Effective inactivation of food pathogens Listeria monocytogenes and Salmonella enterica by combined treatment of hypericin-based photosensitization and high power pulsed light

Aims: The aim of this study was to evaluate the inactivation efficiency of Listeria monocytogenes ATC_L3C 7644 and Salmonella enterica serovar Typhimurium strain DS88 by combined treatment of hypericin (Hyp)‐based photosensitization and high power pulsed light (HPPL). Methods and Results: Cells were incubated with Hyp (1 × 10^?5 or 1 × 10^?7 mol l^?1) in PBS and illuminated with a light λ = 585 nm. For the combined treatment, bacteria were, after photosensitization, exposed to 350 pulses of HPPL (UV light dose = 0·023 J cm^?2). Fluorescence measurements were performed to evaluate optimal time for cell–Hyp interaction. Results indicate that Hyp tends to bind both Listeria and Salmonella. After photosensitization treatment, Listeria population was reduced 7 log, whereas Salmonella was inactivated just 1 log. Electron photomicrograps of Salmonella and Listeria confirmed that photosensitization induced total collapse of the Listeria cell wall, but not that of Salmonella. After combined photosensitization–HPPL treatment, the population of Listeria was diminished by 7 log and Salmonella by 6·7 log. Conclusions: Listeria can be effectively inactivated by Hyp‐based photosensitization (7 log), whereas Salmonella is more resistant to photosensitization and can be inactivated just by 1 log in vitro. Combined treatment of photosensitization and pulsed light inactivates effectively (6·7–7 log) both the Gram‐positive and the more resistant to photosensitization Gram‐negative bacteria. Significance and Impact of the Study: A new approach to combat Gram‐positive and Gram‐negative bacteria is proposed, combining photosensitization with high power pulsed light.     

DNA repair inhibition by UVA photoactivated fluoroquinolones and vemurafenib

Cutaneous photosensitization is a common side effect of drug treatment and can be associated with an increased skin cancer risk. The immunosuppressant azathioprine, the fluoroquinolone antibiotics and vemurafenib—a BRAF inhibitor used to treat metastatic melanoma—are all recognized clinical photosensitizers. We have compared the effects of UVA radiation on cultured human cells treated with 6-thioguanine (6-TG, a DNA-embedded azathioprine surrogate), the fluoroquinolones ciprofloxacin and ofloxacin and vemurafenib. Despite widely different structures and modes of action, each of these drugs potentiated UVA cytotoxicity. UVA photoactivation of 6-TG, ciprofloxacin and ofloxacin was associated with the generation of singlet oxygen that caused extensive protein oxidation. In particular, these treatments were associated with damage to DNA repair proteins that reduced the efficiency of nucleotide excision repair. Although vemurafenib was also highly phototoxic to cultured cells, its effects were less dependent on singlet oxygen. Highly toxic combinations of vemurafenib and UVA caused little protein carbonylation but were nevertheless inhibitory to nucleotide excision repair. Thus, for three different classes of drugs, photosensitization by at least two distinct mechanisms is associated with reduced protection against potentially mutagenic and carcinogenic DNA damage.     

In situ evidence for the involvement of superoxide anions in cutaneous porphyrin photosensitization

Dihematoporphyrin ether, also known as Photofrin-II (Pf-II) is currently used in the diagnosis and management of a variety of epithelial neoplasms, in a modality known as photodynamic therapy (PDT). A major drawback of these porphyrins for PDT is their ability to evoke prolonged cutaneous photosensitization. The mechanism of tumor ablation and cutaneous photosensitization by these photosensitizers is thought to relate to the generation of one or more reactive oxygen species such as superoxide anion, singlet oxygen and hydroxyl radical. However, the role of these oxygen species has not been established unequivocally. In this study, the mechanism of Pf-II-mediated cutaneous photosensitization was examined using murine ear swelling as a marker. The mice treated with Pf-II and light demonstrated two-fold enhancement of ear swelling whereas animals treated with the SOD mimic, beta-carotene and dimethyl sulfoxide (DMSO) had considerably less ear swelling (p less than 0.01). The observed protective effect was dependent on the dose of each quencher and followed the pattern SOD mimic DMSO beta-carotene. The histopathologic alterations caused by Pf-II photosensitization were significantly alleviated by pretreatment with SOD mimic whereas beta-carotene and (DMSO) were less effective. Inhibitors of superoxide dismutase (sodium diethyldithiocarbamate) and catalase (hydroxyl amine and 3, amino 1,2,4-triazole) augmented Pf-II-mediated cutaneous photosensitization. These data provide the first in vivo evidence for the involvement of superoxide anion in cutaneous porphyrin photosensitization.     

