MECHANISM OF HIGH POWER PICOSECOND LASER UV INACTIVATION OF VIRUSES AND BACTERIAL PLASMIDS

Abstract— The mechanism of inactivating action of high-power picosecond laser UV radiation (λ= 266 nm) on the λ and φX174 bacteriophages and the pBR 322 plasmid has been studied. It has been shown that at UV radiation intensities from 10¹¹ to 10¹³ W/m², inactivation of viruses and bacterial plasmids occurs mainly on account of single-strand breaks in the DNA chain unlike the case of less powerful UV radiation where the inactivation is associated with the formation of pyrimidine dimers.     

ANTIVIRAL ACTIVITY OF THE PHOTOACTIVE THIOPHENE α-TERTHIENYL

The naturally occurring thiophene, α-terthienyl, was investigated for phototoxicity against several viruses and a line of mouse cells. The compound was extremely phototoxic to the two-membrane-containing animal viruses, murine cytomegalovirus (MCMV) and Sindbis virus (SV). Antiviral activity was detected at 10⁵μg/m in the presence of UVA. However, no effect was seen in the absence of UV-A, even at 0.1 μg/m of αT. Mouse cells were much more resistant to αT, as was the bacterial virus T4, which does not contain a membrane. Murine CMV, which had been inactivated by αT and UVA, penetrated mouse cells efficiently; but the viral DNA could not replicate, and late viral proteins were not made. Thus viral gene expression was inhibited in the photoinactivated virus. In order to account for all these data we suggest that αT may interact with viral proteins in addition to membrane lipids.     

Inactivation of Trypanosoma cruzi Trypomastigote Forms in Blood Components with a Psoralen and Ultraviolet A Light

Inactivation of the blood-borne parasite Trypanosoma cruzi by UVA and 4'-aminomethyl-4,5',8-trimethylpsor-alen (AMT) was studied in the blood components fresh frozen plasma (FFP) and platelet concentrate (PC). The AMT was utilized at a concentration of 50 μg/mL and the inactivation procedure included the flavonoid rutin (at 0.35 mM), a quencher of type I and type II photo-reactants, which we have previously found to maintain platelet integrity during this treatment regimen. Within both FFP and PC, complete inactivation of the infective form of T. cruzi , the trypomastigote, was achieved at a UVA (320–400 nm radiation) fluence of 4.2 J/cm². We note that while the infectivity of the parasite is eliminated at 4.2 J/cm^Z the trypomastigote motility continues for at least 16 h post-treatment and is inhibited only after much higher light doses. Isolation of total DNA from the parasite cells after treatment in the presence of ³H-AMT indicated that at the lethal UVA fluence about 0.5 AMT adducts per kilobase pairs occurred. These results suggest that this psoralen plus UVA methodology, which shows promise in enhancing the viral safety of PC, may in addition eliminate bloodborne T. cruzi , the causative agent of Chagas disease.     

INACTIVATION OF LIPID-CONTAINING VIRUSES BY HYDROPHOBIC PHOTOSENSITIZERS AND NEAR-ULTRAVIOLET RADIATION

Abstract—The hydrophobic photosensitizers acridine and phenothiazine inactivate the lipid-contnining viruses PM2,φ6, and herpes simplex when samples are illuminated with near-UV radiation. φ23–1- a . which is insensitive to organic solvents and presumably contains no lipids. is not inactivated under comparable conditions. For acridinc, the inactivation of virus requires that oxygen be present and is inhibited by sodium azide, implicating the involvement of singlet oxygen. For phenothiazine, oxygen is not required for photosensitized inactivation. Treatment of PM2 with acridine and near-UV light caused a complete disruption of the virion, as determined by sucrose gradient analysis of treated and untreated samples. These data and related observations suggest that lipid-containing viruses are inactivated through photosensitized membrane damage.     

