A novel cancer immunotherapy utilizing autologous tumour tissue.

BACKGROUND: With the recent interest in personalized medicine for cancer patients and immune therapy, the field of cancer vaccines has been resurrected. Previous autologous, whole cell tumour vaccine trials have not produced convincing results due, in part to poor patient selection and inactivation methos that are harsh on the cells. These methods can alter protein structure and antigenic profiles making vaccine candidates ineffective in stimulating immune response to autochthonous tumour cells. MATERIALS AND METHODS: We investigated a novel method for inactivating tumour cells that uses UVA/UVB light and riboflavin (vitamin B2) (RF + UV). RF + UV inactivates the tumour cells' ability to replicate, yet preserves tumour cell integrity and antigenicity. RESULTS: Our results demonstrate that proteins are preserved on the surface of RF + UV-inactivated tumour cells and that they are immunogenic via induction of dendritic cell maturation, increase in IFNγ production and generation of tumour cell-specific IgG. Moreover, when formulated with an adjuvant ('Innocell vaccine') and tested in different murine tumour primary and metastatic disease models, decreased tumour growth, decreased metastatic disease and prolonged survival were observed. In addition, immune cells obtained from tumour tissue following vaccination had decreased exhausted and regulatory T cells, suggesting that activation of intra-tumoural T cells may be playing a role leading to reduced tumour growth. CONCLUSIONS: These data suggest that the RF + UV inactivation of tumour cells may provide an efficacious method for generating autologous whole tumour cell vaccines for use in cancer patients.

Analysis of photoreactivity and phototoxicity of riboflavin's analogue 3MeTARF.

Recent studies focus on usage of blue light of λ = 450 nm in combination with photosensitizers to treat surface skin disorders, including cancers. In search of convenient therapeutic factor we studied riboflavin analogue 3-methyl-tetraacetylriboflavin (3MeTARF) as potential sensitizer. Riboflavin (Rfl) itself, non -toxic in the darkness, upon absorption of UVA and blue light, may act as photosensitizer. However, Rfl efficiency is limited due to its susceptibility to photodecomposition. Riboflavin's acetylated analogue, 3MeTARF, bears substituents in ribose chain, which inhibit intramolecular processes leading to degradation. Upon excitation, this compound, reveals higher photochemical resistance, remaining a good singlet oxygen generator. Thus, being more stable as the sensitizer, might be much more efficient in photodynamic processes. The objective of undertaken study was to elucidate mechanisms of 3MeTARF photoreactivity under the irradiation with blue light in comparison to its mater compound, riboflavin. We approached this goal by using spectroscopic methods, like direct singlet oxygen phosphorescence detection at 1270 nm, EPR spin trapping and oximetry. Additionally, we tested both riboflavin and 3MeTARF phototoxicity against melanoma cells (WM115) and we studied mechanism of photodynamic cell death, as well. Moreover, 3MeTARF induces apoptosis in melanoma cells at ten times lower concentration than riboflavin itself. Our studies confirmed that 3MeTARF remains stable upon blue light activation and is more efficient photosensitizer than Rfl.

Preventive role of lens antioxidant defense mechanism against riboflavin-mediated sunlight damaging of lens crystallins.

The main components of sunlight reaching the eye lens are UVA and visible light exerting their photo-damaging effects indirectly by the aid of endogenous photosensitizer molecules such as riboflavin (RF). In this study, lens proteins solutions were incubated with RF and exposed to the sunlight. Then, gel mobility shift analysis and different spectroscopic assessments were applied to examine the structural damaging effects of solar radiation on these proteins. Exposure of lens proteins to direct sunlight, in the presence of RF, leads to marked structural crosslinking, oligomerization and proteolytic instability. These structural damages were also accompanied with reduction in the emission fluorescence of Trp and Tyr and appearance of a new absorption peak between 300 and 400nm which can be related to formation of new chromophores. Also, photo-oxidation of lens crystallins increases their oligomeric size distribution as examined by dynamic light scattering analysis. The above mentioned structural insults, as potential sources of sunlight-induced senile cataract and blindness, were significantly attenuated in the presence of ascorbic acid and glutathione which are two important components of lens antioxidant defense system. Therefore, the powerful antioxidant defense mechanism of eye lens is an important barrier against molecular photo-damaging effects of solar radiations during the life span.

