Oxygen toxicity

Oxygen has been known since the eighteenth century to be poisonous.1 All organisms that use oxygen and are therefore exposed to it and to its various reduction products must protect themselves against oxygen toxicity. It is hardly necessary to point out that oxygen is toxic to anaerobic microorganisms, though its effect on these varies from immediate cell death to bacteriostasis. It is less obvious, however, that oxygen is poisonous to aerobic organisms, as well. High concentrations of oxygen, above the normal atmospheric level of 21%, slow plant growth2, kill cultured animal cells3, and increase the rate of occurrence of blindness in premature infants4. Conversely, lowering ambient oxygen levels below 21% speeds the growth rate of both plants and cultured animal cells. Hyperbaric oxygen causes chromosome breakage in grain5 and mutations in bacteria6. Breathing pure oxygen is so toxic that there has been at least one human fatality resulting from medical treatment with hyperbaric oxygen7.

Figure 1. The partial reduction products of oxygen.8


Free radical theory of oxygen toxicity

The causes of the poisonous properties of oxygen were obscure before the publication of Gershman's free radical theory of oxygen toxicity, which states that the toxicity of oxygen is due to partially reduced forms of oxygen9. The unusually high concentrations of oxygen found in tissues during exposure to pure oxygen encourages the formation of these reduced oxygen species. The toxic species are formed by one-electron reductions of oxygen by compounds and enzymatic reactions occurring in vivo8(fig. 1). These reduction products include superoxide (O2-), hydrogen peroxide (H2O2), and the hydroxyl radical (OH.). These are commonly referred to as active oxygen species. Ordinary dioxygen is a biradical, since its two free electrons exist in two different orbitals and have parallel spins8. Dioxygen's reactivity, however, is greatly lowered by the spin restriction: both of these electrons are of the same spin, so that if oxygen is to oxidize another molecule by accepting a pair of electrons from it, that pair must be of antiparallel spin, which cannot occur if both electrons occupy the same orbital. The spin restriction is removed when dioxygen is excited to the state known as singlet oxygen. In the cell, the spin restriction is removed enzymatically by complexing the dioxygen with transition metals, which results in a delocalization of the electrons. However, the most common biological reductions of oxygen occur by the transfer of single electrons.10 All of these forms of oxygen are reactive as oxidants and reductants to a greater or lesser extent with a wide variety of compounds, both in vitro and in vivo.


Sources of superoxide

Superoxide is one of the species that has been implicated in the toxicity of oxygen. Generation of O2- in vitro has been observed in a number of different experimental systems. These systems include the enzyme xanthine oxidase acting upon the substrates xanthine or hypoxanthine11,12, and the reduction of flavin mononucleotide and flavodoxin by ferredoxin-TPN+ oxidoreductase13. Aqueous O2- may also be generated in vitro by dissolving tetramethyl-ammonium superoxide or potassium superoxide in water. Subcellular components such as cell nuclei14, 15, chloroplasts16,17, mitochondria18, human monocytes and neutrophils19, macrophages20, and microsomes21 have been demonstrated to produce O2- in vitro. The generation of O2- by phagocytic cells has a microbicidal action essential to normal phagocytosis22. The autoxidation of many biological compounds results in the production of O2-; among these compounds are oxyhemoglobin, which becomes methemoglobin23, oxymyoglobin, which becomes metmyoglobin24, clostridial and spinach ferredoxins25, ascorbate, catecholamines, phenols, and reduced flavins26. Other circumstances in which O2- is generated include the photolysis of water27, and the excitation of carboquone, mitomycin c, and streptonigrin by visible light in the presence of oxygen28. Superoxide is also produced by redox-cycling compounds in vivo. As an example, bipyridylium herbicides, such as paraquat, are reduced by an NADPH-dependent cytoplasmic diaphorase in E. coli, and the monocation radicals so produced reduce dioxygen to O2- 29.

Reactions of superoxide

Superoxide has been shown to be reactive to a number of compounds in vitro, as has its conjugate acid, the perhydroxyl radical, HO2. While less than 1% of O2- will be present as HO2 at the physiological pH of 7.4, the concentration increases dramatically as pH decreases. Since the pH is lower in the microenvironment adjacent to a membrane surface or in lysosomes, both the protonated and the unprotonated forms of O2- should be considered. Among the reactions of O2- observed in vitro are reactions with small molecules: ascorbate reacts with a rate constant of 1.5 x 105 M-1s-1 at pH 9.030. a-tocopherol is oxidized by O2- to 8a-hydroxy-a-tocopherone, which spontaneously forms a-tocopherol quinone31. NADH is oxidized by HO2 and O2-, when bound to lactate dehydrogenase, with rate constants at 235C of 1.2 x 106 M-1s-1 and 3.6 x 106 M-1s-1, respectively32. Superoxide also oxidizes and reduces many transition metal ions and their complexes33. For example, it oxidizes Mn2+ with a rate constant of 6 x 106 M-1s-1 34, and it reduces Fe3+ complexed to EDTA with a rate constant of 1.3 x 106 M-1s-1; it reduces Fe3+ complexed to DTPA very slowly, if at all35. The reduction of cytochrome c by xanthine and xanthine oxidase is mediated by O2- 36, and this reduction is used as the basis of assays for both O2- and superoxide dismutase (SOD). The rate constant for the reaction of O2- with ferricytochrome c is 2.6 x 105 M-1s-1, at pH 9.0.30 Other assays for O2- exploit the fact that it reacts with nitroblue tetrazolium, at a rate constant of 5.9 x 104 M-1s-1.30,37. The perhydroxyl radical has been shown to react in vitro with fatty acids; the rate constant for the reaction with linoleic acid is 1.2 x 103 M-1s-1; with linolenic acid, the rate constant is 1.7 x 103 M-1s-1; and with arachidonic acid, it is 3.0 x 103 M-1s-1.38 Sulfur compounds such as dithiothreitol, 2-mercaptoethanol, reduced glutathione, and ethyl mercaptan are oxidized in air; superoxide has been shown to play a part in these reactions39 and in the oxidation of phenols40 and catecholamines.41 Superoxide also reacts with certain proteins in vitro, inactivating them; among these are the enzyme catalase42, and the selenium-containing glutathione peroxidase43. Sinovial fluid, the breakdown of which is part of the pathology of arthritis44, has been shown in vitro to be degraded by a O2--generating system45. In addition, O2- may react with the lipids of intact cell membrane46, 47. The O2--generating reaction of xanthine oxidase oxidizing acetaldehyde damages resealed erythrocyte ghosts containing lipid hydroperoxides, while the enzyme superoxide dismutase protects against this damage.48 DNA strand scission is yet another form of damage caused by O2- generation in vitro49, 50, 51.

In vivo effects of superoxide

Further, O2- has been demonstrated to cause some deleterious effects in vivo. Superoxide generated extracellularly by a xanthine oxidase/purine system has been shown to kill the bacterium Staphylococcus epiderimidis52. Potassium superoxide, dissolved in water to produce 2 nmoles/ml O2-, caused 50% cell mortality in cultured Chinese hamster ovary cells, and caused an increase of mutants in a dose-related fashion53. O2--generating systems have also been shown to cause mutations and cancerous tumors. C3H mouse fibroblast 10T1/2 cells, exposed either to human neutrophils which had been stimulated to synthesize reactive oxygen intermediates, or to hypoxanthine and xanthine oxidase, then placed in tissue culture, were malignantly transformed in vitro.54 When injected into nude mice, the cells resulted in the production of malignant and benign tumors in vivo 55.

Arguments against the toxicity of superoxide

Despite the aforementioned data concerning the reactivity of superoxide, certain investigators contend that O2- is not toxic.56,57,58 Their conclusions are based largely on the limited lifespan of O2- in aqueous media59, and its unreactivity in aprotic solvents due to its relative insolubility in them33. Furthermore, the reactivities of a number of specific compounds with O2- in aqueous solution are low. The buffer Tris, the chelator EDTA, and certain metabolites such as a-ketoglutarate, pyruvate, and succinate are all fairly unreactive, with second-order rate constants ranging from .001 to 0.3 M-1s-1.30 Superoxide is not very reactive toward amino acids, and neither is its protonated form, HO260. For HO2, the rate constants with amino acids range from 10 M-1s-1 for the aliphatic amino acids to about 60 M-1s-1 for the aromatic amino acids. For O2-, the range is from about 0.1 to 20 M-1s-1.60 Thus, it is evident that O2- is not a universally reactive species.

Hydrogen peroxide

While it is possible that O2- is not itself reactive enough to explain all of the biological damage resulting from systems that produce O2-, it is certainly evident that the damage does occur. Some reaction rates of O2- given above are high enough to indicate that the species is potentially damaging in itself; additional damage may be caused by secondary species formed from O2-. One such potentially damaging species is H2O2. Hydrogen peroxide will be present whenever O2- is formed, because the dismutation of O2- generates molecular oxygen plus H2O2, reaction (1):

O2- + O2- + 2 H+ ---> H2O2 + O2 (1)

The spontaneous rate for this reaction at physiological pH is 5 x 105 M-1s-1, and the enzyme superoxide dismutase, which catalyzes this reaction, increases it by a factor of 104.44 Thus, much of the damage that has been attributed to O2- may actually have been caused by H2O2 that was formed from the O2- that was initially generated.

Biological sources of hydrogen peroxide

Biological sources of H2O2 include lung mitochondria61, whose release of H2O2 is increased ten-fold as the ambient oxygen concentration rises from 21% to 100% at one atmosphere pressure62. Activation of phagocytic cells also results in H2O2 production through the dismutation of O2- generated by a membrane-bound NADPH oxidase.22 Hydrogen peroxide is also formed during the one and two electron oxidations of numerous phenols, thiols, and catecholamines.63 Insulin stimulates intracellular H2O2 production in rat epidydimal fat cells; H2O2 has been proposed to act as a second messenger for this hormone64.