Adenosine Diphosphate Ribosylation of Dinitrogenase Reductase and Adenylylation of Glutamine Synthetase Control Ammonia Excretion in Ethylenediamine-Resistant Mutants of Azospirillum brasilense Sp7

Three bacterial strains—Escherichia coli, Acinetobacter calcoaceticus, and the A. calcoaceticus RecA(−) mutant—underwent photosensitization by a low-concentration (0.73 μmol/L) tetramethyl pyridyl porphine (a cationic hydrophylic photosensitizer) and a 4-J/cm² dose of 407 to 420 nm blue light. The viability of the first two strains decreased by approximately 60%. and that of the RecA(−) strain decreased by 90%. Increasing the amount of photosensitizer to 14.6 μmol/L at the same dose of blue light resulted in a 95% to 98% decrease in viability of the three strains. Very little damage to the bacterial DNA was observed after this treatment. Increasing the concentration photosensitizer under the same illumination conditions also resulted in very little damage to the DNA. Western blotting demonstrated that the low photosensitization procedures enhance RecA production for mending the damaged chromosomal DNA. RecA production as a result of low-dose photosensitization was confirmed and demonstrated by immunofluorescent staining and gold immunolabeling. Although DNA is not the primary target for photosensitization, this process of RecA production may provide a certain degree of DNA mending and may also affect the survival of bacterial cells on low-intensity photosensitization.     

Caspase-resistant vimentin suppresses apoptosis after photodynamic treatment with a silicon phthalocyanine in Jurkat cells

Oxidative stress, such as photodynamic therapy, is an apoptosis inducer. Apoptosis, as well as photosensitization, have been associated with disruption of the cytoskeletal network. The purpose of the present study was to assess the role of vimentin, a major cytoskeletal protein, in apoptosis after photodynamic treatment (PDT) with the silicon phthalocyanine Pc 4 in human Jurkat T cells. Here we show for the first time that photosensitization with Pc 4 initiates vimentin cleavage and that this event precedes poly(ADP-ribose) polymerase (PARP) degradation. Similar findings were obtained in the presence of C2-ceramide, an inducer of oxidative stress and apoptosis. In the presence of benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone, a pan-caspase inhibitor, Pc 4-PDT-induced vimentin and PARP cleavage were abolished. In Jurkat cells transfected with a caspase-resistant vimentin apoptosis was partly suppressed and delayed post-Pc 4-PDT. We suggest that the full-length vimentin confers resistance to nuclear apoptosis after PDT with Pc 4.     

Spectroscopic studies on Zn(II)-phthalocyanine in homogeneous and microheterogeneous systems

The absorption and fluorescence properties of Zn2+-phthalocyanine (Zn-Pc) have been characterized in homogeneous and microheterogeneous media. By our preparation procedure, the phthalocyanine can be associated in a monomeric state with cationic micelles, unilamellar liposomes, and low-density lipoproteins; the distribution of Zn-Pc in the hydrophobic phases appears to be controlled by the nature of the lipid environment. The potential use of liposome-bound Zn-Pc for photosensitization studies in aqueous media and for phototherapeutic applications in vivo is briefly discussed.     

Factors influencing the susceptibility of Gram-negative bacteria to toluidine blue O-mediated lethal photosensitization

AIMS: Bacteria can be killed by red light in the presence of a photosensitizer. The purpose of this study was to evaluate the effect of physiological and environmental factors on the susceptibility of some bacteria associated with oral infections in immunocompromised patients to killing by the photosensitizer toluidine blue O (TBO). METHODS AND RESULTS: Suspensions of Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae in human saliva, horse serum or saline were exposed to light from a helium/ neon laser in the presence of TBO. Additional suspensions at various growth phases and pHs were treated in an identical manner. Survivors were enumerated by viable counting. All three species were susceptible to lethal photosensitization under all of the conditions tested. The presence of serum and, to a lesser extent, saliva decreased the level of kill attained. The bactericidal effect was reduced at acid pHs but was unaffected by the growth phase of the organism. CONCLUSIONS: The composition and pH of the fluid in which bacteria are suspended influenced the effectiveness of TBO-mediated lethal photosensitization, whereas killing was unaffected by the growth phase of the organism. SIGNIFICANCE AND IMPACT OF THE STUDY: Environmental factors operating in the mouths of patients with mucositis could reduce the effectiveness of TBO-mediated lethal photosensitization of bacteria associated with this condition.     