Evaluating the Toxicity/Fixation Balance for Corneal Cross-Linking With Sodium Hydroxymethylglycinate (SMG) and Riboflavin-UVA (CXL) in an Ex Vivo Rabbit Model Using Confocal Laser Scanning Fluorescence Microscopy.

PURPOSE: To develop methods to delineate the relationship between endothelial cell toxicity and tissue fixation (toxicity/fixation) using sodium hydroxymethylglycinate (SMG), a formaldehyde releaser, and riboflavin-UVA photochemical corneal cross-linking (CXL) for therapeutic tissue cross-linking of the cornea. METHODS: Eleven fresh cadaveric rabbit heads were used for ex vivo corneal cross-linking simulation. After epithelial debridement, the tissue was exposed to 1/4 max (9.8 mM) or 1/3 max (13 mM) SMG at pH 8.5 for 30 minutes or riboflavin-UVA (CXL). The contralateral cornea served as a paired control. Postexposure, cross-linking efficacy was determined by thermal denaturation temperature (Tm) and endothelial damage was assessed using calcein AM and ethidium homodimer staining (The Live/Dead Kit). Confocal laser scanning fluorescence microscopy was used to generate live/dead cell counts using a standardized algorithm. RESULTS: The ΔTm after CXL, 1/3 SMG, and 1/4 SMG was 2.2 ± 0.9°C, 1.3 ± 0.5°C, and 1.1 ± 0.5°C, respectively. Endothelial cell damage was expressed as the percent of dead cells/live + dead cells counted per high-power field. The values were 3 ± 1.7% (control) and 8.9 ± 11.1% (CXL) (P = 0.390); 1 ± 0.2% (control) and 19.5 ± 32.2% (1/3 max SMG) (P = 0.426); and 2.7 ± 2.4% (control) and 2.8 ± 2.2% (1/4 max SMG) (P = 0.938). The values for endothelial toxicity were then indexed over the shift in Tm to yield a toxicity/fixation index. The values were as follows: 2.7 for CXL, 14 for 1/3 max, and 0.1 for 1/4 max. CONCLUSIONS: Quarter max (1/4 max = 9.8 mM) SMG effectively cross-linked tissue and was nontoxic to endothelial cells. Thus, SMG is potentially a compound that could achieve both desired effects.

Riboflavin-overproducing strains of Lactobacillus fermentum for riboflavin-enriched bread.

Lactobacillus fermentum isolated from sourdough was able to produce riboflavin. Spontaneous roseoflavin-resistant mutants were obtained by exposing the wild strain (named L. fermentum PBCC11) to increasing concentrations of roseoflavin. Fifteen spontaneous roseoflavin-resistant mutants were isolated, and the level of vitamin B2 was quantified by HPLC. Seven mutant strains produced concentrations of vitamin B2 higher than 1 mg L⁻¹. Interestingly, three mutants were unable to overproduce riboflavin even though they were able to withstand the selective pressure of roseoflavin. Alignment of the rib leader region of PBCC11 and its derivatives showed only point mutations at two neighboring locations of the RFN element. In particular, the highest riboflavin-producing isolates possess an A to G mutation at position 240, while the lowest riboflavin producer carries a T to A substitution at position 236. No mutations were detected in the derivative strains that did not have an overproducing phenotype. The best riboflavin overproducing strain, named L. fermentum PBCC11.5, and its parental strain were used to fortify bread. The effect of two different periods of fermentation on the riboflavin level was compared. Bread produced using the coinoculum yeast and L. fermentum PBCC11.5 led to an approximately twofold increase of final vitamin B2 content.