ANALYSIS OF VIRAL DNA, PROTEIN AND ENVELOPE DAMAGE AFTER METHYLENE BLUE, PHTHALOCYANINE DERIVATIVE OR MEROCYANINE 540 PHOTOSENSITIZATION

Abstract— Although numerous photosensitizers have been used experimentally to decontaminate viruses in cellular blood components, little is known about their mechanisms of photoinactivation. Using M13 bacteriophage and vesicular stomatitis virus (VSV) as model viruses, we have investigated alteration of the viral genome, protein and envelope after phototreatment. Methylene blue (MB) and aluminum phthalocyanine tetrasulfonate (AlPcS₄) phototreatment inactivated bacteriophage M13 and decreased the fraction of single-stranded circular genomic DNA (sc-DNA) by converting it to linear form. This conversion was enhanced by treating the extracted DNA with piperidine at 55°C. Piperidine-labile breaks were well correlated to phage survival (5.1% sc-DNA at 1.7% phage survival for MB) under conditions where only minor differences were seen in the relative abundance of M13 coat protein on sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE). Neither aluminum phthalocyanine (AlPc) nor merocyanine 540 (MC540) inactivated M13 nor were there significant changes observed in DNA and coat protein. Methylene blue, AlPcS₄ and AlPc inactivated VSV and inhibited fusion of the virus envelope to Vero cells at pH 5.7 (i.e. with plasma membrane). However, the degree of this inhibition was small compared to the extent of virus inactivation (43% inhibition vs 4.7 log10 or 99.998% inactivation, for MB). In contrast, an antibody to VSV G-spike protein inhibited fusion at pH 5.7 by 52% with a concomitant decline in VSV infectivity of 0.15 log₁₀ (30%). Few changes were observed in the relative abundance of G protein for MB and AlPcS₄ phototreated samples and no additional protein bands were observed on SDS-PAGE. Phototreatment did not appreciably change the relative fusion ability at pH 7.2 (via the endocytotic pathway). These results collectively suggest that nucleic acid may be an important target for photoinactivation of these model viruses by MB and AlPcS₄.     

DETERMINATION OF RESIDUAL 4-AMINOMETHYL-4,5',8-TRIMETHYLPSORALENAND MUTAGENICITY TESTING FOLLOWING PSORALEN PLUS UVA TREATMENT OF PLATELET SUSPENSIONS

Abstract— Psoralens and UVA light have been used in the laboratory to study the inactivation of viruses that may be infrequently present in platelet concentrates that are prepared for transfusion. In order to evaluate safety aspects of the treatment of platelet suspensions with 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), we have investigated the residual levels and mutagenic potential of AMT after UVA phototreatment. 4'-aminomethyl-4,5',8-trimethylpso-ralen, at a final concentration of 40 μg/mL, was added to platelet suspensions which contained 16% plasma and a synthetic medium. Platelet suspensions containing AMT were irradiated with up to 7.2 J/cm² UVA light under normal oxygen levels. Residual levels of AMT were determined by HPLC and a bioassay based on bacteriophage 0.6 inactivation. The photodestruction of AMT or its activity by UVA was characterized by a D₃₇ value of 0.6 and 0.3 J/cm² with HPLC or bioassay, respectively. At 2.4 J/cm² UVA, which results in approximately 5 log₁₀ inactivation of vesicular stomatitis virus (VSV) and retention of platelet in vitro properties, 12% (HPLC) to 9% (bioassay) AMT remained. Like other psoralens, AMT was found to bind to serum proteins as shown by ultrafiltration. Results are consistent with approximately 36% of the initial drug load binding primarily to serum albumin. It was determined using ³H-AMT that 9 to 18% of radioactivity was bound to platelets in the absence of irradiation. Similar fractions (13 to 18%) of AMT were bound to platelets after 3.6 J/cm² UVA irradiation, and 8 to 10% of total AMT was associated with saline-washed irradiated platelets and is presumably tightly bound. Mutagenicity testing (Ames test, in the absence of UVA) was also carried out on the UVA irradiated platelet samples. With Salmonella tester strains which detect primarily base substitution mutations (TA100, TA1535 and TA102), no increase from background mutagenesis levels was observed with any of the samples. However, tester strains which detect frameshift mutations (TA98, TA1537, and TA1538) displayed significant increases in histidine revertants over background levels for irradiated and non-irradiated AMT-containing samples tested in the presence of S9 microsomal enzymes. In the absence of S9 activation, a mutagenic response was observed only with tester strain TA1537. All frameshift tester strains exhibited decreased numbers of induced revertants with lower residual AMT concentrations (which correlated with higher UVA dose). Significant mutagenesis was still observed for platelet suspensions irradiated with virucidal levels of UVA which maintain platelet in vitro function (2.4 J/cm²). These results suggest that residual available AMT is mutagenic in the Ames test and that the observed frameshift mutations may be caused by binding of AMT or its metabolites to nucleic acids in the absence of UVA light.     