Rapid induction of apoptosis in human keratinocytes with the photosensitizer QLT0074 via a direct mitochondrial action

QLT0074 is a newly introduced, porphyrin-derivative for use in photodynamic therapy (PDT). In the current study, the intracellular distribution of QLT0074 and the mode of cell death induced by photosensitization with this compound in vitro were assessed for transformed human HaCaT keratinocytes. Fluorescence microscopy studies indicated a distribution of the drug to the cytoplasm, nuclear membrane and mitochondria of these cells. In the absence of light, QLT0074 produced no evidence of apoptosis-related biochemical changes or affected cell viability. When combined with blue light exposure, cytotoxicity was exerted in a QLT0074- and light-dose-related manner. Appearance of the mitochondrial protein cytochrome c in the cytosolic fraction and expression of the apoptosis-associated mitochondrial 7A6 antigen were demonstrable following photosensitization at nano-molar levels of QLT0074. Evidence of processing of the apoptosis-effector molecules caspase-3, -6, -7, -8 and -9 as well as cleavage of the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) were demonstrable subsequent to cytochrome c release after PDT. Treatment with the anti-oxidant pyrrolidine dithiocarbamate (PDTC) inhibited cytochrome c release, caspase-3 activation and PARP cleavage associated with PDT thereby supporting the contention that QLT0074 induces apoptosis through the generation of reactive oxygen species upon light activation. QLT0074 is a potent photosensitizer with the capacity to directly initiate apoptosis by acting upon mitochondria.     

Vanillin as an antioxidant in rat liver mitochondria: Inhibition of protein oxidation and lipid peroxidation induced by photosensitization

Using rat liver mitochondria, as model systems, we have examined the ability of the natural compound and the food-flavoring agent, vanillin to protect membranes against oxidative damage induced by photosensitization at concentrations normally used in food preparations. Vanillin, at a concentration of 2.5 mmol/L, has afforded significant protection against protein oxidation and lipid peroxidation in hepatic mitochondria induced by photosensitization with methylene blue plus light. The effect observed was both time- and concentration-dependent. The inhibitory effect is similar to ascorbic acid and the singlet oxygen quencher, diazabicyclo[2.2.2]octane (DABCO) but less effective than sodium azide and glutathione. Examination of possible mechanisms responsible for the observed protection, showed that vanillin has a significant ability to quench singlet oxygen (¹O₂), a reactive species responsible for damage induced during photosensitization by Type II mechanism. Hence, this flavoring compound, due to its antioxidant ability, may have potential to prevent oxidative damage to membranes in mammalian tissues and thereby the ensuing diseased states.     

Haematoporphyrin derivative photosensitization and gamma-radiation damage interaction in Chinese hamster ovary fibroblasts

The interaction of haematoporphyrin derivative (HPD) photosensitization and gamma-irradiation was studied with regard to clonogenicity of Chinese hamster ovary (CHO) fibroblasts. Exposure to either treatment alone resulted in shouldered response curves. Exposure to 4.2 Gy gamma-radiation immediately before graded doses of visible light had no effect on the shape of the visible-light survival curve; similarly, exposure to 8.75 kJ/m2 light immediately before graded doses of gamma-radiation had no effect on the shape of the gamma-radiation response curve. These data indicate that damage due to gamma-radiation and HPD photosensitization did not interact, suggesting that the mechanisms of cell killing are different.     