Corneal collagen crosslinking in vitro: inhibited regeneration of human limbal epithelial cells after riboflavin-ultraviolet-A exposure.

PURPOSE: To assess the hypothesis that during corneal crosslinking (CXL) treatment, riboflavin and ultraviolet-A (UVA) may have a toxic effect on human limbal epithelial cells. SETTING: Center for Eye Research, Department of Ophthalmology, Oslo University Hospital Ullevål, Oslo, Norway. DESIGN: Experimental study. METHODS: In this vitro study, limbal biopsies from corneoscleral rims collected after corneal transplantation were treated with the following combinations: riboflavin-UVA, riboflavin only, or UVA only; a control group received no treatment. After 3 weeks of cell culture, outgrowth of epithelium from the biopsies was evaluated by measuring the area of cell expansion and the number of cell layers. The explanted biopsies were analyzed for proliferation using immunohistochemistry marker Ki-67 and for apoptosis using the terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling (TUNEL) assay. RESULTS: The mean outgrowth from the biopsies was 2.25 mm(2) ± 6.90 (SD) in the riboflavin-UVA group, 181.4 ± 94.8 mm(2) in the riboflavin-only group, 128.5 ± 129.5 mm(2) in the UVA-only group, and 176.2 ± 114.0 mm(2) in the control group. There were no statistically significant between-group differences in the number of cell layers except in the riboflavin-UVA group, in which no cells were found. Detection of apoptosis with the TUNEL-assay was found in the riboflavin-UVA group only (4/5 sections). The proliferation marker Ki-67 was positive in some sections in all groups. CONCLUSION: Cytotoxicity and reduced cell expansion of human limbal epithelial cells occurred after riboflavin-UVA treatment in vitro, emphasizing the importance of avoiding riboflavin-UVA on the limbus during CXL.

The efficacy of simulated solar disinfection (SODIS) against Ascaris, Giardia, Acanthamoeba, Naegleria, Entamoeba and Cryptosporidium.

The antimicrobial activity of simulated solar disinfection (SODIS) in the presence and absence of riboflavin against various protozoa and helminth organisms was investigated in this study. Assays were conducted in transparent 12 well microtitre plates containing a suspension of test organisms in the presence or absence of 250 µM riboflavin. Plates were exposed to simulated sunlight at an optical irradiance of 550 Wm(-2) (watts per square metre) delivered from a SUNTEST™ CPS+ solar simulator. Aliquots of the test suspensions were taken at set time points and the viability of the test organisms was determined by either culture, microscopy or flow cytometry where applicable. With Acanthamoeba, Naegleria, Entamoeba and Giardia exposure to SODIS at an optical irradiance of 550 Wm(-2) for up to 6h resulted in significant inactivation of these organisms. The addition of riboflavin to this system significantly increased the level of inactivation observed with cysts of A. castellanii. With Cryptosporidium oocysts and Ascaris ova exposure to SODIS in the presence and absence of riboflavin for 6-8h resulted in a negligible reduction in viability of both organisms. In this present study we have been able to show that SODIS is effective against a variety of previously untested waterborne organisms and with A. castellanii cysts the addition of micro-molar concentrations of riboflavin can enhance cyst inactivation. However, care must be taken as Ascaris larvae continue to develop inside the ova after exposure to SODIS and Cryptosporidium remain impermeable to propidium iodide staining indicating they may still be infectious.

[Effects of excess vitamin B1 or vitamin B2 on growth and urinary excretion of water-soluble vitamins in weaning rats].