DETERMINATION OF PARAMETERS OF EXCITED STATES OF DNA and RNA BASES BY LASER UV PHOTOLYSIS

Abstract— The mechanism of photodecomposition of nucleic acid bases in a neutral aqueous solution upon two-step excitation of high-lying electronic states by a powerful laser UV radiation is discussed. Experimental dependences of photodecomposition efficiency versus UV radiation intensity are measured both under picosecond and nanosecond laser UV irradiations. By comparison of experimental dependences with a theoretical model, we obtain some characteristics of excited states, such as lifetime t₁ of the first electronic excited state S₁ intersystem crossing yield φ, photosensitivity from an intermediate excited state and others for all five nucleic acid bases.     

INACTIVATION OF A RHIZOBIUM BACTERIO-PHAGE BY ULTRAVIOLET RADIATION OF DIFFERENT WAVE-LENGTHS

Abstract— The action spectrum for inactivation of a Rhizobiurn bacteriophage by U.V. radiation follows the shape of the absorption spectrum of DNA between the wave-lengths of 240 and 290 mμ (where inactivation probably reflects damage to the nucleic acid only), and deviates sharply upwards at wave-lengths shorter than 240 mμ (where inactivation may depend on damage to both the nucleic acid, and protein of the phage). The rate of inactivation follows first order kinetics approximately at all wave-lengths tested. Infectivity of the phage is halved when each mg of the phage nucleic acid has absorbed about 0 08 J of radiation energy at any wave-length between 240 and 290 mμ.
The bacteriopharge can be photoreactivated after inactivation at any wave-length between 230 and 290 mμ, but less so after inactivation at 230 mμ than at any wave-length above 240 mμ. No evidence was found to suggest that dimerization of thymine residues of the phage-DNA plays any part in the mechanism of inactivation of the bacteriophage by U.V. radiation.     

PHOTOPHYSICAL AND PHOTOSENSITIZING PROPERTIES OF 2-AMINO-4 PTERIDINONE: A NATURAL PIGMENT

Abstract— The characteristics of the fluorescence and phosphorescence emission of 2-amino-4 (3H) pteridinone (or pterin) in aqueous solutions are pH dependent. The room temperature fluorescence quantum yield is low and is maximum at pH = 10 (φ_F∼ 0.057). The 77K phosphorescence emission consists of two overlapping emissions originating from τ* triplet states. In agreement with low temperature results, the 353nm laser flash photolysis makes it possible to detect at pH 9.2, two transient triplet absorptions (τ₁∼ 0.3 μs and τ₂∼ 2.3 μs). The longer lived triplet is characterized by φ_TM∼ 0.20 and ∼ (550nm) = 2000 M ⁻¹ cm⁻¹. It reacts with the solvent forming the semireduced pterin with a quantum yield φ_R∼ 0.06. The photosensitizing properties of pterin have been studied by laser flash spectroscopy and steady state irradiations. Photoreactions implying singlet oxygen formation are shown to occur. Laser flash spectroscopy indicates that the pterin triplet is reduced by amino acids and nucleic acid bases. Corresponding bimolecular reaction rate constants have been measured.     