Novel approach to control Salmonella enterica by modern biophotonic technology: photosensitization

Aims:  Salmonellosis is one of the most common foodborne diseases in the world. The aim of this study was to evaluate the antibacterial efficiency of 5-aminolevulinic acid (ALA) based photosensitization against one of food pathogens Salmonella enterica .
Methods and Results:  Salmonella enterica was incubated with ALA (7·5 mmol l⁻¹) for 1–4 h and afterwards illuminated with visible light. The light source used for illumination of S. enterica emitted light λ  = 400 nm with energy density 20 mW cm⁻². The illumination time varied from 0 to 20 min and subsequently a total energy dose reached 0–24 J cm⁻². The data obtained indicate that S. enterica is able to produce endogenous photosensitizer PpIX when incubated with ALA. Remarkable inactivation of micro-organisms can be achieved (6 log) after photosensitization. It is obvious that photosensitization-based inactivation of S. enterica depends on illumination as well as incubation with ALA time.
Conclusion:  ALA-based photosensitization can be an effective tool against multi-drug resistant Gram-negative bacteria S. enterica serovar Typhimurium.
Significance and Impact of the Study:  Experimental data and mathematical evaluations support the idea that ALA-based photosensitization can be a useful tool for the development of nonthermal food preservation technology in future.     

Novel approach to the microbial decontamination of strawberries: chlorophyllin-based photosensitization

Aims: This study is focused on the possibility to control microbial contamination of strawberries by chlorophyllin (Na‐Chl)‐based photosensitization. Moreover, photosensitization‐induced effects on key quality attributes of treated strawberries was evaluated. Methods and Results: Strawberries were inoculated with Listeria monocytogenes ATC_L3C 7644, soaked in 1 mmol l^?1 Na‐Chl for 5 min and illuminated for 30 min with visible light (λ = 400 nm, energy density 12 mW cm^?2). Results indicated that the decontamination of strawberries using photosensitization was 98% compared to control sample. Naturally occurring yeasts/microfungi and mesophiles were inhibited by 86 and 97%, respectively. The shelf life of treated strawberries was extended by 2 days. The total antioxidant activity of treated strawberries increased by 19%. No impact on the amount of phenols, anthocyanins or surface colour was detected. Conclusions: Photosensitization may be an effective, nonthermal and environmentally friendly microbial decontamination technique which expands the shelf life of strawberries without any negative impact on antioxidant activity, and phenols, anthocyanins or colour formation. Significance and Impact of the Study: Experimental data support the idea that Na‐Chl‐based photosensitization can be a useful tool for the future development of nonthermal food preservation technology.     

Inactivation of food pathogen Bacillus cereus by photosensitization in vitro and on the surface of packaging material

Aims: The study was focused on the possibility to inactivate food pathogen Bacillus cereus by 5‐aminolevulinic acid (ALA) – based photosensitization in vitro and after adhesion on the surface of packaging material. Methods and Results: Bacillus cereus was incubated with ALA (3–7·5 mmol l^?1) for 5–60 min in different environment (PBS, packaging material and wheat grains) and afterwards illuminated with visible light. The light source used for illumination emitted light at λ = 400 nm with energy density at the position of the cells, 20 mW cm^?2. The illumination time varied from 0 to 20 min, and subsequently a total energy dose was between 0 and 24 J cm^?2. The obtained results indicate that B. cereus after the incubation with 3–7·5 mmol l^?1 ALA produces suitable amounts of endogenous photosensitizers. Following illumination, micro‐organism inactivated even by 6·3 log. The inactivation of B. cereus after adhesion on the surface of food packaging by photosensitization reached 4 log. It is important to note that spores of B. cereus were susceptible to this treatment as well; 3·7‐log inactivation in vitro and 2·7‐log inactivation on the surface of packaging material were achieved at certain experimental conditions. Conclusions: Vegetative cells and spores of Gram‐positive food pathogen B. cereus were effectively inactivated by ALA‐based photosensitization in vitro. Moreover, the significant inactivation of B. cereus adhered on the surface of packaging material was observed. It was shown that photosensitization‐based inactivation of B. cereus depended on the total light dose (illumination time) as well as on the amount of endogenous porphyrins (initial ALA concentration, time of incubation with ALA). Significance and Impact of the Study: Our previous data, as well as the one obtained in this study, support the idea that photosensitization with its high selectivity, antimicrobial efficiency and nonthermal nature could serve in the future for the development of completely safe, nonthermal surface decontamination and food preservation techniques.     

Studies on the photochemical and photocytotoxic properties of the new PDT photosensitizer aluminum sulfonated phthalocyanine.