To determine the tolerable upper intake levels of vitamin B(1) and vitamin B(2) in humans, we investigated the effects of excess thiamin or riboflavin administration on body weight gain, food intake, tissue weights, and urinary excretion of B-group vitamins in weaning rats. The weaning rats were freely fed ordinary diet containing 0.0006% thiamin-HCl or the same diet with 0.006%, 0.03%, 0.18% or 1.0% thiamin-HCl for 30 days, or the diet containing 0.0006% riboflavin or the same diet with 0.1%, 0.5 or 1.0% riboflavin for 22 days. Mild diarrhea was seen only in the rats fed with 1.0% thiamin-HCl diet. Excess thiamin-HCl or riboflavin did not affect body weight gains, food intake or tissue weights. The urinary excretions of water-soluble vitamins also did not differ among the diets. These results clearly showed that feeding a diet containing up to 1.0% thiamin-HCl or 1.0% riboflavin did not induce apparent adverse effects, and the no-observed-adverse-effect-levels (NOAELs) for thiamin-HCl and riboflavin in rats might be 1.0% in diet, corresponding to 900 mg/kg body weight/day.

Efficacy and safety of blue-light scleral cross-linking.

PURPOSE: To evaluate the efficacy of blue-light scleral cross-linking as well as its safety in preventing retinal damage beneath the treated sclera. METHODS: Six rabbits were unilaterally treated with topical riboflavin (0.5%) and blue light (465 nm) on the equatorial sclera using a light emitting diode source with an exposure area of 9 mm in diameter. Four weeks after the treatment, the animals were euthanized and the exposed sclera and contralateral eye sclera excised for comparative testing of biomechanical rigidity and histologic retinal cellular damage. Extensiometry was performed to evaluate the stress-strain curve of treated versus untreated sclera, and light microscopy of the treated sclera and underlying retina were also comparatively evaluated. RESULTS: Blue-light scleral cross-linking showed a three-fold increased stiffening in all tested animals in the stress-strain curve. Histological investigation revealed no retinal damage in any of the treated eyes. CONCLUSIONS: Scleral cross-linking with riboflavin and blue light (465 nm) has a stiffening effect on the sclera without histological tissue damage to the retina.

Toxicity testing of a novel riboflavin-based technology for pathogen reduction and white blood cell inactivation.

The Mirasol PRT System (Gambro BCT, Lakewood, CO) for platelets and plasma uses riboflavin and UV light to reduce pathogens and inactivate white blood cells in donated blood products. An extensive toxicology program, developed in accordance with International Organisation for Standardisation (ISO) 10993 guidelines, was performed for the Mirasol PRT system. Test and control articles for most of the reported studies were treated (test) or untreated (control) blood products. For some studies, pure lumichrome (the major photoproduct of riboflavin) or photolyzed riboflavin solution was used. Systemic toxicity was evaluated with in vivo animal studies in the acute and subchronic settings. Developmental toxicity was evaluated with an in vivo animal study. Genotoxicity and neoantigenicity were evaluated with in vitro and in vivo tests. Hemocompatibility and cytotoxicity were assessed with standard, in vitro assays. The pharmacokinteics, excretion, and tissue distribution of (14)C-riboflavin and its photoproducts was evaluated with an in vivo animal study. The possible presence of leachable or extractable compounds (from the disposable set) was evaluated with novel assays for measuring these compounds in blood. No treatment-related toxicity was observed in any of the studies.

Cross-linking of scleral collagen in the rabbit using riboflavin and UVA.