Damage produced by hydrogen peroxide

A number of forms of biological damage have been shown to be caused by H2O2. One of several sites at which damage has been clearly shown to be due to H2O2, and not to O2- or OH., is the lens of the eye in organ culture65. The autoxidation of oxymyoglobin to metmyoglobin, mentioned above as a source of O2-, has been shown to be inhibited by catalase, the enzyme which breaks down H2O2, suggesting that peroxide may initiate this oxidation.24 DNA is damaged by H2O2 in the presence of metals66, 67, 68, 69; low concentrations of H2O2 induces resistance of procaryotic cells to degradation of DNA by a future challenge with H2O2.67 It also causes cell death in fibroblasts22 and bacteria70, mutagenicity in bacteria,70 and it has been shown to cause tumors in Drosophila embryos71. Hydrogen peroxide also slowly but irreversibly inactivates the enzyme superoxide dismutase72.

Metal catalyzed generation of the hydroxyl radical

Hydrogen peroxide is not the only reactive oxygen species that may be formed from O2-. Haber and Weiss mentioned in a 1934 paper73 the earlier proposal by Haber and Willstätter that O2- could react with hydrogen peroxide to form OH.:

O2- + H2O2 ---> H2O + OH- + OH. (2)

This reaction has become known as the Haber-Weiss reaction. Hydroxyl radical, too, could be the source of part of the damage that has been attributed to O2-. However, the Haber-Weiss reaction as written is exceedingly slow, with a reaction rate of 3.0 M-1s-1 74 at pH 7.3. Chelated iron, on the other hand, which is frequently present in biological fluids, catalyzes this reaction, increasing the reaction rate to a significant degree8,75. Iron is chelated in vivo with phosphate esters such as ADP, ATP, GTP, and pyrophosphate76, and other compounds such as citrate and oxalate77. In vitro chelators such as EDTA and nitrilotriacetic acid (NTA) also allow iron to catalyze the reaction. In contrast, chelators that prevent iron catalysis of reaction (2) are diethylenetriaminepentaacetic acid (DTPA),78 bathophenanthroline sulphonate (BPS),79 phenanthroline,79 and bipyridine.80 The reaction for the iron catalyzed Haber-Weiss reaction is as shown in equations (3) and (4).44

Fe+++ + O2- --> Fe++ + O2 (3)

Fe++ + H2O2 --> Fe+++ + OH- + OH. (4)

The rate constant for equation (3) when the iron is chelated with EDTA has been determined to be k2 = 1.3 x 106 M-1s-1 at pH 7.0.35 The rate constant for equation (4), which is known as the Fenton reaction, for EDTA-chelated iron is given as k3 = 104 M-1s-1 at pH 7.081. Thus, the catalytic properties of iron make the Haber-Weiss reaction quite possible in vivo and the hydroxyl free radical (OH.) is probably formed in vitro and in vivo mainly through the iron-catalyzed Haber-Weiss reaction (reactions 3 and 4).

Evidence for the Haber-Weiss reaction

Evidence for the occurrence of the iron-catalyzed Haber-Weiss reaction in vitro includes the observation that more OH. is detected from a xanthine/xanthine oxidase system when more iron or iron-EDTA is present, but that the iron-chelator DTPA, in suppressing the formation of the OH. signal, maintains the presence of the O2- signal. This was shown by using electron spin resonance to detect products of the reaction of OH. with compounds known as spin traps, such as 5,5'-dimethylpyrroline-N-oxide (DMPO).82 The hydroxylation of salicylate was used as an assay for OH. formed by the xanthine/xanthine oxidase system, relying on the ability of OH. to hydroxylate salicylate and form o-diphenols. Hyaluronic acid's depolymerization was found to be inhibited by inhibitors of the production or continued presence of OH., namely the iron chelators BPS and DTPA, the enzymes catalase and SOD, and the OH. scavengers mannitol and formate.79 The in vivo formation of single strand breaks in DNA by H2O2 was concluded to be mediated by the Haber-Weiss reaction when phenanthroline and bipyridine, strong chelators of iron, were found to protect the DNA from breakage by H2O2 in human fibroblasts.80 DNA binds iron (II) in a manner such that OH. formation from H2O2 is enhanced as compared to the amount formed without DNA83. DNA strand scission in a system containing xanthine and xanthine oxidase increased as a function of iron concentration. Strand scission was stimulated by EDTA and inhibited by DTPA, the OH. scavengers mannitol and formate and the enzymes SOD and catalase.49 The iron carrying enzymes lactoferrin and transferrin have been found to catalyze the Haber-Weiss reaction when fully loaded with iron84, but lactoferrin that is not fully loaded with iron was found to inhibit the reaction by inhibiting iron catalysis85. Reduction of paraquat has been shown by gas chromatography to result in the production of OH.86.

Other chemical sources of hydroxyl free radical

Non-Haber-Weiss production of OH. is also possible. The antitumor drug tallysomycin produces OH. without the production of intermediate O2- or H2O2.87 The cardiotoxic antitumor drug adriamycin semiquinone reacts with H2O2, requiring neither metal catalyst nor O2- for the production of OH.88. The Fenton reaction produces OH. whenever Fe++ and H2O2 are present together.

Biological sources of hydroxyl free radical

Biological sources of OH. include liver microsomes89, human neutrophils,90 and human monocytes during the phagocytosis of opsonized zymosan.90 Interestingly, monocytes from a patient with chronic granulomatosis disease, a hereditary disorder in which the patient suffers persistent and multiple infections, failed during phagocytosis to generate the ethylene gas from methional used as an assay for OH. in this experiment.91

Reactivity of hydroxyl free radical

The hydroxyl radical is extremely reactive with a wide variety of compounds. Second-order reaction rates are commonly on the order of 108 to 109 M-1s-1 92; the reaction rate of OH. with the enzyme catalase is 2.6 x 1011 M-1s-1. As would be expected from its extreme reactivity, the OH. is capable of causing extensive and indiscriminate biological damage. It has been found to degrade DNA,67 form crosslinks between DNA molecules and between DNA and proteins,93 oxidize thymidine,94 oxidize and hydroxylate amino acids,92 kill bacterial cells,95 and mediate the induction of diabetes in mice by alloxan.96,97 Hydroxyl radical also mediates the degradation of the wood polymer lignin by the fungus Phanerochaete chrysosporium.98 Toxic oxygen species such as OH. may be involved in the normal senescence of leaf tissue.99

Hydroxyl radical scavengers

Scavengers of OH. are compounds that react with OH. as rapidly as or even more rapidly than other compounds, and which thus, when added to a model systemor biological system in high enough concentrations, prevent other OH. -mediated reactions from occurring. To be suitable for this purpose, compounds must not be especially reactive with substances other than the OH.; furthermore, the products of their reaction with OH. must not be particularly damaging in themselves. Substances commonly used as OH. scavengers include ethanol, mannitol, formate, benzoate, thiourea, and dimethylsulfoxide

Table I. Bimolecular Rate Constants for Reactions Between Scavengers

and the Hydroxyl Radical and Singlet Oxygen.


Scavenger Rate Constant (M-1sec-1)


Hydroxyl Radical92 Singlet Oxygen100,101


Sodium Azide 7.5 x 109 5 x 108

Dimethylsulfoxide 7.0 x 109 ~ 103

Sodium Benzoate 6.0 x 109 3 x 103

Thiourea 5.0 x 109 8 x 105

Sodium Formate 2.8 x 109 ~103

Ethanol 1.5 x 109 ~103

Urea ~106 ~103


(DMSO). The latter two scavengers are particularly useful for in vivo experiments, as they are capable of crossing cell membranes. Table I gives a more complete list of OH. scavengers, along with the rate constants of their reaction with OH.. Urea, a poor OH. scavenger, is frequently added as a control; the rate of its reaction with OH. is less than 7.0 X 105 M-1s-1. Some OH. scavengers, such as azide, also scavenge 1O2, but DMSO and ethanol only react with OH..

Oxygen toxicity summary

In conclusion, the poisonous properties of oxygen to all organisms is due to the reactivity of oxygen species with many cellular components. Oxygen in the molecular state is not very reactive. However, partially reduced forms of oxygen, known as active oxygen species, are considerably more reactive. The toxicity of oxygen is mediated by these species, which comprise O2-, H2O2, and OH.. The active oxygen species are generated as byproducts of cellular metabolism, as well as by the activity of toxic agents. The reduction of molecular oxygen to form the more active species is effected by escaped electrons from the electron transport chain in respiring cells, by the action of enzymes such as the microbicidal oxidases of phagocytic cells, by the autoxidation of compounds such as hemoglobin, catecholamines, and reduced flavins, and by the redox cycling properties of certain herbicides and antibiotics.

Defenses against Oxygen toxicity

In vivo methods of studying the photodynamic effect can make great use of manipulations of the cell's natural defenses. Discovering the necessary natural defenses also yields information on what forms of damage and damaging agents contribute to lethality.

