skin. The calculations indicate that competition for phototherapy light by hemoglobin in the skin is the predominant factor that defines the spectrum of light absorbed by bilirubin. If the latter is correct, a valid physical analog of the calculated spectrum is the excitation spectrum of bilirubin in blood. The fluorescence excitation spectrum was recorded and, indeed, found to be very similar to the calculated spectrum. Both spectra exhibit maxima near 476 nm and widths at half height of about 50 nm. This result supports the conclusion that light between 460 and 490 nm is most effective for phototherapy of neonatal jaundice.     

8-METHOXYPSORALEN-PHOTOINDUCED DNA CROSSLINKS AS DETERMINED IN YEAST BY ALKALINE STEP ELUTION UNDER DIFFERENT REIRRADIATION CONDITIONS. RELATION WITH GENETIC EFFECTS

Abstract— DNA damage induced by 8-methoxypsoralen (8-MOP) plus near UV light (UVA) was analyzed in diploid yeast using the alkaline step elution technique. The presence of 8-MOP and UVA induced DNA interstrand cross-links was revealed by the increase of DNA retained on elution filters as compared to untreated controls. The fraction of DNA retained on filters increased linearly with UVA dose. The amount of cross-links was estimated from the fraction of DNA retained on filters using a dose of -radiation leading to a number of DNA strand breaks at least equivalent to the number of 8-MOP induced photoadducts.
When 8-MOP treated cells were exposed to monochromatic light, 365 nm light induced monoadducts and cross-links whereas 405 nm light induced only monoadducts. When submitting 8-MOP plus 405 nm light treated cells to 365 nm irradiation, after removal of unbound 8-MOP by washing, a portion of 8-MOP plus 405 nm light induced monoadducts was converted into cross-links. The amount of monoadducts transformed into cross-links was dependent on the dose of 365 nm irradiation up to a maximum likely to correspond to the number of suitably positioned furan-side monoadducts that could be converted into biadducts. When 8-MOP plus 365 nm light treated cells were reirradiated with 365 nm light, following the same protocol, the maximum level of cross-linking obtainable in yeast was lower than that obtained with 8-MOP in a 405 nm plus 365 nm reirradiation protocol.
In the presence of 8-MOP single exposures to 405 nm light were found to be only slightly genotoxic. However, when followed by second exposures to 365 nm light, a dose-dependent increase in genetic effects, i.e. mutation and gene conversion, was observed in parallel to the induction of DNA crosslinks. These results stress again the prominent role of DNA cross-links in the genotoxicity of 8-MOP.     

LED Phototherapy with Gelatin Sponge Promotes Wound Healing in Mice

Tiny but highly efficient, a light‐emitting diode (LED ) can power a therapy device, such as a phototherapy device, and, at the same time, decrease the device's size requirements. In this study, a LED phototherapy device was designed to investigate the possible impact on wound healing using a mouse model and a cell line exposed to red and blue light. To enhance wound phototherapy, a gelatin sponge was fabricated. Results showed that the red and blue lights promoted cell growth and wound healing, while the blue light with a gelatin sponge protected the wound from infection in the early stages of wound healing. The LED phototherapy device combined with the gelatin sponge, therefore, has potential significance in clinical application for wound healing.     

Genotoxic properties of 2-dodecylcyclobutanone, a compound formed on irradiation of food containing fat

When food containing fat is treated by ionizing radiation, a group of 2-alkylcyclobutanones is formed. These components contain the same number of carbon atoms as their precursor fatty acids and the alkyl group is located in ring position 2. Thus, from palmitic acid 2-dodecylcyclobutanone is derived. To date, there is no evidence that the cyclobutanones occur in unirradiated food. Therefore, these components cannot be considered inherent to food, and for questions pertaining to risk assessment of irradiated food it would be advisable to determine the genotoxic and toxic potentials of cyclobutanones. Measurements of DNA damage in cells exposed to 2-dodecylcyclobutanone, employing the single cell microgel electrophoresis technique, have been carried out. In vitro experiments using rat and human colon cells indicate that 2-docylcyclobutanone in the concentration range of about 0.30 – 1.25 mg/ml induces DNA strand breaks in the cells. Simultaneously, a concentration related cytotoxic effect is observed as was determined by trypan blue exclusion. To which extent these in vitro findings are of relevancy for the in vivo human exposure situation needs to be investigated in further studies. In vivo tests in rats are in progress.     