The properties of photosensitization of sulfonated aluminum phthalocyanine (ALSPC), a new photosensitizer of potential use in cancer photodynamic therapy (PDT) was studied on both the molecular and cellular levels. The mechanism of ALSPC photosensitization on the molecular level was investigated by testing its efficiency of singlet oxygen (1O2) production, using the method of tryptophan degradation and that of ESR spectroscopy and observing the enhancing effect of D2O and the quenching effect of NaN3. Results of all these experiments confirmed the important role of the Type II or 1O2 mechanism in ALSPC photosensitization. In our in-vitro experiments, ALSPC's incorporation into cells and its photocytotoxic effect were investigated on a human liver cancer cell line. The cell incorporation was illustrated by the laser-excited fluorescence spectra emitted both from cell homogenate and cell monolayers incubated with ALSPC aqueous solution. The position of fluorescence peak observed, implied that ALSPC exists in the cells mainly as monomers. The efficiency of cell killing of ALSPC photosensitization was estimated by counting surviving cells with the method of trypan blue staining and by the method of radioisotope labelling. Experiments using the latter method also showed DNA damage caused by ALSPC photosensitization.     

Photosensitization and the nervous system in the planarian Dugesia gonocephala

Summary Photosensitization phenomena may be induced in planarias by eosin and hematoporphyrin, and as a result, dopamine agonistic behavior (screw-like hyperkinesia) is set up in the animal. Histochemical, ultrastructural and pharmacological investigations have shown that this hyperkinesia is of postsynaptic origin in eosin photosensitization, and of pre-synaptic origin in hematoporphyrin photosensitization. The authors suggest an hypothesis to explain the different activity of the two photosensitizers, and discuss the validity of the experiment with regard to human porphyria.     

Interaction of the photosensitization products of oestrone with DNA: comparison with the horseradish peroxidase catalyzed reaction

The interaction with DNA of [4-14C]oestrone upon photosensitization with hematoporphyrin (HP) as a photosensitizer has been investigated. By means of Sephadex LH-20 gel filtration and extraction with dichloromethane it was found that, after irradiation (lambda greater than 425 nm) of a solution of HP, DNA and [4-14C]oestrone 21% of the radiolabel was associated with DNA. If DNA was added after irradiation 23% was bound to DNA, whereas 25% of the oestrone remained after photoreaction under the conditions applied. The binding occurs via the reactive 10 beta-hydroperoxy-1,4-estradien-3,17-dione, which is the only product after photosensitization of oestrone. The hydroperoxide has a strong interaction with DNA compared with that of other steroids. By repeated precipitation with 5 M NaCl and ethanol the association can be broken. It is reported, that binding of oestrone to protein induced by both photosensitization and horseradish peroxidase (HRPO)/H2O2 is irreversible, but that the amount of binding to DNA is dependent on the method of determination. However, neither the hydroperoxide nor its reduced product, a p-quinol, is intermediate or product in the HRPO catalyzed reaction of oestrogens. The tight association of the hydroperoxide product of oestrone with DNA, which may proceed via hydrogen bonding between the -OOH group and oxygen atoms of the backbone phosphate groups or of the furanose ring, might be a cause of chemical modification of DNA and of mutagenic effects.     

Proton magic angle spinning NMR reveals new features in photodynamically treated bacteria

The bacterium Propionibacterium acnes is light-sensitive due to porphyrin-induced photosensitization. The light sensitivity increases with incubation of 5-aminolevulinic acid, ALA. For the first time, 1H magic angle spinning NMR spectroscopy is used to describe the photoinduced changes in the bacterium after ALA incubation. Successful photosensitization was performed with light-emitting diodes in the blue and red regions (430 and 654 nm, respectively). The irradiation setup, suitable for irradiation of bacterium suspensions in petri dishes is described. For NMR studies blue light diodes with about 90 micromol/m2s were chosen. After blue light irradiation, the endogenous glycine betaine, proline, glutamate and choline levels in P. acnes decreased with increasing irradiation time. For sublethal light doses (50% survival fraction), the endogenous glycine betaine level decreased 80% on average. The corresponding percentages for proline, choline and glutamate were about 40, 25 and 10, respectively. It is hypothesized that the irradiation, inducing porphyrin photosensitization amplified by ALA incubation, leads to elimination of the osmolyte glycine betaine and possibly also proline by so-called regulatory volume decrease (RVD) mechanisms. These mechanisms are known to be active in several prokaryotic and eukaryotic cells when exposed to hypotonic stress. They are also known to be present in several eukaryotic cells during photodynamic therapy (PDT) exposure leading to hypotonoc stress. The findings contribute to the knowledge of the inactivation mechanisms of P. acnes in photosensitization, and could therefore be of interest in the efforts to use PDT as treatment of the acne disease.     