Cells maintain a variety of defenses against oxygen toxicity. Among these are an array of enzymes that have evolved to deal with oxidative stress, including superoxide dismutase and catalase (described below).44 In animals, additional defensive enzymes include methionine sulfoxide reductase, which repairs methionine residues in proteins that have been damaged by OH., glutathione peroxidase, and the glutathione reductase which regenerates the cofactor for the glutathione peroxidase. Still other defensive enzymes are endonucleases, exonucleases, and DNA polymerase, which repair single-stranded breaks and modified bases in DNA caused by oxidative and other stresses. In eucaryotes, uric acid, a-tocopherol, ascorbic acid, and b-carotene are among the compounds that function in vivo to scavenge active oxygen species and other organic radicals formed in the cell by reactions with the oxygen radicals; both procaryotes and eucaryotes contain high levels of glutathione, a scavenger of OH. and 1O2.44

Superoxide dismutases

Superoxide dismutase (SOD) catalyzes the reaction between two molecules of superoxide to form hydrogen peroxide and molecular oxygen (reaction 1, repeated below):

O2- + O2- + 2 H+ ---> H2O2 + O2 (1)

Superoxide dismutases are ubiquitous in aerobic organisms.102 Eucaryotes contain, typically, both a copper and zinc-containing superoxide dismutase (CuZnSOD) and another SOD that contains manganese(MnSOD); the two are unrelated, and the latter is compartmentalized in the mitochondria, while the former is in the cytoplasm103. Bovine copper-zinc SOD has a molecular weight of 32,500104 and contains two atoms of copper and two atoms of zinc per molecule.105 It is composed of two identical subunits joined by at least one disulfide bond.105 The human CuZnSOD is similar to the bovine CuZnSOD.106 The mechanism of action of CuZnSOD involves alternate reduction and reoxidation of the Cu2+ at the active site during successive interactions with O2-.102 The mitochondrial SOD, MnSOD, contains four subunits, each with a molecular weight of 20,000; it is located in the matrix of the chicken liver mitochondrion.102

Procaryotes such as E. coli contain both an MnSOD and an iron-containing superoxide dismutase (FeSOD). The procaryotic MnSOD shows homology to the eucaryotic enzyme but contains two subunits rather than four and is consequently half as large, with a molecular weight of 40,000. The FeSOD is partially homologous to the MnSOD.107 It has two subunits, one Fe3+ ion per molecule of enzyme, and a molecular weight of 39,000. The mechanism of the Mn and FeSODs is probably similar to that of the eucaryotic CuZnSOD.102 Superoxide dismutases increase the spontaneous rate of O2- removal according to equation (1) from about 5 x 105 M-1s-1 at physiological pH44 to about 1.8 X 109 M-1s-1 for the MnSOD at pH 7.8 and about 1.6 x 109 M-1s-1 for the CuZnSOD between pH 5.3 and 9.5.44

Regulation of SOD levels

Of the two E. coli superoxide dismutases, FeSOD is constitutively expressed, while MnSOD is inducible. Studies on the activity of E. coli SODs indicate that levels of the FeSOD can be increased somewhat with iron supplementation, and decreased by iron chelators.108 Levels of the MnSOD are increased more dramatically by chelating agents, Mn(II), paraquat108 or oxygenation, and decreased by anaerobic conditions. It has been proposed that oxygenation and intracellular O2- production induce MnSOD because O2- oxidizes Mn(II) to Mn(III). Mn(II) has a higher affinity for the manganese-free apoMnSOD, and successfully competes with Fe(II) that would otherwise combine with the apoMnSOD to produce an inactive enzyme. This results in increasing MnSOD levels.108 Maximum levels of total SOD in E. coli are induced by growth in minimal media that has been supplemented by a combination of 100mM Fe(II), 100 mM Mn(II), 10 mM paraquat, and 100 mM 8-hydroxyquinoline; under these conditions, as much as ten percent of the total cellular protein is superoxide dismutase.

Compounds that are capable of entering cells and inducing superoxide dismutase include paraquat (methyl viologen), pyocyanine, phenazine methosulfate, streptonigrin, juglone, menadione, methylene blue, and azure c, all known redox-cycling compounds.109

Low levels of the MnSOD are characteristically seen when cells have been grown in a glucose minimal medium. High levels of SOD are produced by growth in TSY, or by growth in minimal medium to which 100 mm iron(II), 100 mm manganese(II), 100 mm 8-hydroxyquinoline, and 10 mm paraquat have been added; levels are enhanced thirty-fold by the latter treatment.108 Similar induction levels are observed by the addition of paraquat and manganese alone at similar levels.


The catalases belong to the family of enzymes which contains the hydroperoxidases and peroxidases. Catalase catalyzes the reaction:

2 H2O2 ---> 2 H2O + O2, (5)

whereas peroxidase catalyzes the reaction:

AH2 + H2O2 ---> A + 2 H2O (6)

where A is an electron donor such as dianisidine or guiacol.110

There are two main enzymes with catalase activity in E. coli; one, hydroperoxidase II (HPII), possesses only catalatic activity. The other enzyme, which migrates more slowly than HPII during electrophoresis, hydroperoxidase I (HPI), is both a catalase and a broad spectrum peroxidase. When electrophoresed on a polyacrylamide gel, HPI can be visualized as two isozymes, HPI-A and HPI-B.111 Peroxidase (HPI) has a molecular weight of 337,000, is composed of four subunits of equal size, and contains two molecules of protoheme IX per tetramer.110 Catalase (HPII) has a molecular weight of 240,000 and consists of four polypeptides, each of which is associated with a ferric protoporphyrin IX.112

Factors that affect expression of hydroperoxidases

Catalase is induced by peroxide and ascorbate.113 It is repressed by glucose in a classical example of catabolite repression.114 Catalase is important in protecting the cell against ultraviolet lethality; in fact, it was some time after the discovery of nur, a gene named for its conferring resistance to near-ultraviolet light, that it was found to code for a hydroperoxidase.115 Exogenous catalase provides protection against lethality due to acridine orange as well as to phenazine methosulfate.109 Catalase is inducible by treatment of log-phase cells for thirty minutes with 500 mM H2O2, or with 5 mM ascorbate, which gives rise to H2O2; catalase levels are enhanced as much as six-fold by this treatment116 and twenty- to thirty-fold by treatment with phenazine methosulfate and pyocyanine.109

Low levels of catalase are observed in cells that have been grown in glucose minimal medium; levels four- to five-fold higher are seen when cells have been exposed for thirty minutes to 0.5 mm hydrogen peroxide or 5 mm ascorbic acid.111 The increase in catalase level is due to the induction of hydroperoxidase I, the broad spectrum peroxidase of E. coli. Enhancement of intracellular catalase is also seen when cells are pretreated with paraquat and manganese117.

The oxyR regulon

Christman et al.118 have discovered a regulatory element, oxyR, which appears to regulate a group of enzymes that defend against oxidative stress, particularly HPI. Constitutive mutants in oxyR overproduce nine of the proteins that are induced by oxidative stress, including HPI-A and HPI-B, MnSOD, and glutathione reductase.

Cloning of superoxide dismutase and catalase genes

The genes for three of the E. coli enzymes of defense against oxidative stress, HPI (katG), MnSOD (sodA), and FeSOD (sodB) have been cloned onto multicopy plasmids. Loewen et al.116 screened the Clarke and Carbon E. coli colony bank by plating out the colonies and looking for high catalase levels, as evidenced by an increase in the colony's bubbling in response to H2O2. One strain, JA200/pLC36-19, proved to have a basal catalase level about twice the normal basal level.116 Loewen and Triggs have subcloned this gene to form plasmids BT22 and BT28, which lead to an increase in catalase and peroxidase levels of twenty to thirty-fold, compared to the parent strains.119 Nettleton et al.120 identified two clones in the Clarke and Carbon bank that contained an extra copy of the iron SOD gene, using a synthetic oligonucleotide probe constructed from information on the amino acid sequence of the protein. They found that the probe binds exclusively to a fragment contained in common by clones JA200/pLC18-11 and JA200/pLC13-47.120 Sakamoto and Touati also cloned the FeSOD gene, using immunoprecipitation of crude extracts to screen a cosmid bank they had constructed121; they found that their cosmid contained a 6.6 kb Pst I fragment which carried sodB. Touati similarly cloned and mapped the MnSOD gene from the same cosmid bank and subcloned it into plasmid DT1-5; crude extracts of a strain containing this plasmid contain four to five times as much superoxide dismutase as the parent strain when grown in rich media to late log phase.122

DNA repair

The defensive enzymes superoxide dismutase, hydroperoxidase, and catalase act to remove toxic oxygen species before they can do intracellular damage. In the instance that these defenses fail, other systems are ready to repair damage, particularly DNA damage, that is done by the toxic oxygen species.

Kinds of DNA damage

The existence of stress agents such as ultraviolet light, chemical mutagens, intracellular oxygen or organic radicals that damage DNA requires cells to maintain enzymes to repair damaged DNA. DNA damage may include not only single- and double-stranded breaks, but also the production of damaged bases, such as thymine dimers or guanine residues that have been depurinated or hydroxylated, resulting in alkali-labile phosphodiester bonds.

Kinds of DNA damage that happens under the influence of light include altered or missing bases, single-strand breaks, double-strand breaks, and cross-linking. At least three kinds of damage are commonly defined: far ultraviolet kill, which seems mainly to result from the formation of thymine dimers; chemically-assisted ultraviolet kill (with psoralens for example), which results in DNA cross-linking; and the kind of kill seen as a result of exposure to near ultraviolet light or visible light in the presence of dyes, photodynamic kill, which includes modified bases, apurinic/apyrimidinic sites, and single stranded breaks. The differences in kinds of damage produced by these different forms of light were suggested by their being protected against by different repair systems.

