Phototoxicity

Esa Tyystjärvi

I. Introduction

271

II. Chemical and Biochemical Basis of Phototoxicity

272

III. Phototoxic Compounds and their Roles in Nature

273

IV. Herbicides and Heavy Metals Mediating Phototoxic

Responses

277

V. Is Photoinhibition of Photosynthesis a Phototoxicity

Mechanism?

277

VI. Phototoxicity and Programmed Cell Death

278

Acknowledgments

278

References

278

I. Introduction A. History of Phototoxicity

Phototoxicity can be defined as a phenomenon in which a substance becomes damaging when activated by light. Primary phototoxins are photosensitizers, i.e. light absorption by the toxin makes it poisonous. Secondary phototoxins affect the metabolism of the target organism, causing the accumulation of an endogenous photosensitizer.

Study on phototoxicity started in autumn 1897, when Professor von Tappeiner from Pharmacological Institute of Ludwig-Maximilians University, Munich, Germany gave Oscar Raab a topic for his thesis: to study whether acridine would kill paramecia (Paramecium caudatum). Due to the weather conditions, the illumination in the laboratory varied from day to day, and soon Raab found that the killing efficiency of acridine depends on light (Raab, 1900). Phototoxic poisoning of grazing animals has long been of practical importance. In the 16th century, only black sheep were raised in Tarentine fields infested with the phototoxic herb Hypericum (Blum, 1941), because dark fur and skin afford tolerance by limiting the passage of activating light. Plants containing phototoxic furanocoumarins have been used to treat vitiligo for at least 3300 years (Fitzpatrick and Pathak, 1959).

This chapter focuses on phototoxicity reactions in which a plant is either the source of the phototoxin or the subject of the phototoxic poisoning. Light-activated veterinary diseases

Plant Cell Death Processes

Copyright 2004, Elsevier, Inc. All rights reserved

(Ivie, 1982) and human porphyrias (Kappas, 1995) will not be discussed here. The rapidly growing area of the medical use of phototoxic reactions (Dougherty et al, 1998; Bethea et al., 1999) is also out of the scope of this review. Even within the subject, phototoxicity in plants, literature coverage is selective.

B. Targets and Sources of Phototoxins

Phototoxic poisoning occurs in all types of organisms including viruses, bacteria, fungi, plants, and animals. Indiscriminate toxicity results from the ubiquity of the biomolecules, mainly membranes and DNA, readily attacked by phototoxins. The plant kingdom is the most important source for phototoxins, with 75-100 compounds isolated from flowering plants. For reviews on phototoxicity, see Towers, 1984;Laustriat, 1986; Spikes and Straight, 1987; Downum, 1992; Berenbaum, 1995; and Arfsten etal., 1996.

II. Chemical and Biochemical Basis of Phototoxicity

A. Chemistry of Reactive Oxygen Species (ROS)

Phototoxicity is closely associated with the production of reactive oxygen species (ROS), mainly free radicals and singlet oxygen (xO2). Oxygen toxicity and antioxidative defenses have been reviewed extensively (Elstner, 1982; Foyer et al., 1994; Ahmad, 1995; Bartosz, 1997; Halliwell and Gutteridge, 1999; see also Chapter 13). ROS include both highly reactive species like the hydroxyl radical HO' and singlet oxygen *O2 and the partially reduced forms of oxygen (H2O2, O22-, O2- and HO2). XO2 is reactive towards compounds containing double bonds, oxidizing histidine, tryptophan, methionine and cysteine, cholesterol, unsaturated fatty acids, NADPH, and the bases of DNA (Halliwell and Gutteridge, 1999). XO2 can also transfer its excitation energy to a quencher molecule such as «-tocopherol, j-carotene or azide (Foote, 1987). The lifetime of XO2 is 1-4 ^s in H2O, 55-68 ^s in D2O (Ogilby and Foote, 1983; Valenzeno, 1987), and 20-25 ^s in lipid micelles (Valenzeno, 1987). Superoxide is toxic mainly because its presence can lead to formation of the highly reactive hydroxyl radical.

