Endogenous Photosensitizers Research Articles

Ultraviolet light-induced generation of vitamin E radicals and their recycling. A possible photosensitizing effect of vitamin E in skin.

Vitamin E (alpha-tocopherol) is the major lipid-soluble chain-breaking antioxidant of membranes. Its UV-absorbance spectrum (lambda max 295 nm) extends well into the solar spectrum. We hypothesize that in skin alpha-tocopherol may absorb solar UV light and generate tocopheroxyl radicals. Reduction of tocopheroxyl radicals by other antioxidants (e.g. ascorbate, thiols) will regenerate (recycle) vitamin E at the expense of their own depletion. Hence, vitamin E in skin may act in two conflicting manners upon solar illumination: in addition to its antioxidant function as a peroxyl radical scavenger, it may act as an endogenous photosensitizer, enhancing light-induced oxidative damage. To test this hypothesis, we have illuminated various systems (methanol-buffer dispersions, liposomes and skin homogenates) containing alpha-tocopherol or its homologue with a shorter 6-carbon side chain, chromanol-alpha-C6 with UV light closely matching solar UV light, in the presence or absence of endogenous or exogenous reductants. We found that: (i) alpha-tocopheroxyl (chromanoxyl) radicals are directly generated by solar UV light in model systems (methanol-water dispersions, liposomes) and in skin homogenates; (ii) reducing antioxidants (ascorbate, ascorbate+dihydrolipoic acid) can donate electrons to alpha-tocopheroxyl (chromanoxyl) radicals providing for vitamin E (chromanol-alpha-C6) recycling; (iii) recycling of UV-induced alpha-tocopheroxyl radicals depletes endogenous antioxidant pools (accelerates ascorbate oxidation); (iv) beta-carotene, a non-reducing antioxidant, is not active in alpha-tocopherol recycling, and its UV-dependent depletion is unaffected by vitamin E.

ACTION SPECTRA FOR THE GENERATION OF SINGLET OXYGEN FROM MITOCHONDRIAL MEMBRANES FROM SOYBEAN (Glycine max) HYPOCOTYLS

Abstract— The action spectrum for the generation of singlet oxygen (1O2) from mitochondrial membranes under aerobic conditions was measured at wavelengths between 360 and 600 nm, using sub‐mitochondrial particles (SMP) prepared from soybean hypocotyls. The spectrum, showing a peak at about 420 nm, remarkably resembles the absorption spectra of the Fe‐S centers of nonheme iron proteins. Disruption of the Fe‐S centers by treating SMP with mersalyl acid resulted in a substantial decrease in the efficiency of 1O2 generation, leaving an action spectrum whose pattern is significantly similar to the absorption spectrum of flavins, at least in the region of near UV and blue light wavelengths. Estimating the contribution of the Fe‐S centers to the generation of 1O2 from SMP, we suggest that the Fe‐S centers act as very important endogenous photosensitizers in plant cells, in so far as the type II mechanism is concerned. Possible involvement of mitochondrial flavoproteins in the generation of 1O2 is also discussed.

Bacterial Genes Involved in Response to Near-Ultraviolet Radiation

A model of the possible pathways of activities following NUV treatment was presented in Section I and in Fig. 1. Some of the components are firmly established, some are speculative, and many are difficult to evaluate because of insufficient experimental information. Perhaps the most relevant experiments, especially concerning ozone depletion, would be to determine the mutational specificity of NUV. By selecting lacI mutants after exposing cells to NUV, and sequencing the bases of this gene, this is now feasible. There are some problems, however. The mutation frequency is normally so low that it might be difficult to distinguish NUV mutants from spontaneous mutants. However, by irradiating cells having a uvrA or uvrB mutation, the frequency of mutation above background can be increased considerably. There remains the problem as to what fraction of the observed mutations results from oxidative damage. Some of this could be clarified by comparing mutation spectra of cells treated with NUV and cells subjected to excess oxidative damage and determining what fraction results from other avenues of lesion formation in DNA. Different species of reactive oxygen could cause different kinds of DNA lesions, and, fortunately, use of appropriate mutants should allow us to sort out any differences in specificity of lesions. Also, by appropriate manipulation of quantities of endogenous photosensitizers, it might be possible to sort out the specific mutations that are caused by photodynamic action. Another avenue of research is to explore the pathways by which NUV lesions are repaired, and whether such repair is error prone or error free. Again, the use of mutants such as xthA, uvr, and polA should assist in our understanding of the specificity of the mutational events. There are now a number of examples of global control mechanisms whereby cells abruptly shift their protein synthesis pattern under environmental stress. It is important to understand whether NUV stress results in induction of one or more of the known regulatory genes, or whether another regulon might be involved. One particular aspect of regulation that remains unsolved is the role of the katF gene, which is known to regulate the xthA and katE, but it may also regulate other genes as well. A number of striking physiological events occur even at very low fluences of NUV irradiation of cells. In part, this may be related to regulon induction. However, some of these events are in need of special exploration, such as changes at the membrane level.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of Chloroplast Development on the Activation of the Diphenyl Ether Herbicide Acifluorfen-Methyl