Apurinic/apyrimidinic (AP) sites are generated spontaneously and by treatment with acid or as a result of chemical alkylation of deoxyguanosine, which weakens the N-glycosidic bond that attaches the base to the sugar-phosphate backbone of the DNA.123 They are also generated by DNA glycosylases, which recognize and cleave N-glycosidic bonds of damaged nucleotide residues in DNA.124

When the DNA backbone is broken by ionizing radiation or hydrogen peroxide, the ends contain 3' terminal phosphoglycolaldehydes125 or thymine glycols,126 or 3' terminal phosphates,125 which cannot act as substrates for DNA polymerase I or DNA ligase. These blocked 3' ends must be removed before repair of the DNA can take place, starting with the 3'-OH group of the primer DNA.127

DNA repair enzymes

As E. coli is the best studied of all organisms with respect to DNA repair,128 this discussion will concentrate on the repair systems in this organism. There is some redundancy of the DNA repair systems, which allow mutants in any one of the enzymes to function reasonably well under normal physiological conditons; this indicates their importance to the survival of the organism. Some strains of E. coli are more sensitive to photodynamic kill than others; E. coli B/s is ten times more sensitive than E. coli K12 to photodynamic and paraquat toxicity.129 It is possible that the responsible difference between the two strains involves differential DNA repair ability.

DNA repair systems in E. coli include photoreactivation repair, excision repair, recombination repair, and SOS repair. Photoreactivation involves the cleavage of thymine dimers into a pair of normal thymines by photolyase, an enzyme activated by visible light.130

Excision repair is a process in which two cuts are made in the sugar-phosphate backbone of the DNA on either side of a distortion caused either by a thymine dimer or by a base with which no nucleoside can form a base-pair, producing a 3'-OH group on the 5' side. This allows DNA polymerase I to synthesize a new strand, displacing the defective strand; DNA ligase joins the new strand to the original strand. The original incisions are made in E. coli by endonuclease I. 130

Recombination repair, also called daughter-strand gap repair, involves by-passing a block to DNA polymerase III, allowing replication to continue, leaving a gap opposite the block (e.g., a thymine dimer). The unpaired gaps are filled by excising the homologous piece of undamaged sister strand and inserting it into the gap. DNA polymerase I and DNA ligase then join the inserted piece to adjacent regions and fill in the gap left in the donor segment.130

In SOS repair, binding of the recombination A (RecA) gene product to single-stranded DNA in the region of a thymine dimer or other distortion allows the insertion of adenines or mismatched bases by DNA polymerase III. The RecA protein also induces a series of other proteins, called din for damage inducible, which are kept repressed by the LexA protein in the absence of single-stranded DNA.130 A model of the SOS regulatory system is shown in figure 2. A fusion of one of the genes for a din protein and the coding region for the ß-galactosidase gene has been made, allowing the induction of the SOS response to be easily studied by assaying for ß-galactosidase levels after submitting the cells to an appropriate inducing stress agent, such as mitomycin c.131 It was thought at one point that the protein rec A protected against both far and near ultraviolet and photodynamic killing in E. coli;132 later studies showed that rec A did not protect at all against photodynamic killing, while nur showed great protection.115

Figure 2. Model of the SOS regulatory system. The open circles represent proteolytically inactive RecA molecules and closed circles represent proteolytically active RecA molecules. The semicircles represent LexA molecules. (From G. Walker, Microbiol. Rev. 48: 60-93.)


The phosphodiester bonds at apurinic/apyrimidinic sites are cleaved by AP endonucleases as the first step in excision repair. These AP endonucleases have been found to be ubiquitous in both procaryotes and eucaryotes.133 At least four AP endonucleases have been identified in E. coli.123 These include exonuclease III , endonuclease III, endonuclease IV, and endonuclease V; little is known about endo V at this time.134 AP sites are not the only kinds of lesions that require repair by these enzymes. Single-stranded breaks must have a free terminal 3' -OH group in order for DNA polymerase I to initiate repair synthesis, so any ends which contain a phosphoglycolaldehyde end, a terminal phosphate, or otherwise lack a 3'-OH group, must be cut out using an exonuclease, before repair synthesis can occur.130 Those enzymes which have this ability include endonuclease III, endonuclease IV, and exonuclease III.134

Exonuclease III

Exonuclease III is the major AP endonuclease in E. coli, with about 85% of E. coli's measurable AP endonuclease activity.133 Its functions include cutting at AP sites, acting as a 3' to 5' exonuclease of double stranded DNA, cutting at the 5' side of a urea residue in DNA, removing 3' phosphates and phosphoglycolate esters, and hydrolyzing the RNA from a hybrid duplex in the 3' to 5' direction.134 Exonuclease III mutants are hypersensitive to H2O2 and to near ultraviolet light, both of which produce AP sites.115 Both exo III and endo IV can efficiently remove 3' terminal phosphoglycolaldehyde from DNA.127

Endonuclease III

Endonuclease III cleaves 3' to AP sites and ring-damaged pyrimidines, especially hydroxylated thymine residues (thymine glycols), generating 3'-OH and 5'-P termini.135 It has no cofactor, so it is active in the presence of EDTA.134 Endo III does not remove terminal PO or phosphoglycoaldehyde. The gene for endonuclease III, nth, has been cloned onto plasmid pRPC53.135

Endonuclease IV

In E. coli, endonuclease IV (endo IV) is a nuclease for AP sites; it catalyzes the cleavage of a phosphodiester bond 5' to an AP site, and has no other known activity.134 It is resistant to EDTA. Both exo III and endo IV can efficiently remove 3' terminal phosphoglycoaldehyde from DNA.127 Endo IV is induced in E. coli by paraquat, which is also known to induce SOD.133 Endo IV was identified as an EDTA-resistant endonuclease activity. Less than 10% of measurable AP endonuclease activity is due to endo IV in E. coli grown aerobically in rich medium, in the absence of an inducing agent such as paraquat.135 Strains mutant in endo IV have an increased sensitivity to alkylating agents and oxidants such as peroxides and bleomycin.134 The gene for endo IV has been cloned into plasmids pLC38-27 and pWB21.135

Uses of Defense and Repair Enzymes

Defense and repair enzymes are useful in the study of damage produced by active oxygen in two ways. First, the existence of the enzymes shows what the cell finds damaging; if a substance is not harmful, the enzyme is unlikely to exist solely to remove it. The discovery of SOD was what led to the proposal that superoxide is harmful.136 Secondly, levels of the enzymes can be manipulated, either by induction under appropriate stress conditions, or by subcloning of their genes; if increased concentrations of an enzyme are protective, then the enzyme's substrate must somehow mediate damage, and this substrate is likely to be produced during a stress episode.

The gene for the E. coli HPI has been placed onto the plasmids pBT22 and pBT28 by Triggs-Raine and Loewen.119 The gene for the E. coli MnSOD was placed onto the plasmid pDT1-5 by Danielle Touati.122 Bernard Weiss's group placed the endonuclease IV (nfo) gene on a plasmid, pWB21.135 All three of these plasmids were used in this study.

The Photodynamic Effect

Oxygen-dependent toxicity that also requires the presence of light and a sensitizing compound is known as the photodynamic effect.137 It was first described by Raab in 1896.137 It affects all aerobic eucaryotic and procaryotic organisms and has been the subject of thousands of studies. The mechanism of the photodynamic effect remains mysterious138. Visible light is not absorbed by most cellular components, and therefore does not usually have the toxic effects associated with X-rays or ultraviolet light. However, a sensitizer that absorbs visible light-a colored substance, or dye-may serve to transfer the energy of the light absorbed, usually through oxidation or reduction reactions, to a species which will then cause damage to cellular components. The wavelengths of light and their activities, as well as the spectra of representative photosensitizing dyes, are shown in figure 3. Photosensitizers are molecules which can absorb light to produce a chemical reaction which would not occur in their absence.139

Biological examples of photosensitization

There are many instances of reactions for which this effect is responsible. For example, skin diseases of phototoxicity in humans,140 livestock, and experimental animals; light-activated pesticides;141 and medical treatments including photochemotherapy142 and phototherapy of jaundice,143 herpes simplex144 and psoriasis.145 Studies have shown that photosensitizers can cause cell death,146 DNA damage,147,148,149 protein damage,150,151 membrane damage,148 mutagenesis,146 and both tumor destruction152 and possibly carcinogenesis.153 It is thought that photodynamic action is responsible for the

Figure 3. The phototoxic spectrum.

conditions that bring about the end of the toxic red tide, as photosensitizing compounds are released by the deaths of creatures killed by the red tide bloom.154

Examples of photosensitizers

Among the chemicals known or suspected to induce skin phototoxicity are sweeteners, both cyclamates and saccharin,155 cosmetics including perfumes156 and lipsticks,157 germicidal soaps,157 industrial products including pitch, liquor picis detergents, and crude coal tar,140 agricultural products such as celery,158 and hypericin, which is found in a toxic agricultural pest eaten by livestock, St. John's wort.159 Other inadvertent inducers of skin phototoxicity are drugs such as the phenothiazines, which are prescribed as antipsychotics;160,161 tetracyclines;162,163,164 sulfonamides, which were the first drugs recognized as having phototoxic side effects;165,166,167 psoralens;168,169 chlordiazepoxide, a tranquilizer that can cause liver damage due to photoproducts formed in the skin;170 phenazines, drugs used for the treatment of psychotic disorders;161 nalidixic acid171, an antibiotic used in the treatment of urinary tract infections;145 methotrexate, an anti-cancer drug;172 and benoxaprofen, an antiinflammatory and analgesic drug.173 Phototoxicity is also observed in diseases that cause the production or accumulation of photosensitizing metabolites, such as hyperbilrubinemia.174 Despite all of these known phototoxic activities, the underlying mechanisms remain poorly understood.138


The photodynamic effect is not restricted to deleterious effects, but may be used therapeutically. Niels Finsen was awarded a Nobel prize in 1903 for the treatment of lupus vulgaris, facial lesions seen in tubercular patients, with a carbon arc lamp.175 Phototherapy has long been used in the treatment of psoriasis, with the application of photosensitizers such as coal tar followed by ultraviolet light exposure.145 Partially refined versions of coal tar are available on prescription under a variety of proprietary names.145 Phototherapy with neutral red144 and proflavin176,177 has been used in the treatment of herpes simplex, although not without harmful side effects.153,178 Phototherapy has also been used in the treatment of idiopathic vitiligo, seborrhea, eczema, and chronic lichen simplex.145

Phototherapy of cancers

A major recent impetus to the study of photodynamic effect has been the potential use of dye photosensitizers in phototherapy of cancers.142 Leukemic cells show greater sensitivity to phototoxicity than normal cells. Tumors in mice treated with photochemotherapy have shown regression of greater than 90%.179 Derivatives of hematoporphorin are at present undergoing extensive clinical trials as sensitizers for photodynamic therapy of neoplasms180. At the present time one of the major problems with photochemotherapy is the current poor understanding of the basic mechanisms involved.152

Photosensitizers in this study

The classes of photosensitizers considered in this study include: acridines; other synthetic dyes including thiazines, xanthenes, and phenazines; porphyrins, and a naphthalimide (see figure 4, structures of dyes). The extinction coefficients, given here parenthetically where known, are from Houba-

Figure 4. Structure of dyes.