B. Type I and Type II Reactions

Several competing reaction routes are available for most phototoxins, and the actual mech-anism(s) by which a given toxin functions in a particular biological system, is never trivial to deduce (Foote, 1987). Photosensitization starts when the sensitizer S absorbs a quantum, and internal conversion to an excited triplet 3S leads to a long-lived reactive molecule. The reactions that follow are classified as Type I reactions starting with electron transfer involving 3S and Type II reactions starting with energy transfer from 3S to 3O2, forming XO2 (Gollnick, 1968) (Fig. 18-1).

Type I reactions. Excited states can enter both oxidative and reductive one-electron chemistry (Foote, 1987), both yielding radical products that are easily oxygenated. Type I reactions are often complex combinations of radical reactions producing an oxygenated or otherwise inactivated substrate (Fig. 18-1). Oxidation of 3S by 3O2, yielding O2-, is a special Type I reaction.

Type II reactions. Excitation energy donation from 3S to another triplet species occurs readily if certain energetic requirements are filled and if the other triplet is close enough. Energy transfer from 3S to molecular oxygen (3O2) produces the singlet excited state of

Figure 18-1. Photosensitization of a biomolecule B. The sensitizer S is excited by light to an excited singlet state S*. Chemical reactions usually start from long-lived triplet excited state 3S which results from intersystem crossing in S*. One-electron transfer reactions between 3S and aBor between 3 S and O2 yield radical products, and oxygenation of B or another biomolecule follows (Type I reactions; dotted arrows). Adduct formation between S and B does not require oxygen. Energy transfer reaction between 3 S and 3O2 yields the reactive singlet oxygen 1O2 which may oxidize several biomolecules (Type II reaction, dashed arrow).

Figure 18-1. Photosensitization of a biomolecule B. The sensitizer S is excited by light to an excited singlet state S*. Chemical reactions usually start from long-lived triplet excited state 3S which results from intersystem crossing in S*. One-electron transfer reactions between 3S and aBor between 3 S and O2 yield radical products, and oxygenation of B or another biomolecule follows (Type I reactions; dotted arrows). Adduct formation between S and B does not require oxygen. Energy transfer reaction between 3 S and 3O2 yields the reactive singlet oxygen 1O2 which may oxidize several biomolecules (Type II reaction, dashed arrow).

oxygen (1O2) and the ground state of S (Fig. 18-1). Cellular membranes are particularly sensitive to Type II oxidation.

Type I and Type II pathways are always in competition in natural systems (Ahmad, 1992). The Type I/II character of a photodynamic reaction can be probed with inhibitors specific to a certain ROS, by comparing reaction rates between aerobic and anaerobic conditions, and by using D2O to affect the lifetime of 1O2. All methods have limitations. Most 1O2 quenchers and chemical traps may also react with oxygen radicals or quench the excited state of the sensitizer. The use of D2O, likewise, is limited to cases where quenching by the H2O or D2O limits the rate of 1O2 decay (Foote, 1987).

Photosensitizers have an extensive n-electron system (Downum, 1992). High absorptivity, high intersystem crossing rate and a long 3 S lifetime are expected to favor phototoxicity. In the polyacetylene group, at least three conjugated triple bonds are required for phototoxicity (Marchant and Cooper, 1987). Most phototoxic effects also require that both the sensitizer and light penetrate the target cell.

III. Phototoxic Compounds and their Roles in Nature

A. Acridine and Xanthene Dyes and Polyaromatic Hydrocarbons

The acridine dyes like acridine orange [1] (see Fig. 18-2 for the formulae) and the xanthene dyes like rose bengal [2] are primarily Type II sensitizers. In light, these compounds inactivate bacteriophage 0X147, kill bacteria (Houba-Herin et al., 1982), cause loss of chlorophyll from plant leaves and inactivate the photosynthetic electron transfer chain (Knox and Dodge, 1985a,b; Chung and Jung, 1995). The absorption maxima are in the visible region, and the quantum yield of 1O2 production in water is very high for rose bengal, methylene blue and eosin Y (Houba-Herin et al., 1982). The toxicity of low molecular weight polyaromatic hydrocarbons (e.g. anthracene [3]) is also enhanced by ultraviolet-A (UVA) light (Arfsten et al, 1996).