PURPOSE: Scleral biomechanical weakness and thinning is known to be one of the main factors in the pathogenesis of progressive myopia. We tried to strengthen rabbit sclera by cross-linking scleral collagen using ultraviolet A (UVA) and the photosensitizer riboflavin. METHODS: Circumscribed 10 x10 mm sectors of the posterior--equatorial sclera of six chinchilla rabbit eyes were treated in vivo using a UVA double diode with 4.2 mW/cm(2) UVA at 370 nm and applying 0.1% riboflavin-5-phosphate drops as photosensitizer for 30 min. 1 day postoperatively biomechanical stress--strain measurements of three treated scleral strips were performed using a microcomputer-controlled biomaterial testing device and compared to non-treated contralateral control sclera. In addition, three treated eyes were examined histologically by light microscopy, TUNEL staining and electron microscopy to evaluate side-effects. RESULTS: Following the cross-linking treatment, the ultimate stress was 11.87+/-1.8 MPa versus 3.63+/-0.40 in the controls (increase of 227.9%, p=0.014), Young's modulus 27.67+/-4.16 MPa versus 4.9+/-.15 MPa in the controls (increase of 464.7%, p=0.021) and ultimate strain 92.2+/-7.43% versus 165.63+/-19.09% in the controls (decrease of 54.52%, p=0.012). Histologically, serious side-effects were found in the entire posterior globe with almost complete loss of the photoreceptors, the outer nuclear layer and the retinal pigment epithelium (RPE). CONCLUSIONS: Our new method of scleral collagen cross-linking proved very effective in increasing the scleral mechanical strength; the new treatment may represent an option for strengthening scleral tissue in progressive myopia. However, serious side-effects were observed in the outer retina. In future studies these side-effects could be avoided by reducing the irradiation dose below the cytotoxic level of the retina. Before its clinical application, the new method should be tested in a myopia animal model.

Corneal endothelial cytotoxicity of riboflavin/UVA treatment in vitro.

Recently, we have developed collagen crosslinking induced by combined riboflavin/UVA treatment, thus increasing the biomechanical rigidity of the cornea to treat progressive keratoconus. The present safety study was performed to evaluate possible cytotoxic effects of combined riboflavin/UVA treatment on the corneal endothelium in vitro. Endothelial cell cultures from porcine corneas were treated with 500 microM riboflavin solution, exposed to various endothelial UVA irradiances (370 nm) ranging from 0.1 to 1.6 mW/cm2 for 30 min and evaluated 24 h later using trypan blue staining and Yopro fluorescence staining. The effect of either treatment alone (UVA irradiation ranging from 0.2 to 6 mW/cm2) was also tested. An abrupt cytotoxic threshold irradiance level was found at 0.35 mW/cm2 after combined treatment with riboflavin plus UVA irradiation and at 4 mW/cm2 with UVA irradiation alone. Riboflavin alone was not toxic. A cytotoxic effect of the combined riboflavin/UVA treatment on corneal endothelial cells is to be expected with a corneal thickness of less than 400 microm. Therefore, pachymetry should be routinely performed before riboflavin/UVA treatment to exclude patients at risk.

Hypoxia potentiates ultraviolet A-induced riboflavin cytotoxicity.

Flavins are thought to be important chromophores for chronic photo-induced skin injury, but the mechanism is not well known. We have reported that the primary cytotoxicity remaining in ultraviolet A-irradiated riboflavin solution is attributable to hydrogen peroxide. Because the dermis is more hypoxic than the atmosphere, we investigated the cytotoxicity of riboflavin solution during and after ultraviolet A irradiation under hypoxia. Riboflavin solution showed stronger cytotoxicity during irradiation under hypoxia than under air. Riboflavin solution that had been irradiated under hypoxia at lower ultraviolet A doses showed stronger cytotoxicity and contained more hydrogen peroxide than solution irradiated under air at the same doses. At higher ultraviolet A doses, however, the cytotoxicity and hydrogen peroxide quantity were similar in riboflavin solutions irradiated under different oxygen conditions. The effect of a singlet oxygen quencher, sodium azide, on the induction of cytotoxicity and production of hydrogen peroxide by ultraviolet A irradiation of riboflavin solution was examined. The presence of sodium azide in the solution during ultraviolet A irradiation suppressed the cytotoxicity and hydrogen peroxide production to similar levels at various ultraviolet A doses regardless of oxygen conditions. At the maximum suppression by sodium azide, hydrogen peroxide production decreased to 10% of the unsuppressed production. About 40% of the oxygen molecules of hydrogen peroxide produced was thought to be derived from oxygen dissolved in the riboflavin solution.

Light-induced vitamin deficiency in Drosophila melanogaster.