Herrin (1982).181 The acridines studied include acridine orange (wavelength maximum 492 nm, extinction coefficient 41,600 M-1cm-1), acridine yellow (wavelength maximum 442 nm, extinction coefficient 29,000 M-1cm-1), and proflavin (wavelength maximum, 444; extinction coefficient, 34,300 M-1cm-1). Xanthenes included were fluorescein (wavelength maximum 496 nm) and rose bengal (wavelength maximum 550 nm, extinction coefficient 99,800 M-1cm-1), which possesses one of the highest absorption coefficients known.182 The phenazine studied was neutral red (wavelength maximum 540 nm). Thiazines included methylene blue (wavelength maximum 665 nm, extinction coefficient 78,000 M-1cm-1), azure c (wavelength maximum 616 nm), thionin (wavelength maximum 598 nm), and toluidine blue (wavelength maximum 626 nm).

Many, but not all, of these dyes are capable of penetrating into the procaryotic cell. Those dyes which cannot enter the cell will be able to damage only the cell membrane, which can itself cause cell death; however, in the absence of a reductant, dyes that cannot enter the cell are not lethal.183 The porin channels in the outer membrane of E. coli freely allow the passage of small positively charged molecules, but keep out large, hydrophobic or negatively charged compounds184 in order to exclude the hydrophobic and anionic bile salts that are a threat to E. coli in its natural habitat.185 In the drawing in figure 4, the thiazines and the phenazine have positive charges, while the other dyes are neutral. In experiments by Martin and Logsdon (1987), the dyes toluidine blue and acridine orange penetrated the cell, while fluorescein and lucifer yellow did not.183 Neutral red and methylene blue are accumulated in E. coli at neutral pH, but rose bengal only at acidic pH.186 In general, the

acridines, thiazines, and phenazine enter the cell, whereas the xanthenes and the napthalimide lucifer yellow do not.

Most of the dyes used in this study intercalate in between the bases of DNA. The acridines, thiazines, and phenazines all have planar, tricyclic structures, as seen in figure 3; however, the xanthenes are bulkier and cannot fit in between the bases of the DNA. In addition, the xanthines are negatively charged and are probably repelled by the sugar-phosphate backbone of the DNA. Xanthenes, unlike acridines, thiazines, and phenazines, do not intercalate into DNA. Since intercalating dyes will be largely intercalated into the DNA once they enter the cell, it is important to study dye reactions when the dyes are in the intercalated state, as well as alone.

Why these dyes are included in this study

These organic dyes have been chosen for this study because they are the most widely studied compounds in photochemistry and the physiology of

photodynamic action. Although they vary widely in their structures they are all capable of oxidation reduction reactions, and, because of this fact, of giving rise to oxygen radicals.183 Most of them are known to generate singlet oxygen (1O2) as well.146 Extensive studies have been done illustrating the effects of these particular dyes in E. coli, before the significance of O2- was known. Many of these dyes are structurally related to known redox active compounds such as pyocyanine and phenazine methosulfate, which are known to give rise to O2-by redox-cycling within E. coli.187 Reasons why so many structural classes of dyes were considered in this study were in order to determine whether their individual toxicities could be explained by a common mechanism; to determine the relative importance of cell localization in determining their toxic effects; and to illustrate that all forms of light, from near ultraviolet through the red, are sufficiently energetic to promote phototoxic effects in the presence of an appropriate photosensitizing agent and to do so through the generation of reduced oxygen species.183

Uses of photosensitizers

The dyes used in this study have a variety of functions and origins. Some acridines occur in coal tar.175 Acridine orange and acridine yellow are used as adjuncts to laser surgery of cells, and as stains for nucleic acids,188,189 while proflavin is used as a topical antiseptic.190 Porphyrins occur as metabolic byproducts, and are used in photochemotherapeutic agents.180 Fluorescein (D & C Yellow No. 7) is commonly used to test for corneal abrasion.190 Rose bengal has become one of the most widely used of all photodynamic sensitizers.191 Rose bengal is used as a biological stain, as a dye for straw and wood chips, as an ingredient in inks, for coloring edible products, and in cosmetics.190 Related xanthenes make up a number of dyes approved by the FDA for use in drugs and foods.192 Neutral red is used as a pH indicator and as a biological stain in Golgi apparatus studies190 and is related structurally to phenazine methosulfate and pyocyanine, potent, known redox cycling compounds.187 Methylene blue is used as a stain in bacteriology, as an antimethemoglobinemic and cyanide antidote in humans, and as an antiseptic and disinfectant in veterinary use. Eye drops containing methylene blue are available over the counter in some countries.193 Thionine is used in general nuclear staining, in the counting of bacteria in milk, and as an antioxidant for linseed oil.190 Azure c and toluidine blue o are used as biological stains.

Organisms affected by the photodynamic effect

The toxicity of photosensitizers is nearly universal; among those organisms sensitive to inactivation by the photodynamic effect are viruses, bacteriophage, bacteria, protista, yeasts, insects, and cultured mammalian cells.

Damage to viruses and bacteriophage by the photodynamic effect

Viruses and bacteriophage are susceptible to inactivation by the photodynamic effect. Phage particles are inactivated by light plus methylene blue,194 toluidine blue,195 and proflavin.196 Other isolated viruses, including adenovirus, vaccinia virus, SV40 papovavirus, influenza A, parainfluenza-3, measles virus, reovirus 1, and herpesvirus are all inactivated by toluidine blue o. Neutral red inactivated measles virus, and herpesvirus, while proflavin inactivated reovirus 1, influenza A, measles virus, and herpesvirus.197

Damage to bacteria by the photodynamic effect

Experiments involving the exposure of bacteria to photosensitizing dyes have shown them to be killed as a result. Rose bengal immobilized on polystyrene beads was able to kill E. coli upon exposure to light.198 Toluidine blue killed E. coli cells.199 Thiazines, xanthenes, phenazines, acridines, and naphthalimides that kill E. coli include azure c and thionin;200 rose bengal200 and fluorescein;183 neutral red;200 proflavin,200 acridine yellow,183 and acridine orange;183 and lucifer yellow.183 Other bacteria suffer similarly; for example, methylene blue inactivated Acholeplasma laidlawii201 and toluidine blue inactivated Micrococcus roseus.202

Damage to yeast cells by the photodynamic effect

Yeasts have been used extensively to study photodynamic lethality. Acridine orange in combination with 410-600 nm light inactivates yeast. The inactivation curves are the same shape as for X-ray and FUV killing of yeast, suggesting that there is at least some overlap between the targets and sites of these three forms of radiation. Ascorbate, a biological reductant, caused an oxygen-dependent enhanced lethality in yeast cells of the thiazines toluidine blue, methylene blue, and thionin, but not the xanthenes rose bengal and eosin yellow, though it had no lethality in absence of dye.203 Proflavin led to inactivation of yeast.146 Rose bengal, thionin, methylene blue, toluidine blue, acridine orange, proflavin, and acriflavin all inactivated yeast cells.146

Damage to single-celled eucaryotes by the photodynamic effect

Other eucaryotic cells have been studied for sensitivity to photodynamic effects, as well. Paramecia are killed by acridine in the light, but not in the dark; Raab's discovery of this effect led to the first published example of the photodynamic effect, in 1900.137 Hematoporphorin derivative photosensitizes DNA strand breakage in Chinese hamster ovary cells.147 The crustacean Artemia salina was immobilized by the photodynamic effect of polycyclic aromatic hydrocarbons.204 The activity of nerve cells is altered by acridine orange plus light,205 as well as by fluorescein206 and lucifer yellow207 while erythrocytes are lysed when exposed to dyes such as rose bengal, methylene blue, and neutral red plus light of the appropriate wavelength.137

Other systems susceptible to the photodynamic effect

Other systems in which damage due to photosensitization occurs include multicellular organisms, both plants and animals. For example, strong illumination with blue and ultraviolet light photolyses rhodopsin, leading to the ultrastructural degeneration of the photoreceptors in the Drosophila eye.208,209 Rose bengal caused breakdown of chlorophyll and carotenoids and a decrease in CO2 uptake in leaves.210 Chlorophyll is among the most effective of photosensitizers for the oxygenation of organic substances.211