Genotoxicity of visible light (400-800 nm) and photoprotection assessment of ectoin, L-ergothioneine and mannitol and four sunscreens

This study was designed to determine the genotoxic effects of visible (400-800nm) and ultraviolet A (UVA)/visible (315-800nm) lights on human keratinocytes and CHO cells. The alkaline comet assay was used to quantify DNA-damage. In addition, photo-dependent cytogenetic lesions were assessed in CHO cells by the micronucleus test. Three protective compounds [ectoin, l-ergothioneine (ERT) and mannitol] were tested with the comet assay for their effectiveness to reduce DNA single-strand breaks (SSB). Finally, the genomic photoprotections of two broad-band sunscreens and their tinted analogues were assessed by the comet assay. The WST-1 cytotoxicity assay revealed a decrease of the keratinocyte viability of 30% and 13% for the highest UVA/visible and visible irradiations (15 and 13.8J/cm(2), respectively). Visible as well as UVA/visible lights induced DNA SSB and micronuclei, in a dose-dependent manner. The level of DNA breakage induced by visible light was 50% of the one generated by UVA/visible irradiation. However, UVA radiations were 10 times more effective than visible radiations to produce SSB. The DNA lesions induced by visible and UVA/visible lights were reduced after a 1-h preincubation period with the three tested compounds. The maximal protective effects were 92.7%, 97.9% and 52.0% for ectoin (0.1mM), ERT (0.5mM) and mannitol (1.5mM), respectively, against visible light and 68.9%, 59.8% and 62.7% for ectoin (0.1mM), ERT (0.5mM) and mannitol (1.5mM), respectively, against UVA/visible light. Thus, visible light was genotoxic on human keratinocytes and CHO cells through oxidative stress mechanisms similar to the ones induced by UVA radiations. The four tested sunscreens efficiently prevented DNA lesions that were induced by both visible and UVA/visible irradiations. The tinted sunscreens were slightly more effective that their colorless analogues. There is a need to complement sunscreen formulations with additional molecules to obtain a complete internal and external photoprotection against both UVA and visible lights.     

Bilirubin photoisomerization products in serum and urine from a Crigler-Najjar type I patient treated by phototherapy

The relative compositions of the photoisomers of bilirubin-1X alpha (4Z, 15Z-bilirubin) in serum and urine of a patient with Crigler-Najjar type I syndrome treated by phototherapy are reported. High-performance liquid chromatography analysis reveals the presence of high serum levels of the configurational bilirubin photoisomer (4Z,15E-bilirubin) before the beginning of phototherapy (between 12 and 16% of the total bilirubin). The configurational photoisomer value increases during phototherapy with blue fluorescent lamps up to a photoequilibrium of about 25%, similar to that obtained in a bilirubin solution in vitro irradiated by the same lamps. This evidence suggests an inefficient serum excretion of the 4Z,15E-bilirubin. Indeed, its average half-life in serum of the Crigler-Najjar patient is found to be about 8 h. No detectable traces of the bilirubin structural isomer, lumirubin, are found in the serum. On the other hand, lumirubin represents the dominant bilirubin isomer excreted in the urine, as both 15Z and 15E configurations. Smaller amounts of 4Z,15E-bilirubin, 4E,15Z-bilirubin and native 4Z,15Z-bilirubin are observed in urine. The presence in urine of 4Z,15Z-bilirubin is probably due to a fast reversion of the configurational photoisomers to their native form. The half-life of the configurational photoisomers in urine kept at 38 degrees C is found to be of the order of a few minutes. Our study indicates that in Crigler-Najjar type I patients, mechanisms exist to excrete all bilirubin photoisomers. The lumirubin pathway seems to contribute markedly to bilirubin excretion in the urine, as occurs in jaundiced babies under phototherapy. However, the contribution of configurational isomers cannot be neglected.     

Photogenotoxicity of Skin Phototumorigenic Fluoroquinolone Antibiotics Detected Using the Comet Assay