NON-NUCLEAR DAMAGE AND CELL LYSIS ARE INDUCED BY UVA, BUT NOT UVB OR UVC, RADIATION IN THREE STRAINS OF L5178Y CELLS

The potential to induce non-nuclear changes in mammalian cells has been examined for (1) UVA1 radiation (340–400 nm, UVASUN 2000 lamp), (2) UVA + UVB (peak at 313 nm) radiation (FS20 lamp), and (3) UVC (254 nm) radiation (GI5T8 lamp). The effects of irradiation were monitored in vitro using three strains of L5178Y (LY) mouse lymphoma cells that markedly differ in sensitivity to UV radiation. Comparisons were made for the effects of approximately equitoxic fluences that reduced cell survival to 1–15%. Depending on the cell strain, the fluences ranged from 830 to 1600 kJ/m² for the UVASUN lamp, 75 to 390 J/m² for the FS20 lamp and 3.8 to 17.2 J/m² for the G15T8 lamp. At the exposure level used in this study, irradiation with the UVASUN, but not the FS20 or G15T8, lamp induced a variety of non-nuclear changes including damage to cytoplasmic organelles and increased plasma membrane permeability and cell lysis. Cell lysis and membrane permeabilization were induced by the UVA1 emission of the UVASUN lamp, but not by its visible + IR components (>400 nm). The results show that the plasma membrane and other organelles of LY cells are highly sensitive to UVA1 but not to UVB or UVC radiation. Also UVA1, but not UVB or UVC radiation, causes rapid and extensive lysis of LY cells. In conclusion, non-nuclear damage contributes substantially to UVA cytotoxicity in all three strains of LY cells.     

SINGLET OXYGEN INVOLVEMENT IN THE INACTIVATION OF CULTURED HUMAN FIBROBLASTS BY UVA (334 nm, 365 nm) AND NEAR-VISIBLE (405 nm) RADIATIONS

The UVA (320-380 nm) radiation inactivation of mammalian cells is dependent upon the presence of oxygen. In order to examine the intermediates involved, we have irradiated cells in the presence of chemical probes which are able to modify the activity of various oxygen species. We have also examined the possibility that UVA inactivates cultured human fibroblasts via generation of intracellular hydrogen peroxide. An iron scavenger (desferrioxamine) and a hydroxyl radical scavenger (dimethylsulfoxide) protect the cells against hydrogen peroxide. Diethyldithiocarbamate (a superoxide dismutase inhibitor) and aminotriazole (a catalase inhibitor) sensitize the cells to this oxidizing agent. These data support previous reports that hydrogen peroxide inactivates as a result of the iron-catalyzed generation of hydroxyl radical. None of these agents significantly alter the fluence-dependent inactivation of cell populations by radiation at 365 nm. In contrast, the cells are sensitized to radiation at 334, 365 and 405 nm in the presence of deuterium (an enhancer of singlet oxygen lifetime) and are protected against radiation at 365 nm by sodium azide (a quencher of singlet oxygen). These results are consistent with the conclusion that the generation of singlet oxygen, but not hydrogen peroxide or hydroxyl radical, plays an important role in the inactivation of cultured human cells by UVA and near-visible radiations.     

THE ANTI-HIV ACTIVITIES OF PHOTOACTIVE TERTHIOPHENES

Abstract— Various synthetic analogues of the naturally occurring terthiophene, α-terthienyl (αT), were evaluated for anti-human immunodeficiency virus (HIV) activity. The compounds were incubated individually with a known amount of the virus, with or without UVA radiation (long-wavelength ultraviolet) and residual virus was monitored for its ability to produce cytopathic effects in cell culture and the production of virus-specific protein (p24). The basic terthiophene structure was essential for good anti-HIV activity, although various side chains, such as alcohols, bromo, methyl, thiomethyl and trimethylsilyl groups, permitted retention of maximum activity. Under optimum conditions, as little as 12 ng/mL of these compounds ( i.e approximately 3 × 10⁻⁸ M ) could inactivate 10³ infectious virions. None of the compounds however were more active than αT itself. In all cases, UVA radiation was essential. Several side chains decreased the antiviral efficacy, and some side chains abrogated the activity.     

Photochemical inactivation of alpha- and poxviruses

The objective of this study was to determine whether photochemical inactivation of viruses could be accomplished with high efficiency while preserving the molecular integrity of viral targets allowing subsequent diagnostic tests to be performed at a lower level of containment and cost. We studied the effect of 5-iodonaphthyl 1-azide (INA) and amotosalen (AMO, also known as S-59), which are photochemicals known to target either viral proteins or nucleic acids, respectively. We found that vaccinia virus (VACV, an orthopox virus with a DNA genome) and pixuna virus (PIXV, an alphavirus with an RNA genome) were stable when irradiated with UVA alone or when exposed to either INA or AMO in the dark. AMO followed by UVA exposure was at least 1000-fold more virucidal than INA/UVA on vaccinia and pixuna viruses treated under similar conditions. Photoinactivation with either INA or AMO at conditions that abolished viral infectivity resulted in only minimal impairment of subsequent ELISA and PCR testing. The results presented in this study should assist in developing methods to inactivate in the field environmental and forensic samples suspected of viral contamination, thus limiting the need for costly security and safety operations after an accidental or intentional viral release.     