Human photoreactions

Some of the dyes used in this studied have been observed for their effects on human skin. Neutral red has been reported to cause acute contact dermatitis in humans.177 Rose bengal, when injected into the skin before light exposure, causes erythema and edema.166 Fluorescein and eosin, applied to the skin and then rubbed off before sun exposure, resulted in whealing.137 Not much is known about the mechanisms of action of photodermatoses.212

Cellular targets of photodynamic damage

The targets of photodynamic damage in the cell have been examined, both in vivo and in vitro, using isolated components. Subcellular components including mitochondria and cell membranes are degraded by photosensitizers plus light. Mitochondria that are exposed to light plus hematoporphyrin or hematoporphyrin derivative (HpD) successively lose coupling, calcium ion transport and respiration.213 Isolated chloroplasts lose their ability to carry out the Hill reaction (photochemical water-splitting process) following photodynamic treatment.214 Acridine plus ultraviolet light damages cell membranes.148 The photodynamic treatment with rose bengal of ribosomes isolated from E. coli causes a rapid loss of their ability to incorporate amino acids into polypeptides.215 Photodynamic damage to stained membranes, which occurs with many dyes in the presence of oxygen and intense illumination, is not well understood and is thought to involve production of free radicals by 1O2.216

Damage to proteins by the photodynamic effect

Among those compounds which have been found to be degraded in vitro are amino acids, such as tryptophan,which is destroyed by methylene blue in the presence of light and oxygen.217 Photosensitization has been shown to degrade proteins,150,151 denaturing proteins such as egg albumin137 and inactivating enzymes such as catalase and invertase.137

Damage to DNA by the photodynamic effect

DNA has been determined to be a major target of the photodynamic damage. From an analysis of yeast survival curves, it was concluded that the principle damage is probably mediated through the DNA.218 Direct evidence of DNA damage in vitro has included loss of viscosity, single stranded breaks, apurination, dimerization, and loss of ability to function as a template for DNA polymerase. The viscosity of calf thymus DNA is reduced by rose bengal and methylene blue in the light.219 Acridine plus ultraviolet light damages DNA., causing it to sediment more slowly.148 Acridine orange plus light causes lowered viscosity, a decreased sedimentation coefficient, and a decrease in the thermal denaturation temperature of salmon sperm DNA. This is due to depolymerization, as shown by CsCl banding experiments; depolymerization is due mainly to single-stranded scission. The damage is maximized when the acridine is bound to DNA; this implies the formation of a short-lived intermediate.218 Selective degradation of the guanine residues might be the primary damage responsible for the formation of both alkali-labile bonds and strand scissions.220 Acridine produces not only thymine dimers but also other lesions.221 Acridine orange and methylene blue activate phage lambda in lysogenic Escherichia coli, which indicates that DNA damage is occurring.222 DNA synthesis is inhibited by acridine orange or methylene blue in the presence of light. Dark reactivation repair cannot repair after damage by acridine orange plus light.222 In the light, methylene blue, rose bengal, eosin Y, thionin, azure c, toluidine blue o, acridine orange, and neutral red all decreased melting temperature of DNA, suggesting that the DNA had been degraded.149 Photosensitization has been shown to destroy deoxyguanosine and deoxyadenosine.223

Proflavin plus light caused single stranded breaks in the bacteriophage FX174, where any single-stranded break is a lethal lesion. The packaging of DNA inside the phage head increases damage over isolated DNA.224 Strand breakage has been demonstrated to be caused by acridine orange,218 methylene blue,219 and ethidium bromide.225

Proflavin nicks inhibited template activity of DNA polymerase in FX174,223 indicating that the broken ends of the DNA do not contain a free 3'-OH group, which is required for the initiation of repair synthesis of the broken strand. The loss of infectivity of FX174 following photodynamic treatment with proflavin can be explained by a block in DNA polymerase reaction; termination occurs one base before a damaged guanine residue.226 The photodynamic reaction of methylene blue with deoxyribonucleic acid led to the rapid loss of ultraviolet absorbance accompanied by the uptake of one mole O2/mole derivative; the reaction occurred with the guanine compounds, while thymine compounds reacted very slowly227. Photodynamic reaction of methylene blue with DNA was more rapid with denatured than with native DNA.228 Strand scission of DNA in vivo has been shown for acridines with bacteria 229 and cultured mammalian cells.230 There is a peak in DNA damage for human cells but not B. subtilis when irradiated at 450 nm even without the addition of exogenous sensitizers, indicating that endogenous riboflavin probably acts as a sensitizer in the human cells.223

Mutations caused by the photodynamic effect

Unsurprisingly, considering the evidence for photodynamic DNA damage, mutations have been detected in photosensitizer-treated bacteriophage, bacteria, yeast, and isolated DNA used in transformations.

Mutations in phage caused by the photodynamic effect

The addition and deletion frameshifts induced by proflavin in phage T4 were used by Crick et al. to determine the nature of the genetic coding unit.231 Proflavin caused a 2-fold increase in rate of mutation in phage T4; the mutations were of the base-substitution type.232 The visible light treatment of bacteriophages sensitized by acridines has been found to induce transitions and transversions.233

Mutations in bacteria caused by the photodynamic effect

Acridine orange causes mutations in bacteria.233,234 Neutral red plus light results in mutations of the base-substitution type in Salmonella typhimurium.;153 methylene blue has the same effect in the same system.235 S. typhimurium Ames strains TA 1535 (base substitution-sensitive) and TA1538 (frameshift mutation-sensitive) were used to show the presence of base-substitution type mutations caused by acridine orange; no frameshift mutations were seen in this study.

Mutation in E. coli to resistance to bacteriophage T5 was induced by visible light (>408 nm) and black light (300-400 nm), causing mutation rates to increase more than 18-fold. In Escherichia coli B strain S, mutagenesis was produced by acridine orange and proflavin, both of which are radiomimetic, i.e., they induce cross-resistance to ultraviolet radiation and to each other; this suggests a similarity in the mechanisms used to protect the cells against the two forms of damage. All radiomimetics are mutagens in E. coli B.236 Acridines induced mutation in E. coli tenfold greater than the normal rate in the absence of dyes.237

Mutation in yeast caused by the photodynamic effect

Rose bengal caused inactivation and gene conversion of yeast;238 as did thionin; proflavin also led to gene conversion of yeast. Acridine orange, proflavin, and acriflavin mutated yeast cells.146 Strong mutagenic action was noted for acridine orange, acridine yellow, methylene blue, and toluidine blue in yeast, as well as in phage and isolated transforming DNA.

Reactions of photosensitizing dyes

The toxic and damaging effects produced by the photosensitizers described above require the presence of light and oxygen. The effect of the interaction of light with a photosensitizer is to add energy to the sensitizer molecule. The energy taken up by the sensitizer changes the energy level of one of its electrons,239 resulting in the formation of an excited singlet state. The singlet state rapidly decays to a triplet state, which has a longer lifespan than the singlet state.240 The primary photochemical reactions of acridine, xanthene and thiazine dyes are the formation of the singlet and then a longer-lived triplet state. For example, the triplet excited state of fluorescein lasts 10-4 second, which is very long compared to the lifetime of the initial singlet excited state, around 10-9 second.241 Secondary reactions include a triplet-triplet electron-transfer process, leading to the semi-oxidized and semi-reduced forms of the dye,242 or the interaction of triplet state dye with oxidants, reductants, or energy transfer compounds (such as the interaction with ground state oxygen (O2) to form singlet oxygen(1O2)). The ability of various dyes to sensitize the photo-dynamic degradation of nucleic acids is correlated with their ability to form electronically excited dye molecules in a metastable state, in which case the nucleic acids act as reductants.149 The excited triplet-state sensitizer is capable of reactions which the ground-state sensitizer would not take part in, or which would occur much more slowly with the ground-state. Photoreduction of excited dye molecules is frequently observed, and the reduced dye donates electrons to other substances, returning to the oxidized state, from which the process may be repeated.239

Type I and type II reactions of photosensitizers

The triplet state photosensitizer may react directly with a target molecule, causing damage in an oxygen-independent oxidation or energy transfer reaction, or react with an oxidizable substrate, gaining an electron (equation 8). Subsequently, the semi-reduced dye reacts with dioxygen to yield O2- (eq. 9),


0dye + hv --> 3dye (7)

3dye + AH2 --> dye.- + AH. + H+ (8)

Type I

dye.- + O2 --> dye + O2- (9)

3dye + O2 --> dye + 1O2 (10)

Type II

3dye + O2 --> dye.+ + O2- (11)

1O2 + NADH --> O2- + NAD. (12)


Figure 5. Dye reactions. "AH2" represents a reductant; "3dye" represents a triplet state dye.

which secondarily damages the target; in either case, the initial reaction with the sensitizer is known as a Type I reaction (fig. 5). Alternatively, the triplet state photosensitizer may be quenched directly by oxygen, yielding either 1O2 or the O2- radical by electron transfer, in what is known as a Type II reaction (equations 10-11). For most sensitizer triplets, even in the absence of a reaction with oxidizable substrate, a small but significant percentage of interactions between the sensitizer and oxygen result in electron transfer, which produces O2-243. Either of the two dioxygen radicals, the singlet oxygen or the O2- anion, may then go on to react with a target molecule. Superoxide, as previously indicated, may dismute to form H2O2 and may ultimately give rise to the OH.. Both species exert deleterious effects in biological systems.