Abstract— The fluoroquinolone(FQ) antibiotics photosensitize human skin to solar UV radiation and are reported to photosensitize tumor formation in mouse skin. As tumor initiation will not occur without genotoxic insult, we examined the potential of ciprofloxacin, lomefloxacin, fle-roxacin, BAYy3118 (a recently developed monofluori-nated quinolone) and nalidixic acid to photosensitize DNA damage in V79 hamster fibroblasts in vitro. Cells were exposed to 37.5 kj/m₂ UVA (320-400 nm; glass filtered Sylvania psoralen + UVA (PUVA) tubes; calibrated Waldmann radiometer) at 4A_oC in the presence of FQ and immediately afterwards embedded in agarose, lysed and placed in an electrophoretic field at pH 12. Under these denaturing conditions, the presence of DNA single-strand breaks (SSB), alkali-labile sites (ALS) and double-strand breaks (DSB) can be visualized as DNA migrating away from the nucleus (characteristic "comet" appearance) after staining with a specific fluorochrome. At FQ concentrations that induced minimal loss of cell viability (neutral red uptake assay) the compounds tested induced comets with a rank order of BAYy3118 norfloxacin ciprofloxacin lomefloxacin fleroxacin nalidixic acid. If cells were incubated after treatment for 1 h at 37_oC, the comet score decreased, suggesting efficient removal of SSB/ALS/DSB. Addition of the DNA polymerase, inhibitor, aphidicolin, to cells treated with either ciprofloxacin alone or ciprofloxacin + UVA resulted in an accumulation of SSB due to the endo/exonuclease steps of excision repair. We have demonstrated that the FQ are photogenotoxic in mammalian cells but that FQ-pho-tosensitized SSB are efficiently repaired. Preliminary evidence that ciprofloxacin photosensitizes the formation of DNA lesions warranting excision repair may indicate production of more mutagenic lesions.     

Bilirubin-sensitized photoinactivation of enzymes in the isolated membrane of the human erythrocyte.

Abstract— Bilirubin has been found to sensitize the photodynamic inactivation of several enzymes in the isolated membrane (ghost) of the human red cell. When ghosts (pH 8.0, 10°C) + bilirubin (0.1 mM) were irradiated with blue light (350 Wm^-2), the activity of glyceraldehyde 3-phosphate dehydrogenase decayed with t_1/2? 15 min. No effect was observed in the absence of pigment or with incident yellow light. Diazabicyclo-octane (DABCO) sharply reduced the inactivation rate, suggesting that ¹O₂ is involved. Sodium dodecyl sulfate-gel electrophoresis of ghosts containing fully inactivated glyceraldehyde 3-phosphate dehydrogenase revealed no change in the polypeptide band corresponding to the subunit of the enzyme. Solubilized enzyme, which was similarly photosensitive, could be partially protected by nicotinamide adenine dinucleotide or glyceraldehyde 3-phosphate. The integral enzymes Mg²⁺-ATPase, Na⁺, K⁺-ATPase, and acetylcholinesterase were also affected. Under the above conditions and bilirubin = 0.37 mM, these enzymes were photoinactivated in first-order fashion, k? 2, 1.2 and 0.2 h^-1, respectively. The rate of decay of total ATPase was found to vary as the square root of the bilirubin concentration over the range 7–370 μM. At a fixed bilirubin concentration (0.37 mM), this rate was also shown to be directly proportional to light intensity. Inasmuch as the —SH content of bilirubin-containing ghosts diminished during irradiation, oxidation of essential cysteine residues could be responsible for the inactivation of some of the enzymes studied.     

Escherichia coli: Dominance of Red Light over Other Visible Light Sources in Establishing Viable but Nonculturable State

In this study, the effect of UV-A and different wavelengths of visible light irradiations combined with or without a photosensitizer (methylene blue, MB) on the establishment of viable but nonculturable (VBNC) state in Escherichia coli was investigated. Survival of the E. coli was investigated by measuring plate counts, respiring cell count (RCC), direct viable count (DVC) and total counts over a period of up to 72 h. The inhibition rates of various light sources in the presence or absence of MB on E. coli in seawater were ranked in the order UV-A>red light>white light>blue light>green light (from greatest to least activation). E. coli survived for 10.2, 19.0, 21.3 and 24.04 h under exposure to red, white, blue and green light and for 6.8 h under exposure to UV-A in the presence of MB according to t ₉₉ . Although the VC declined to undetectable levels in a relatively short time, the RCC showed that some cells were still capable of respiration and, therefore, are assumed to have entered the VBNC phase. This is the first time that red light has been shown to have a stronger effect on E. coli survival and VBNC than white, green and blue light in seawater environment.     

Effects of intranasal phototherapy on nasal mucosa in patients with allergic rhinitis