Illumination by visible light (400 Ix) of cultures containing larvae of Drosophila melanogaster can reduce survival (Bruins et al., Insect Biochemistry 21:535-539, 1991). Here we show that the effect of light depends on the presence of propionic or acetic acid in the food medium. We also show that survival is far more affected by illumination of the yeast food media than by direct illumination of the eggs and developing larvae. It is shown that addition of antioxidants to the food prevents light induced mortality. The action of antioxidants suggests that free radicals are important in light induced mortality. We also showed that both yeast and riboflavin (vitamin B2) solutions illuminated with visible light (400 Ix) generate hydrogen peroxide. Other vitamin and amino acid solutions do not produce peroxide in measurable amounts. However, the concentration of photogenerated hydrogen peroxide is far too low to explain the death of eggs and developing larvae upon exposure to light. A 400 Ix light treatment destroys the capability of yeast food media to support survival of larvae. Addition of vitamin C, carotene, tryptophan, nipagin, uric acid, or sucrose to the light treated medium does not restore viability. It is restored when riboflavin is added to the photo-inactivated yeast. A high concentration of pyridoxine also produced an improvement in survival. When riboflavin is treated with light, it cannot support survival on synthetic food media nor can it restore survival on light treated yeast food media. These results show that riboflavin (or a derivative) is a major light sensitive compound of yeast, which can be degraded by light. Light induced loss of riboflavin leads to mortality, because this is an essential dietary vitamin. The vitamin degradation can be prevented by dietary antioxidants. A chromatographic analysis confirms this conclusion.

[Photosensitized DNA damage: mechanisms and clinical use].

On the basis of our recent investigations by a DNA sequencing technique, mechanisms of photoinduced DNA damage in the presence of various endogenous molecules are summarized with special reference to UV carcinogenesis. In particular, riboflavin and pterin derivatives have been shown to induce DNA damage, mainly 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) formation, specifically at the 5' site of 5'-GG-3' sequences through electron transfer reaction. The involvement of 8-oxodG formation in ras mutation is discussed. In addition, recent works concerning the mechanism of photodynamic therapy and the properties of photoactivatable DNA-cleaving molecules are described.

Genotoxicity of photoilluminated riboflavin in the presence of Cu(II).

Riboflavin is known to generate superoxide anion (O2.-) and other reactive oxygen species in the presence of Cu(II) and light as well as cause fragmentation of DNA and protein in vitro. In the present study we examined the genotoxic effects of photoilluminated riboflavin in the presence of Cu(II). Using the phage inactivation assay, a significant decline in plaque-forming unit (PFU) is seen. Results of Ames testing have suggested that probably a frameshift mutation is caused by a riboflavin-Cu(II)-mediated reaction. Using neocuproine as a Cu(I) sequestering reagent, Cu(I) has been shown to be an essential intermediate generated in the reaction between Cu(II), photoilluminated riboflavin, and DNA. Results obtained with various scavengers of active oxygen species strongly suggest that the species predominantly responsible for DNA damage is oxygen (O2) in the singlet or triplet state, together with H2O2, hydroxyl radical, and hydroxyl ion, to a lesser extent. In the case of riboflavin, a ternary complex of DNA-drug-Cu(II) is presumably formed. A redox reaction, involving riboflavin and Cu(II) in the complex, may then occur with the formation of a DNA-oxidized riboflavin-Cu(I) complex. This probably acts as a catalyst for the oxidation of Cu (I) to Cu(II), during which molecular oxygen is reduced to generate a variety of active oxygen species. The probable mechanism for the generation of these reactive oxygen species has also been proposed.

Allopurinol, indomethacin and riboflavin enhance radiation lethality in mice.