Factors that influence whether type I or type II reactions occur

Whether the type I or the type II pathway will be followed depends on the relative rates of reaction between the sensitizer and substrate244 and the sensitizer and oxygen, since these reactants compete for triplet state dye molecules, which are the common substrates of the two reaction pathways (fig. 5.). Also important are the relative concentrations of oxygen and substrate. In aerobic cells, the concentration of physiological reductants are quite high. For example, the concentration of NAD(P)H is about 1.0 mM, and that of glutathione about 6.0 mM. However, the level of oxygen is limited to 0.24 mM by solubility constraints and is much lower than this in respiring cells. The rates of reaction between dye and physiological substrates have not been determined for many of the dyes being investigated in this study, but for those that have been, reactions proceed with rate constants similar to that of the triplet state dye with O2. For example, while excited triplet thionine reacts with dioxygen at a rate constant of 4.5 x 108 M-1s-1, it reacts with tryptophan at a rate constant of 3.9 x 109 M-1s-1.245 The rate constant of the reaction between excited methylene blue and oxygen is 3 x 109 M-1sec-1.246 The semiquinone form of anthraquinone reacts with oxygen at rate constant of 108M-1sec-1.247

Substrate concentrations in vivo

If the rate constants of reaction between the triplet dye and singlet oxygen are not very different from that of the reaction of triplet dye with an oxidizable substrate, the factor that will determine which reaction predominates will be the concentration of substrate versus that of oxygen. Oxygen is soluble in water to a concentration of 0.24 mM at 375C and atmospheric pressure; in a respiring cell, the level will be much lower, as the oxygen is continually being used up by the electron transport chain. Substrates under investigation in this study include NAD(P)H, whose in vivo level is around 0.4 to 1 mM, and reduced glutathione, whose in vivo level is around 6 mM.248 Clearly, the relative concentration of substrates and oxygen in vivo would tend to encourage type I reactions. Cadet et al. wrote in a recent review (1986) that "the exact role and the relative contribution of these two competitive mechanisms [type I versus type II] in the photodynamic effects remains to be determined at the cellular level."223

Production of singlet oxygen

For many years attention has centered on the type II production of singlet oxygen, which has frequently been suggested to be the major intermediate for photodynamic actions both in vitro and in vivo. Each of the photosensitizing dyes involved in this study is almost certainly capable of the production of singlet oxygen under certain circumstances, especially in organic solvents where the solubility of O2 is high and in which many of the photochemical studies have been carried out. According to Ito,249 while the theory that singlet oxygen is a major intermediate in the photodynamic effect has served as a valuable stimulant to investigations in the field of photobiology, no photodynamic action so far investigated in vivo is solely explained by the singlet O2 mechanism. Toluidine blue,250,251,252 methylene blue,253 thionin,146 acridine yellow,254 proflavin,254 neutral red, and rose bengal255,256 have all been claimed to produce singlet oxygen, while the damage they produce has been claimed to be mediated entirely by this singlet oxygen.

Questionable arguments for singlet oxygen's mediation of photodynamic damage

In the general enthusiasm to determine that singlet oxygen is the major intermediate for photodynamic action, some rather questionable evidence has been pressed into service. Frequently, the shakiness of the argument is produced by the apparent belief that there are only two possibilities to be selected between: the type II reaction in which the only oxygen species produced is singlet oxygen, and a type I reaction in which any damage that occurs is caused only by a direct reaction between the triplet state dye and the target of damage. This scheme ignores altogether the possibility of other oxygen intermediates, such as O2-, H2O2, or OH. being involved. For example, one of the most widely used methods to demonstrate that singlet oxygen is the sole mediator of damage consists of demonstrating that the reaction is inhibited by the presence of azide. Unfortunately, the efficacy of azide as an indicator of singlet oxygen mediated damage is compromised by the even greater rate of reaction to be found between azide and OH.. While azide reacts with singlet oxygen at a rate constant of k=2.2 x108 M-1sec-1,246 azide reacts with OH. fifty times faster, at a rate constant of 1.08 x 1010 M-1sec-1.92 Moreover, none of the techniques used to assay for singlet oxygen involvement in photodynamic effects, including detection of luminescence, spin-trapping, and increasing reaction rate in D2O, is completely specific for singlet oxygen.257

Reactions of azide

The mere inhibition of reaction by azide has frequently been produced as `proof' that singlet oxygen alone is the mediator of phototoxicity. When azide protected against killing by hematoporphorin, neutral red, methylene blue, rose bengal, and fluorescein, it was taken as evidence of singlet oxygen involvement.154 Because azide reacts with singlet oxygen faster than it reacts directly to quench many triplet-state dyes, such as methylene blue, inhibition by azide is explicitly used as proof that the reaction proceeds by the type II pathway.246 Without confirmation from other methods, azide inhibition was used as evidence that singlet oxygen participates as a major intermediate in the photodynamic induction of genetic changes in yeast by acridine orange.258 Even in a recent (1986) paper, Kraljic used inhibition by sodium azide as sole proof that photooxidation occurs "exclusively or predominantly via the singlet oxygen mechanism", "since the triplet state of HP does not react measurably with azide in aerated solution."259

The deuterium oxide effect

The exacerbation of damaging effects of the dyes by the substitution of deuterium oxide for water is often used as evidence that singlet oxygen mediates the damage in question. Singlet oxygen in aqueous solution will be quenched (and removed) by water molecules at a fast rate, 106 M-1sec-1 at a water concentration of about 55 M. This quenching reaction is in direct competition with other reactions involving singlet oxygen. However, deuterium oxide (D2O) reacts with singlet oxygen much more slowly than water does. When deuterium oxide replaces water, the rate of singlet removal by the quenching reaction is very low relative to the rate of singlet reaction with other available targets. The solvent lifetime of singlet oxygen is 10 to 17 times longer in D2O than in H2O, thus allowing it more time to exert any deleterious effects on targets.44, 260, 261

Lack of evidence for singlet oxygen's mediation of photodynamic damage

Frequently, other data has failed to confirm azide's demonstration of singlet oxygen involvement in photodynamic action. As exacerbation of damage by D2O was looked for and not conclusively seen, in 1977 Ito and Kobayashi were unable to conclude that singlet oxygen was solely responsible for the toxic effects of xanthenes, thiazines, and acridines in yeast cells, although azide protected.146 When the deuterium effect was not as large as predicted from in vitro experiments with singlet oxygen, it was hypothesized that natural quenchers in the cell masked the deuterium effect, and that therefore it could still be concluded that singlet oxygen must be the major intermediate for photodynamic actions in acridine orange-sensitized yeast cells.260 Deuterium oxide did not enhance the gene conversion rate caused by acridine orange in yeast cells.260 It has been stated that it is unlikely that the attack of singlet oxygen on DNA will give rise to either single stranded breaks or alkali-labile bonds.223 However, both type I and type II reactions act in the photodynamic destruction of guanine.262

When Nilsson et al. (1978) observed that "until now there has been no conclusive evidence for the participation of singlet oxygen in any biological photooxidation in solution," they claimed that now they had unambiguous evidence for the participation of singlet oxygen in photodynamic oxidation of amino acids, but their unambiguous evidence turned out to rely on the observation of the destruction of histidine, since there is a low rate constant for reaction of histidine with triplet dye-without considering rate constants for alternative reactions of histidine with O2-, peroxide, or OH..263 Singlet oxygen has been assayed as the decrease in tryptophan absorbance at 280 nm, without confirming assays264, when clearly there are more species than singlet oxygen capable of degrading tryptophan, and some of them, such as OH., are very likely to be produced by singlet oxygen-generating systems. In fact, tryptophan degradation has been used as a direct assay for the production of OH.. Deuterium oxide enhancement is very small in toxicity in yeast with acridine orange, proflavin, and acriflavin, though somewhat larger for rose bengal.265 The role of singlet oxygen was investigated but could not be established in the methylene blue-photosensitized strand cleavage of DNA, because there was little increase in reaction in the presence of deuterium oxide.266

Production of oxygen radicals and peroxide by the photodynamic effect

Evidence does exist for the production of O2-, H2O2, and OH. by the photodynamic effect. For example, hemolysis in vitro by light-activated benoxaprofen results in production of free radicals, singlet oxygen and O2- anion.267 Other evidence concerning the production of O2-, H2O2, or OH is given below.

Production of superoxide by the photodynamic effect

Superoxide is difficult to observe directly in photosensitized irradiations since most sensitizers absorb light strongly in the 250-300 nm region where O2- absorbs;262 nevertheless, indirect assays have demonstrated production of O2- by excited, semi-reduced dye molecules. The superoxide dismutase-inhibitable reduction of cytochrome c was used to demonstrate that O2- is produced by acridine yellow, acridine orange, fluorescein, and lucifer yellow, when illuminated.183 A reduced oxygen radical formed from the fluorescein-photosensitized autoxidation of tyrosine was mentioned by Kasche in 1967.268 Kasche and Lindqvist suggested that the reaction between the triplet state of fluorescein and oxygen most likely produces O2-.269 This observation was made before the discovery that oxygen radicals are commonly produced in biological systems. Photochemical generation of O2- by rose bengal in the presence of sulfites was suggested in 1978270. Both singlet oxygen and O2- are produced by polymer-bound rose bengal through direct oxidation of rose bengal by dioxygen.271 Superoxide is produced by the methylene blue-sensitized photooxidation of epinephrine; it was hypothesized to be produced from singlet oxygen,272 but others maintain that O2- is most likely produced by a different path than singlet oxygen.273 However, in the presence of appropriate reductants, singlet oxygen actually proves to be a source of O2-; NADH reacts with singlet oxygen to produce O2- and a semi-reduced NAD radical (fig. 5).274 Though rose bengal and its derivatives have been used extensively as a singlet oxygen source (type II), their strong absorption at ~550 nm suggests a number of other possible photochemical processes, none of which have been fully exploited or explored.182 The photooxidation of rose bengal in non-polar solvents is predominantly type-I.182 Using benzoquinone to detect O2-, Lee and Rodgers (1987) found that about 20% of activated oxygen molecules resulting from reaction with rose bengal in the absence of an oxidizable substrate was O2-.262 The light-activated phenothiazine drug chlorpromazine gave a DMPO-OH adduct indicative of OH. whose intensity was decreased 50% by SOD.275 Psoralens have been observed to produce O2- directly.276,277,168 Both light-activated linear psoralens and isopsoralens produced both singlet oxygen and O2-, in varying proportions. When riboflavin, psoralen, benzoyl peroxide, and hematoporphyrin derivative were irradiated with ultraviolet light, they each produced superoxide.278 When water was replaced with deuterium oxide, the enhancement of guanosine oxidation by the dyes acridine orange, rose bengal, thionin, and methylene blue was less than expected, suggesting a mixture of singlet oxygen and O2- involvement.279 The production of single stranded breaks in FX174 DNA is thought to occur by a two photon process-absorption at 440 nm and between 320 & 360 nm, followed by an electron transfer from the excited dye molecule to the substrate, DNA.220