The activity of acifluorfen-methyl (AFM); methyl 5-(2-chloro-4-[trifluoromethyl] phenoxy)-2-nitrobenzoate in excised cucumber cotyledons (Cucumis sativus L.) was examined. AFM induced membrane disruption, was significantly greater when etiolated cotyledons were illuminated 16 hours at 150 microeinsteins per square meter per second photosynthetically active radiation versus incubation under illumination of 4-fold greater intensity. These results were unexpected since the loss of membrane integrity is initiated by photodynamic reactions. Untreated, etiolated cotyledons were not able to accumulate chlorophyll under the higher light intensity while control and herbicide treated cotyledons greened significantly under the lower intensity illumination suggesting that some process associated with greening stimulated AFM activity. Inhibition of greening by cycloheximide also reduced AFM activity. Intermittent lighting induced greening in AFM treated cotyledons without causing any detectable loss of plasmalemma integrity. Utilization of this system for pretreatment of cotyledons prior to continuous illumination revealed that activity was greater when tissue was greened in the presence of AFM than when herbicide treatments were made after a greening period of the same duration. The results indicate that the pigments in situ in etiolated tissue are sufficient, without greening, to initiate membrane disruption by AFM. However, greening increases the herbicidal efficacy greatly. Furthermore, the stimulation appears to be due to specific interactions between AFM and the developing plastid and is not attributable solely to an increase in endogenous photosensitizers.

Oxidative mechanisms of toxicity of low-intensity near-UV light in Salmonella typhimurium.

The exposure of Salmonella typhimurium to environmentally relevant near-UV light stress has been studied by the use of a low-intensity, broad-band light source. The exposure of cells to such a light source rapidly induced a growth delay; after continuous exposure for 3 to 4 h, cells began to die at a rapid rate. The oxidative defense regulon controlled by the oxyR gene was involved in protecting cells from being killed by near-UV light. This killing may be potentiated by the overexpression of near-UV-absorbing proteins. These results are consistent with near-UV toxicity involving the absorption of light by endogenous photosensitizers, leading to the production of active oxygen species. We have shown, however, that one such species, H2O2, is not a major photoproduct involved in killing by near-UV light. Strains lacking alkyl hydroperoxide reductase were more sensitive to near-UV light, indicating that such hydroperoxides may be photoproducts. Near-UV exposure induced sensitivity to high salt levels, indicating that membranes may be a target of near-UV toxicity and a possible source of alkyl hydroperoxides. The demonstration of the inactivation of the heme-containing protein catalase indicates that direct destruction of UV-absorbing macromolecules could be another factor in near-UV toxicity. Cells which have been exposed to near-UV light for long, but sublethal, periods of time (up to 4 h) can recover and resume growth if the UV exposure is stopped but become progressively more sensitive to further stresses, such as H2O2. This result indicates that cells gradually accumulated damage during near-UV exposure until toxic levels were reached.

Light on the persistent light reaction—photosensitivity dermatitis—actinic reticuloid syndrome

In this article are reviewed the various hypotheses concerning the etiology of the persistent light reaction and photosensitivity dermatitis, which occurs among older people and is characterized by an extreme photosensitivity that sometimes evolves into a pseudolymphoma, that is, actinic reticuloid. An etiologic agent can sometimes be demonstrated, but the precise pathogenetic mechanism is unknown. Clinical experience and experimental research indicate that the cause is most probably multifactoral: contact allergenic, photoallergenic, phototoxic, immunologic, and metabolic factors are involved, but their precise roles in the origin of the extreme photosensitivity are still unclear. Localized persistent light reactivity could result from the continued effects of the original photoallergen, but generalized photosensitivity is more difficult to account for. Among the many different hypotheses, two are of special interest: autosensitization of skin proteins with endogenous photosensitizers and cellular hypersensitivity to light, as is manifested by fibroblast cultures of actinic reticuloid patients.