Two widely used drugs, allopurinol and indomethacin, and the vitamin riboflavin increased the response of mice to ionizing radiation. In mice a dose of 10.5 Gy of gamma rays from a 60Co source resulted in a dose-dependent shortening of survival times after pretreatment with the three agents, applied at doses which were well tolerated alone. When the dose dependency of these drugs on the influence on survival was tested, two response patterns emerged. Indomethacin (25 mg/kg) shifted the survival curve to the left and reduced the LD50 from approximately 6.5 Gy to approximately 4.5 Gy. Allopurinol (100 mg/kg) diminished the survival rate to approximately 50% irrespective of the radiation dose (ranging from 0.75 to 6.0 Gy). A similar though less striking trend was seen with riboflavin (120 mg/kg), which reduced the survival rate to approximately 65% in the dose range from 3 to 6 Gy. Mortality in mice treated with allopurinol or riboflavin and irradiated with nonlethal exposures (from radiation alone) occurred within the first few days after irradiation, suggesting a different type of injury than is usually associated with radiation death. Although doses of the three drugs used clinically are clearly lower than those providing enhanced radioresponse in our experiments, subtle and nonovert injury caused by combined exposure to the drugs and radiation cannot be completely excluded.

Photoreceptor damage following exposure to excess riboflavin.

Flavins generate oxidants during metabolism and when exposed to light. Here we report that the photoreceptor layer of retinas from black-eyed rats is reduced in size by a dietary regime containing excess riboflavin. The effect of excess riboflavin was dose-dependent and was manifested by a decrease in photoreceptor length. This decrease was due in part to a reduction in the thickness of the outer nuclear layer, a structure formed from stacked photoreceptor nuclei. These changes were accompanied by an increase in photoreceptor outer segment autofluorescence following illumination at 328 nm, a wavelength that corresponds to the excitation maxima of oxidized lipopigments of the retinal pigment epithelium.

8-Hydroxydeoxyguanosine formation at the 5' site of 5'-GG-3' sequences in double-stranded DNA by UV radiation with riboflavin.

DNA damage caused by UV radiation in the presence of riboflavin or hematoporphyrin was characterized by the DNA sequencing technique using 32P-labeled DNA fragments and the analysis of 8-hydroxydeoxyguanosine (8-OH-dG) formation in calf thymus DNA. Exposure of double-stranded DNA to 365 or 302 nm radiation in the presence of riboflavin induced the sequence-specific DNA cleavage which is different from that caused by 302 or 254 nm irradiation in the absence of a sensitizer. The specific cleavage sites were the guanine residues located 5' to guanine. On the other hand, when denatured single-stranded DNA was irradiated at 365 nm with riboflavin or hematoporphyrin, cleavages occurred at most guanine residues. With D2O, the sequence-specific damage of double-stranded DNA by riboflavin was not enhanced, whereas the damage to single-stranded DNA by hematoporphyrin was greatly enhanced. Photodynamic action of riboflavin caused the formation of 8-OH-dG in double-stranded DNA. The enhancing effect of D2O on 8-OH-dG formation was not observed with riboflavin. By contrast, hematoporphyrin plus 365-nm light induced the 8-OH-dG formation only in denatured single-stranded DNA and the 8-OH-dG yield was increased about 2-fold in D2O. ESR spin destruction experiments suggested that photoexcited riboflavin reacts with dGMP to produce riboflavin anion radical and guanine cation radical, but not with other mononucleotides. The estimated ratio of 8-OH-dG yield to total guanine loss indicates that the photoexcited riboflavin induces 8-OH-dG formation specifically at the guanine residue located 5' to guanine through electron transfer. The mechanism was discussed in relation to UV carcinogenesis.

Assessment of the genotoxic potential of riboflavin and lumiflavin. B. Effect of light.

On exposure to visible light, riboflavin and lumiflavin produced reactive oxygen species such as singlet oxygen and superoxide radicals. The reaction was found to be time- and concentration-dependent. Both riboflavin and lumiflavin, upon illumination, showed mutagenic response in the umu test as well as in the Ames/Salmonella assay with Salmonella typhimurium TA102. The mutagenic response was partially abolished by superoxide dismutase while sodium azide did not have any effect. No mutagenicity was observed if the compounds were not illuminated. The results suggested the involvement of superoxide radicals in light-induced mutagenicity of riboflavin as well as lumiflavin.