RATIONALE: Rhinophototherapy has been shown to be effective in the treatment of allergic rhinitis. Considering that phototherapy with ultraviolet light (UV) induces DNA damage, it is of outstanding importance to evaluate the damage and repair process in human nasal mucosa. METHODS: We have investigated eight patients undergoing intranasal phototherapy using a modified Comet assay technique and by staining nasal cytology samples for cyclobutane pyrimidine dimers (CPDs), which are UV specific photoproducts. RESULTS: Immediately after last treatment Comet assay of nasal cytology samples showed a significant increase in DNA damage compared to baseline. Ten days after the last irradiation a significant decrease in DNA damage was observed compared to data obtained immediately after finishing the treatment protocol. Difference between baseline and 10 days after last treatment was not statistically significant. Two months after ending therapy, DNA damage detected by Comet assay in patients treated with intranasal phototherapy was similar with that of healthy individuals. None of the samples collected before starting intranasal phototherapy stained positive for CPDs. In all samples collected immediately after last treatment strong positive staining for CPDs was detected. The number of positive cells significantly decreased 10 days after last treatment, but residual positive staining was present in all the examined samples. This finding is consistent with data reported in skin samples after UV irradiation. Cytology samples examined two months after ending therapy contained no CPD positive cells. CONCLUSION: Our results suggest that UV damage induced by intranasal phototherapy is efficiently repaired in nasal mucosa.     

ANALYTICAL MODEL OF PHOTOTHERAPY FOR NEONATAL JAUNDICE—I. THE INITIAL Z-E TRANSIENT

A simple model of phototherapy (PT) for neonatal jaundice is presented. Two coupled systems are considered: the "skin" (upper skin layers and vascular network where phototherapy light may penetrate) and the "body" (i.e. the "dark" ensemble of blood vessels and organs where circulation and metabolization of biliribin (BR) and its photoisomers occur).
The mathematics necessary to analyze the PT process is presented together with an appropriate optical model of skin based on a multilayered system to which the theory of radiation transfer in isotropically scattering and absorbing media is applied. A simple matrix formalism is introduced to determine analytically the light distribution in the multilayer skin. The results are. then, used to analyze the initial transient of PT, i.e. configurational isomerization up to photoequilibrium in the superficial skin layers under blue (450 nm) and green (500 nm) light irradiation within a time interval sufficiently short to neglect diffusion of pigments and formation of structural isomers of BR.
Strong coupling among the various BR layers due to the time variation of diffuse transmittance and reflectance is found, resulting in complicated time patterns with initial and final single-exponential behavior. The initial decay constant of BR concentration depends sensibly on the depth of the layer, while the final one is independent of it. As expected the amount of BR conversion at photoequilibrium is larger with blue than with green light. However, at sufficiently larger depths green light is more efficient than blue light to produce the initial conversion of ZZ to ZE isomers of BR.
The present analysis has some relevance for the understanding of the different efficiencies of coloured fluorescent lamps in clinical PT. The application of the complete model ("skin"+"body") requires the knowledge of the diffusion constants and absorption coefficients of pigments and of the optical parameters of deep cutaneous tissues.     

Genotoxicity of visible light (400–800 nm) and photoprotection assessment of ectoin, l-ergothioneine and mannitol and four sunscreens

This study was designed to determine the genotoxic effects of visible (400–800 nm) and ultraviolet A (UVA)/visible (315–800 nm) lights on human keratinocytes and CHO cells. The alkaline comet assay was used to quantify DNA-damage. In addition, photo-dependent cytogenetic lesions were assessed in CHO cells by the micronucleus test. Three protective compounds [ectoin, l-ergothioneine (ERT) and mannitol] were tested with the comet assay for their effectiveness to reduce DNA single-strand breaks (SSB). Finally, the genomic photoprotections of two broad-band sunscreens and their tinted analogues were assessed by the comet assay. The WST-1 cytotoxicity assay revealed a decrease of the keratinocyte viability of 30% and 13% for the highest UVA/visible and visible irradiations (15 and 13.8 J/cm², respectively). Visible as well as UVA/visible lights induced DNA SSB and micronuclei, in a dose-dependent manner. The level of DNA breakage induced by visible light was 50% of the one generated by UVA/visible irradiation. However, UVA radiations were 10 times more effective than visible radiations to produce SSB. The DNA lesions induced by visible and UVA/visible lights were reduced after a 1-h preincubation period with the three tested compounds. The maximal protective effects were 92.7%, 97.9% and 52.0% for ectoin (0.1 mM), ERT (0.5 mM) and mannitol (1.5 mM), respectively, against visible light and 68.9%, 59.8% and 62.7% for ectoin (0.1 mM), ERT (0.5 mM) and mannitol (1.5 mM), respectively, against UVA/visible light. Thus, visible light was genotoxic on human keratinocytes and CHO cells through oxidative stress mechanisms similar to the ones induced by UVA radiations. The four tested sunscreens efficiently prevented DNA lesions that were induced by both visible and UVA/visible irradiations. The tinted sunscreens were slightly more effective that their colorless analogues. There is a need to complement sunscreen formulations with additional molecules to obtain a complete internal and external photoprotection against both UVA and visible lights.