Inactivation of Bacillus cereus by Na‐chlorophyllin‐based photosensitization on the surface of packaging

Aims: This study was focused on the possibility to inactivate food‐borne pathogen Bacillus cereus by Na‐chlorophyllin (Na‐Chl)‐based photosensitization in vitro and after attachment to the surface of packaging material. Methods and Results: Bacillus cereus in vitro or attached to the packaging was incubated with Na‐Chl (7·5 × 10^?8 to 7·5 × 10^?5 mol l^?1) for 2–60 min in phosphate buffer saline. Photosensitization was performed by illuminating cells under a light with a λ of 400 nm and an energy density of 20 mW cm^?2. The illumination time varied 0–5 min and subsequently the total energy dose was 0–6 J cm^?2. The results show that B. cereus vegetative cells in vitro or attached to the surface of packaging after incubation with 7·5 × 10^?7 mol l^?1 Na‐Chl and following illumination were inactivated by 7 log. The photoinactivation of B. cereus spores in vitro by 4 log required higher (7·5 × 10^?6 mol l^?1) Na‐Chl concentration. Decontamination of packaging material from attached spores by photosensitization reached 5 log at 7·5 × 10^?5 mol l^?1 Na‐Chl concentration. Comparative analysis of different packaging decontamination treatments indicates that washing with water can diminish pathogen population on the surface by <1 log, 100 ppm Na‐hypochlorite reduces the pathogens about 1·7 log and 200 ppm Na‐hypochlorite by 2·2 log. Meanwhile, Na‐Chl‐based photosensitization reduces bacteria on the surface by 4·2 orders of magnitude. Conclusions: Food‐borne pathogen B. cereus could be effectively inactivated (7 log) by Na‐Chl‐based photosensitization in vitro and on the surface of packaging material. Spores are more resistant than vegetative cells to photosensitization‐based inactivation. Comparison of different surface decontamination treatments indicates that Na‐Chl‐based photosensitization is much more effective antibacterial tool than washing with water or 200 ppm Na‐hypochlorite. Significance and Impact of the Study: Our data support the idea that Na‐Chl‐based photosensitization has great potential for future application as an environment‐friendly, nonthermal surface decontamination technique.     

Photosensitization in cattle grazing on pastures of Brahciaria decumbens Stapf infested with Pithomyces chartarum (Berk. & Curt.) M.B. Ellis

Aspects of photosensitization in bovines grazing on pastures of Brachiaria decumbens Stapf infested with Pithomyces chartarum (Berk. & Curt.) M.B. Ellis infested all pastures 45(2):117-136, 1978. This paper reports experimental studies on photosensitization in bovines grazing on different pastures of Brachiaria decumbens Stapf in the "Cerrados" region (Planaltina, DF). Climatic conditions, zinc content and occurence of fungi on pastures were investigated. Pithomyces chartarum (Berk. & Curt.) M.B. Ellis infested all pastures examined. Photosensitization was observed in one animal maintained on a pasture of B. decumbens formed with seeds from Australia. Clinical and necropsy data were similar to those related in literature for sporidesmin-intoxicated animals. An isolate of P. chartarum and samples of bovine bile were assayed for sporidesmin presence.     

Specificity of mutations induced by riboflavin mediated photosensitization in the supF gene of Escherichia coli

Riboflavin-mediated photosensitization has been shown to produce 8-hydroxyguanine (oh⁸Gua) in DNA. We investigated the specificity of mutation of photosensitized supF gene induced in Escherichia coli. The oh⁸Gua repair deficient E. coli mutant mutM and mutY were transformed with plasmid pUB3 carrying the supF gene irradiated with white light in the presence of riboflavin. Under these conditions, riboflavin photosensitization increased the amounts of oh⁸Gua in pUB3 DNA. Three types of a single base substitution occurring at G:C pairs were detected in both wild-type and mutM mutant strains. Almost all base substitutions were transversions to T:A or C:G pairs occurring at a similar extent in both wild-type and mutM strains. Mutations derived from mutY strain transformed with photosensitized DNA were only G:C to T:A transversions. These G:C to T:A transversions observed in the mutY strain were suggested to be the result of mispairing of oh⁸Gua with adenine. Riboflavin-mediated photosensitization may also produce lesions on DNA causing G:C to C:G changes by unknown mechanisms.