Carotenoids quench evolution of excited species in epidermis exposed to UV-B (290-320 nm) light.

Abstract— Reactions involving singlet oxygen and other free radicals have been identified in epidermis containing either exogenous or endogenous photosensitizers. soaked in a singlet oxygen/free radical trap, and then exposed to visible or UV‐A (320‐400 nm) light. Such reactions can be quenched by the presence of the carotenoid pigments β‐carotene and canthaxanthin which accumulate in epidermis after oral administration. We report here that the carotenoid pigments β‐carotene. canthaxanthin and phytoene accumulating in epidermis can also quench to some degree those photochemical reactions involving singlet oxygen and free radicals that occur when epidermis is exposed to the sunburn spectrum of light (UV‐B. 290–320 nm).

Different (direct and indirect) mechanisms for the induction of DNA-protein crosslinks in human cells by far- and near-ultraviolet radiations (290 and 405 nm).

Abstract— Apparent DNA‐protein crosslinking induced by monochromatic 290 and 405 nm Tadiations was measured in cultured human P3 teratocarcinoma cells with DNA alkaline elution techniques. The rates of the induction of crosslinks by 290 nm radiation were the same when the cells were irradiated either aerobically or anaerobically or when the cells were in an H2O or D2O aqueous environment. With 405 nm radiation, anaerobic irradiation reduced the induction of the crosslinks (dose modifying factor is about 0.2), and about twice as many crosslinks were observed when the cells were irradiated in an environment of D2O rather than H2O. The results are consistent with the hypothesis that far‐UV radiation induces DNA‐protein crosslinks by a direct mechanism, whereas near‐UV radiation induces crosslinks via indirect photodynamic photosensitizations in which unidentified cellular endogenous photosensitizers and reactive species of oxygen are used.

The effect of visible radiations on the germination and outgrowth of Bacillus spores

The effect of visible radiations on the germination and outgrowth of spores of Bacillus subtilis MD2 and Bacillus subtilis var. niger was determined by direct observation of populations irradiated on the surface of nutrient agar. Little effect on germination (phase darkening) was found but white light prevented outgrowth of some and retarded it for all spores. Different wavebands in the visible spectrum differed in their effect on outgrowth, the greatest retardation being found for the shorter wavelengths, 410–570 nm. Outgrowth in dark controls was always greater both in number of spores outgrown and rate of outgrowth. The results are consistent with others, suggesting an effect of singlet oxygen generated from endogenous photosensitizers by visible radiation.

Photosensitivity of pigmented and nonpigmented strains ofUstilago violacea

White, pink, pumpkin, orange, and yellow strains ofUstilago violacea containing high and low levels of cytochrome c and various carotenes were subjected to high light intensities in the presence and absence of the endogenous photosensitizer toluidine blue (TB). The white strain 15.10 and the cytochrome-c-accumulating pink strain 1.C429 were completely lacking carotenes and were the most sensitive to visible light under all conditions. The phytoene-containing white strain 2.C419, the carotene-containing pink strains AB278a-1, 1.C421, and 2.C425, and pumpkin strain 2.C415 were more resistant to photodestruction in the presence and absence of TB than strains 15.10 and 1.C429. Consistently, the most light resistant strains were the high carotene, low cytochrome-c-accumulating orange, and yellow strains. The photosensitivity of strains ofU. violacea was related to the level of carotene, cytochrome c, the presence or absence of TB, and the age of the culture.

Distribution of cytochrome b photoreductions mediated by endogenous photosensitizer or methylene blue in fractions from corn and cauliflower

Light‐induced absorbance changes [LIAC; measured as Δ(A428–A410)] reflecting the reduction of a b‐type cytochrome and mediated by an endogenous blue light absorbing receptor have been proposed to be related to blue light physiology of fungi and higher plants. It has also been suggested that the same cytochrome specifically can be reduced by red light in the presence of methylene blue. We have investigated the distribution of LIAC between different membrane fractions from corn (Zea mays L.) coleoptiles and cauliflower (Brassica oleracea L.) inflorescences. The membrane fractions were obtained by differential centrifugation followed by partition in an aqueous polymer two‐phase system. By this procedure fractions rich in plasma membrane were obtained from both mitochondrial and microsomal fractions obtained by centrifugation. LIAC was by far most enriched in fractions also enriched in plasma membranes (identified by silicotungstic acid staining), but LIAC could be obtained also in other fractions. Our conclusion is that LIAC undoubtedly is caused by a b‐cytochrome bound to the plasma membrane, but that LIAC also may be due to other b‐cytochromes, one of which is probably located in the endoplasmic reticulum. Thus, the two assay procedures used for LIAC (blue and red light induced) could not disciminate between different b‐cytochromes giving rise to LIAC.