Comparison of the efficiency and the specificity of DNA-bound and free cationic porphyrin in photodynamic virus inactivation

The risk of transmitting infections by blood transfusion has been substantially reduced. However, alternative methods for inactivation of pathogens in blood and its components are needed. Application of photoactivated cationic porphyrins can offer an approach to remove non-enveloped viruses from aqueous media. Here we tested the virus inactivation capability of meso-Tetrakis(4-N-methylpyridyl)porphyrin (TMPyP) and meso-Tri-(4-N-methylpyridyl)monophenylporphyrin (TMPyMPP) in the dark and upon irradiation. T7 bacteriophage, as a surrogate on non-enveloped viruses was selected as a test system. TMPyP and TMPyMPP reduce the viability of T7 phage already in the dark, which can be explained by their selective binding to nucleic acid. Both compounds proved to be efficient photosensitizers of virus inactivation. The binding of porphyrin to phage DNA was not a prerequisite of phage photosensitization, moreover, photoinactivation was more efficiently induced by free than by DNA bound porphyrin. As optical melting studies and agarose gel electrophoresis of T7 nucleoprotein revealed, photoreactions of TMPyP and TMPyMPP affect the structural integrity of DNA and also of viral proteins, despite their selective DNA binding.     

The Photobiological Differences of Gilvocarcins V and M Are not Related to their Transient Intermediates and Triplet Yields

Abstract— The transient absorption spectra of the intermediates produced by the 355 nm laser excitation of gilvocarcin derivatives have been investigated in various solvents. The spectra consist of a triplet-triplet absorption in the visible region and a residual absorption observed between 340 and 700 nm due to a long-lived species, assigned to the radical cation. A broad-fast decaying band with a maximum at around 700 nm attributed to the solvated electron is also seen in solutions containing a low DMSO/water volume ratio and at 266 nm irradiation of a 50% methanol/water solvent mixture. The molar absorption coefficient of the triplet state of gilvocarcin V (GV) and gilvocarcin M (GM), determined by the energy transfer method, is independent of the solvent properties and has a value of 3.0 × 10⁴/ M cm. The triplet decay rate constants for both drugs are between 1 and 5 × 10⁴/s. A similar initial yield and triplet decay rate constant of GV were observed in the presence of 3.4 m M thymine. Thus, a quenching rate constant of the GV's triplet state by thymine is estimated to be lower than 10⁶/Ms. The triplet quantum yields of both antibiotics determined by using the comparative method are higher in dimethylsulfoxide (DMSO) (0.18) than are those corresponding to 25% DMSO/water (0.06). The decrease in φ_T in the presence of water could be attributed to an enhanced internal conversion rate constant from the S₁ state or to an increase in the photoionization yield. The similarity of the transient intermediates and their yields for GV and GM suggest that their photobiological differences are due to other factors such as DNA binding constants, preferential localization of the drugs in the cell or the enhanced reactivity of the vinyl group toward cellular components.     

SINGLET OXYGEN GENERATION BY HEMATOPORPHYRIN IX, UROPORPHYRIN I and HEMATOPORPHYRIN DERIVATIVE AT 546 nm IN PHOSPHATE BUFFER and IN THE PRESENCE OF EGG PHOSPHATIDYLCHOLINE LIPOSOMES

Abstract— The quantum yield of singlet oxygen generation (φ) was measured at 546 nm with the p -nitrosodimethylaniline (RNO) method. The results obtained in pH 7.4 phosphate buffer (PB) were: φ(HP) = 0.44 ± 0.05: φ= 0.71 ± 0.07: φ(HpD-A) = 0.06 ± 0.02. The value of φ was constant from 3 to 67 μM , attributed to the dominance of HP dimers; φ (HP) increased to 0.77 ± 0.13 in the presence of small egg phosphatidylcholine liposomes (SUV), attributed to solubilization and monomerization: φ (HpD-A) increased to 0.87 ± 0.17 in the presence of SUV. attributed to monomerization of the impurity porphyrins and unfolding of the covalent dimer constituents. The quantum efficiency of the RNO system (100 μ M RNO plus 10 mM histidine) was approximately 0.015 at pH 7.4 and increased significantly at lower pH.     