Production of hydrogen peroxide by the photodynamic effect

Specific evidence for H2O2 production in photodynamic effects has also been found. The toxicity of ultraviolet-irradiated tryptophan has been shown to be largely due to H2O2.280 The formation of H2O2 by methylene blue in the presence of ascorbate has been observed.281 Hydrogen peroxide is produced by the reaction between hematoporphyrin derivative and ascorbate in the presence of light.282 This is of physiological significance since some of the tissues targeted for photochemotherapy contain ascorbate at levels of 2-3 mM-such as the mammalian lens. Hydrogen peroxide is produced by xanthene and thiazine dyes in the presence of oxidizable substrates.283 It has been suggested that the same mechanism is responsible for near ultraviolet inactivation and photodynamic inactivation of E. coli;115 the H2O2 scavenger catalase, when incorporated into plating medium protects against near ultraviolet lethality284 and ammeliorates photodynamic killing by acridines and thiazines.183 The proflavin-sensitized breakdown of DNA may occur by a `radical pathway', which requires a two-photon absorption and leads to the formation of peroxide free radicals in the DNA moiety of the DNA-proflavin complexes, leading to strand breakages.285

Production of hydroxyl free radical by the photodynamic effect

The production of OH. has also been observed in dye photooxidation. Butylated hydroxytoluene's observed ability to protect against photodermo-toxicity had been explained on the basis of a hypothesized ability to change the structure of the skin, but when this was disproved its antioxidant effects were suggested to be responsible.286 This suggests that free radicals are responsible for the phototoxic effect in vivo. Vitamin E is changed to its radical form in the presence of hematoporphyrin derivative and light, indicating that it has been attacked by a free radical.287 Both singlet oxygen and OH. have been shown to be produced by HpD in vitro.147 The OH. scavengers DMSO, sodium benzoate, and thiourea protected E. coli B against the toxic effects of illuminated acridine orange.183 Both singlet oxygen and hydrogen abstraction mechanisms are involved in the photosensitization of substituted phenylalanines and tyrosines, but the hydrogen abstraction mechanism, a typical OH. reaction, predominates at pH 8.288

The formation of OH. by methylene blue in the presence of ascorbate has been observed.289 When exposed to the spin trap compound DMPO, photoexcited psoralens were found to produce a DMPO-OH EPR signal that is abolished when the reaction is carried out in the presence of OH. scavengers.168 Hydroxyl free radicals are produced by hematoporphyrin derivative, ascorbate, and light.290 In the dye-sensitized photooxidation in methanol of polyunsaturated fatty acids, using the dyes methylene blue, erythrosin, hematoporphyrin, and riboflavin, the isomer product distribution was interpreted in terms of a dual singlet oxygen and radical mechanism.142 In the photoactivated reaction of tartrazine, which is mutagenic only in the presence of light, there is no singlet oxygen participation, according to the cholesterol reaction assay, in which singlet oxygen is detected by its reaction with cholesterol, which results in reaction products specific to this reaction. However, superoxide dismutase & catalase inhibited OH. production, as measured by EPR using the spin-trap DMPO. 291

Other photodynamic reactions that produce the hydroxyl radical

Assorted other reactions appear to be dependent on photodynamically-produced OH., whether or not the photosensitizer has been identified. When hematoporphyrin derivative was illuminated in the presence of methionine or tryptophan and Fe-EDTA, OH. were detected using both the salicylate and thiobarbituric acid assays292. Tryptophan yielded a photoproduct that could substitute for H2O2 in the iron-catalyzed Haber-Weiss reaction, while porphyrin radical could replace O2-.292 Light-induced chromatid damage in cells being cultured for other studies was found to be prevented by the H2O2 scavenger catalase or the OH. scavenger mannitol; after filters were installed to protect cells and medium from light of wavelengths less than 500 nm, the unwanted damage to the cultured cells was eliminated.293 DNA stand-breaking activity of lipid peroxides is dependant on metal ions and partly inhibited by catalase294. The formation of fluorescent products from DNA is increased by metal ions and ascorbate.295,296

Dye photosensitization reactions in vitro may produce singlet oxygen or O2-, or both species, depending on the prevailing reaction conditions. In order to determine the identity of oxidizing species in vivo, it is important to 1) use in vitro model systems that mimic in vivo conditions as closely as possible, with regard to presence and concentration of targets, substrates, chelators, and trace metals; and 2)utilize in vivo methods of study as much as possible.

I would like to emphasize why we need to know, in as much detail as possible, about the identity of the oxidizing species and how they are produced. Without such information, our attempts to understand the biological role of these species and then to modify them are likely to fail. This is particularly important for strongly oxidizing species that act like the hydroxyl radical.


Choice of organism

The reasons why Escherichia coli is the organism of choice for these studies have to do with the ease of manipulating it and its extensive use in previous work on oxygen toxicity. The bacterial cell is a single compartment (or at most a dual compartment) organism whose intracellular targets are all approximately equally accessible by oxygen radicals. E. coli is the best studied of all systems with respect to the regulation of SOD and catalase, as well as the role of SOD and catalase in preventing oxygen toxicity, and has been a very useful organism for previous studies because the cellular levels of the enzymes superoxide dismutase and catalase are easily manipulated by the choice of media or additions to the media. E. coli is also the best-studied system as far as the role of redox-cycling compounds in generating oxygen radicals is concerned, as well as systems of defensive enzymes. The genes for one of the catalases, both of the superoxide dismutases, and nucleases involved in DNA repair have already been cloned onto multicopy plasmids. E. coli is an organism that is easily grown and counted, one that can be easily manipulated, both physiologically and genetically. Finally, E. coli has a long history of study in connection with the phototoxicity of dyes used in this work.298,299,300 As all aerobic organisms are subject to similar problems as a result of oxygen toxicity, and their defense mechanisms have much in common, E. coli should provide a useful model for all aerobic cells.


Studies on the photodynamic effect have concentrated on singlet oxygen for historical reasons. The observation that oxygen was necessary for the toxicity of the photodynamic effect led naturally to the conclusion that an activated form of oxygen was the mediator.301 The study of singlet oxygen chemistry antedates the discovery of oxygen radicals and their effects on biological systems. McCord and Fridovich's discovery of superoxide dismutase in 1969 led to the recognition of the toxic effects of the superoxide radical.302 These toxic effects were subsequently found to be at least in part due to the production of hydrogen peroxide and hydroxyl radicals via the iron-catalyzed Haber-Weiss reaction.80 These two bodies of knowledge, dye-mediated photosensitization and oxygen radical toxicity, have grown up in parallel without much cross-fertilization. This study was undertaken in order to show that many biological effects previously attributed to singlet oxygen can be explained by free radical mechanisms. The work described in this thesis follows up on observations already made by our lab in this respect183 and also extends previous observations on photosensitization.

Lethality due to oxygen radicals is an interesting phenomenon worthy of study; so is the production of these oxygen radicals by the photodynamic effect. This investigation is intended to shed light on the identity of cellular targets attacked by toxic oxygen species, on which of the oxygen species are involved in toxicity, and on factors involved in the photodynamic effect's production of oxygen species.

A major cellular target attacked by toxic oxygen species is likely to be DNA. Single-stranded nicking of DNA will be examined in this study in order to determine which factors are involved in this damage. Among the factors to be examined are the effects of scavengers of superoxide, hydrogen peroxide, and of hydroxyl free radical on the DNA damage; effectiveness of a wide range of dyes in classes including the xanthenes, acridines, thiazines, and a phenazine; effect of different amounts of iron and copper and of a wide range of biologically significant potential iron chelators; and the effects of a series of biological reductants on the production of DNA damage by the photodynamic effect.

The production of toxic oxygen species will be examined in this study by means of a series of assays. This study will use assays for oxidation of NADH and reduction of cytochrome c. It will examine the spectral shifts caused by intercalation into DNA of individual dyes to study intercalation, and the dyes' ability to reduce cytochrome c and oxidize NADH even when intercalated will be investigated. Production of hydroxyl radicals as assayed by two different assays will be examined in the presence of various scavengers of O2-, H2O2, and OH., as well as in the presence of different metal concentrations, different dyes, different chelators, and different reductants. Nicking of DNA will be examined similarly. Cellular levels of one biologically important reductant, glutathione, will be measured after exposure of the cells to dyes. Amelioration of kill levels will be determined for the presence of plasmids coding for catalase, SOD, and endonuclease IV, as well as preinduction of protective enzymes by PQ and Mn and the presence of thiourea. Finally, in vivo breakage of plasmid DNA will be looked at.

on to Chapter 2

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on to Chapter 2