Chemical Phototoxicity in Humans12

Phototoxic reactions may be induced by endogenous or exogenous chemicals. Endogenous photosensitizers made by the body include porphyrin molecules which are responsible for the photocutaneous syndromes noted in the porphyrias. Exogenous photosensitizers may arrive on the skin through topical applications or may be distributed through the vasculature. Topical photosensitizers are found in cosmetics, medications, plants, and industrial and air pollutant emissions. Systemic photosensitizers consist primarily of therapeutic agents. Acute and chronic phototoxic reactions may occur. The acute response is generally characterized by erythema and edema followed by hyperpigmentation and desquamation. The end point of chronic damage may be cutaneous cancer formation. The mechanisms of these changes may well relate to nucleic acid injury, protein and membrane damage, and alterations in other nuclear and cytoplasmic molecular structures. However, translation of experimental information into human responses remains to be clarified. A great deal of investigation is still needed in developing assays for detecting phototoxic chemicals and for defining mechanisms involved in phototoxic events in human skin.

Photokilling of Micrococcus roseus.

Abstract. Previous work showed that the bacterium Micrococcus roseus is killed by photodynamic action when an exogenous photosensitizer is present, but when a photosensitizer is not added the organism survives long exposure to high intensity (22,000ft‐c, 348 J/s/m2) white light. Experiments designed to demonstrate the presence of a mechanism to repair damage caused by photodynamic action failed to reveal such a mechanism. However, the organism was killed by light of a very high intensity (32,000ft‐c, 506 J/s/m2) in the absence of added photosensitizer, indicating that cells have an effective endogenous photosensitizer(s). Two carotenoid‐deficient mutants were killed via photodynamic action more rapidly than the fully pigmented wild‐type in the presence or absence of an exogenous photosensitizer. Thus, resistance of M. roseus to photodynamic action is not due to a repair mechanism, nor to lack of an effective endogenous photosensitizer, but to the protective action of carotenoid pigments.

Photosensitization of human diploid cell cultures by intracellular flavins and protection by antioxidants.

Abstract— The damaging effects of near ultraviolet and visible light on WI‐38 human diploid lung fibroblasts were investigated. WI‐38 cells in culture were killed by light doses ranging from 2 to 10 × 103 W/m2 h. There was an inverse correlation between culture age, i.e. population doubling level and photosensitivity. However, this effect could not be related to capacity for DNA synthesis and cell division.Flavins were clearly implicated as endogenous photosensitizers, and antioxidants such as d, l‐α‐tocopherol (vitamin E), BHT and ascorbic acid were found to afford the cells protection from light damage. Furthermore, products of lipid peroxidation could be detected in cell homogenates irradiated in the presence of ribofiavin.

Action of visible light on enzymes in cell envelopes of Micrococcus roseus.

Abstract— Envelopes were isolated from the carotenogenic bacterium Micrococcus roseus, which is subject to photodynamic killing in the presence of a photosensitizing dye but not in the absence of such a dye. Envelope preparations contained 88 per cent of the total cellular carotenoids, 20% of the NADH oxidase, 100 per cent of the ATPase and 30% of the succinic dehydrogenase activity. NADH oxidase activity in envelopes was stimulated 2–5‐fold by light in the presence of dye; this was followed by inactivation. In the presence of dye, ATPase was inactivated by light and 25 per cent of the succinic dehydrogenase activity was lost. In the absence of dye, responses were extended over a longer period of time, but similar patterns were observed for the three enzymes, indicating that envelopes contain an endogenous photosensitizer(s). Carotenoid‐deficient cells were obtained after growth in medium containing diphenylamine. But all three enzymes showed evidence of instability in envelope preparations, indicating that diphenylamine affects membrane structure in addition to inhibiting synthesis of colored carotenoids.

Intracellular pH.

Intracellular pH.

Lethal photosensitization of microorganisms with light from a continuous-wave gas laser.