THE PHOTOACTIVE ANTIMICROBIAL PROPERTIES OF EUDISTOMINS FROM THE CARIBBEAN TUNICATE Eudistoma olivaceum

Abstract— Five eudistomins, β-carboline derivatives isolated from a Caribbean tunicate, were tested for phototoxicity against several viruses, bacteria, yeast, and mammalian cells. The five compounds showed varying degrees of UVA dependant phototoxicity (i.e. long wavelength UV dependant) against murine cytomegalovirus (MCMV), Sindbis virus (SV) and mouse 3T3 cells, although the relative order of potency was the same for these three organisms. Eudistomin N was the most active (approximately the same as the β-carboline, harmine), while eudistomins M and O were moderately phototoxic, and H and I had little activity. To some degree the relative phototoxicity was correlated with fewer side chain substituents. A similar relative order of phototoxic potency was seen against phage T4, but in this case the magnitude of the effect was considerably reduced, in contrast to harmine. The antibacterial and antifungal activities were not correlated with antiviral effects, and some UVA-independent activities were seen. Thus the eudistomins may possess different mechanisms of action against different organisms, depending upon the presence or absence of UVA.     

CYTOTOXICITY AND MUTAGENICITY OF OPHTHALMIC SOLUTION PRESERVATIVES AND UVA RADIATION IN L5178Y CELLS

Four chemical preservatives commonly used in ophthalmic solutions were tested for their toxic and mutagenic potential in mouse lymphoma cells with and without exposure of the cells to ultraviolet A (UVA) radiation. The preservatives tested were benzalkonium chloride (BAK), chlorhexidine, thimerosal and ethylenediaminetetraacetic acid (EDTA). Cell survival and mutagenesis were measured using the L5178Y mouse lymphoma (TK +/-) system. Cells were exposed to varying amounts of preservatives for 1 h at 37 degrees C, and then aliquots were irradiated with UVA radiation (during the exposure to preservative). Cells were then assayed for survival, and for mutagenesis at the thymidine kinase (TK) locus. In concentrations commonly found in ophthalmic solutions, BAK, chlorhexidine, and thimerosal were toxic to cells, and thimerosal was slightly mutagenic. When cells were exposed to preservative and UVA radiation, chlorhexidine was mutagenic and the mutagenic activity of thimerosal was enhanced.     

KINETICS OF SIMIAN VIRUS 40 AND LAMBDA INACTIVATION BY PHOTOADDITION OF PSORALEN DERIVATIVES

Abstract The kinetics of psora/en photoinactivation of two distinct DNA viruses, bacteriophage λ and the papovavirus SV40 were investigated. When λ is treated with near ultraviolet light (UVA, 320-400 nm) and 4,5',8-trimethylpsoralen (TMP) at 1 μg/m/, the phage is rapidly inactivated. The survival curve exhibits a distinct shoulder indicating second or higher-order kinetics. SV40, on the other hand, is much more resistant to psoralen photoinactivation and the survival curve is linear, reflecting first order or'pseudo-first order'kinetics. Two TMP derivatives with increased solubility in aqueous solutions, 4'-aminomethyl-TMP and 4'-hydroxymethyl-TMP, were similarly tested. In both virus systems, TMP was much more effective. In experiments designed to examine the role of psoralen cross-link formation in virus inactivation, treated samples were irradiated a second time in the absence of drug. Since reirradiation causes a decline in λ infectivity as great as that observed in continuously irradiated samples, cross-links are implicated as the primary lethal event. In the case of SV40, the results of such a protocol suggest that both monoadducts and cross-links may be lethal or that monoadduct formation may be rate-limiting.