Abstract— Eleven strains of microorganisms were examined for their susceptibility to death by irradiation with 21–30 mW of light at 6328 Å from a continuous‐wave gas laser. Washed cells of seven species were rapidly killed when irradiated aerobically in aqueous solutions of toluidine blue: Sarcina lutea (two strains), Escherichia coli, Chromobacterium violaceum, Arthrobacter atrocyanus, Pseudomonas aeruginosa, Saccharomyces cerevisiae, and Rhodotorula glutinis. Cells remained viable when irradiated anaerobically in toluidine blue. This is not proof that oxygen is required for death since toluidine blue is subject to reduction to the leuco form under anaerobic conditions. None of the microbes tested, including cells of Bacillus cereus, Rhodospirillum rubrum, and Euglena gracilis, were killed in aerated suspensions without toluidine blue. Thus, it is unlikely that any strain has an endogenous photosensitizer capable of acting at 6328 Å. However, more than 95 per cent of the cells of R. rubrum lost their motility. A colorless mutant of S. lutea began dying 10 min faster than the wild strain, which is known to be protected from photosensitized death by carotenoids for at least 2 hr when irradiated in toluidine blue with 1000 ft candles of tungsten light. Since toluidine blue is bleached in irradiated cell suspensions, logarithmic death ceases within an hour with 2·5 × 10=6 M toluidine blue. Yeasts are less susceptible than bacteria, exhibiting longer lags before death begins. The size of colonies produced by irradiated yeast cells decreases with irradiation time. This suggests that multiple hits are required for death.

Light-induced lysis and carotenogenesis in Myxococcus xanthus.

Burchard, Robert P. (University of Minnesota, Minneapolis), and Martin Dworkin. Light-induced lysis and carotenogenesis in Myxococcus xanthus. J. Bacteriol. 91:535-545. 1966.-Myxococcus xanthus, grown vegetatively in the light, developed an orange carotenoid after the cells entered stationary phase of growth; pigment content increased with age. Cells grown in the dark did not develop carotenoid and could be photolysed by relatively low-intensity light only during stationary phase; rate of photolysis increased with age. Photolysis adhered to the reciprocity law, was temperature-independent and oxygen-dependent, and required the presence of nonspecific, monovalent cations; it was inhibited by one of several divalent cations. Logarithmic-phase cells were photosensitized by 100,000 x g pellet preparations of sonic-treated stationary-phase cells grown in the light and dark. A porphyrin with a Soret band at 408 mmu was isolated from photosensitive cells; logarithmic-phase cells contained about 1/16 the amount of porphyrin of stationary-phase cells. The purified material had spectral and chemical properties of protoporphyrin IX and photosensitized logarithmic-phase cells. Its spectrum was similar to the action spectrum for photolysis. We concluded that protoporphyrin IX is the natural endogenous photosensitizer. Carotenogenesis was stimulated by light in the blue-violet region of the visible spectrum and was inhibited by diphenylamine, resulting in photosensitivity of the cells. Photoprotection by carotenoid was lost in the cold. A mutant which synthesized carotenoid in the light and dark was photosensitive only after growth in diphenylamine. The ecological significance of these phenomena is discussed.

The function of the carotenoid pigments of Sarcina lutea.

It has been demonstrated that the carotenoid pigments of Sarcina lutea prevent photodynamic killing by either exogenous or endogenous photosensitizers. Evidence has been presented which suggests that the locus of the lethal photooxidation is in or on the cell membrane.

Function of carotenoids in photosynthetic bacteria.

A RELATION between photosensitivity and the loss of carotenoid pigments has been observed in Rhodopseudomonas 1, Chlorella 2, Chromatium 3, Chlamydomonas 4, Rhodospirillum 5, Corynebacterium 6 and Zea mays 7. Sistrom et al. have suggested that photosensitization in the carotenoidless mutant of Rhodopseudomonas spheroides ‘… is a specific consequence of the replacement of carotenoid pigments with a colourless polyene.’ and that ‘… the primary function of carotenoid pigments in phototrophs is to act as chemical buffers against photo-oxidation of other cell constituents by chlorophyll, thus conferring a high degree of immunity to endogenous photosensitization.’8 The carotenoid may carry out this buffering function by acting as a preferentially photo-oxidizable substrate. The effectiveness of such a photochemical buffering system would depend on the existence of an enzymatic mechanism which could reduce the photo-oxidized carotenoid. Inhibition of this enzymatic reduction should result in photosensitization. Accordingly, the effect of low temperature on the photosensitivity of wild type R. spheroides (2.4.1) and Rhodospirillum rubrum was investigated.