B. Plant Secondary Metabolites

Furanocoumarins (psoralens) and related compounds. Furanocoumarins, found mainly in Apiaceae and Rutaceae (Pathak et al., 1962) are the most intensively studied group

Figure 18-2. Structural formulae of some photosensitizers. 1, Acridine orange; 2, Rose bengal; 3, Anthracene;

4, Psoralen (Ri=H; R2=H); 8-Methoxypsoralen (Ri=H; R2=OMe); 5-Methoxypsoralen (R1=OMe; R2=H); 5, Angelicin; 6, Khellin (R1=R2=OMe); Visnagin (R1=OMe; R2=H); 7, Coriandrin; 8, Hypericin; 9, Cercosporin; 10, Phenylheptatriyne; 11, a-Terthienyl; 12, Citral; 13, Curcumin; 14, Protoporphyrin IX; 15, Phylloerythrin.

Figure 18-2. Structural formulae of some photosensitizers. 1, Acridine orange; 2, Rose bengal; 3, Anthracene;

4, Psoralen (Ri=H; R2=H); 8-Methoxypsoralen (Ri=H; R2=OMe); 5-Methoxypsoralen (R1=OMe; R2=H); 5, Angelicin; 6, Khellin (R1=R2=OMe); Visnagin (R1=OMe; R2=H); 7, Coriandrin; 8, Hypericin; 9, Cercosporin; 10, Phenylheptatriyne; 11, a-Terthienyl; 12, Citral; 13, Curcumin; 14, Protoporphyrin IX; 15, Phylloerythrin.

of phototoxins. Linear furanocoumarins (e.g. psoralen, 8-methoxypsoralen or 8-MOP and 5-methoxypsoralen [4]) or angular ones (e.g. angelicin [5]) absorb UVAlight (320-400 nm), and numerous variations of the psoralen theme are known (Pathak et al, 1962; Becker et al, 1993). Furanocoumarin-containing plants like Ammi majus are known to cause photosensi-tization of livestock and poultry (Clare, 1955; Dollahite et al., 1978; Ivie, 1982). Handling of furanocoumarin-containing plants like celery (Ashwood-Smith et al., 1985) or parsnip (Pedersen and Arles, 1997) may also sensitize human skin to UVA; the reaction is often followed by increased pigmentation of the irritated area (Pathak et al., 1962). The phototoxicity of furanocoumarins has been demonstrated in bacteria (Fowlks et al., 1958) and viruses (Hudson et al., 1993). Interestingly, 8-MOP caused UVA-dependent wilting of a fern when fed in the transpiration stream, while Herachleum, an 8-MOP-containing plant, was insensitive (Camm et al., 1976). Furanochromones, e.g. visnagin and khellin [6] and furoisocoumarins like coriandrin [7] resemble furanocoumarins in their biological action (Abeysekera et al, 1983; Hudson et al, 1993).

Phototoxicity of furanocoumarins is largely based on their reactions with DNA (Mathews, 1963; Ivie, 1987; Bethea et al., 1999). Furanocoumarins intercalate, or bind reversibly between two pyrimidine bases of a double-stranded DNA. Under UVA light, a covalent bond is formed between a furanocoumarin and one or two pyrimidine bases; the diadduct may form an interstrand cross-link. Angular furanocoumarins form mainly monoadducts (Cimino et al., 1985). These Type I photosensitization reactions do not require oxygen.

As expected from their reactions with DNA, 8-MOP and 5-MOP cause mutations in bacteria (Mathews, 1963). Treatment of psoriasis with furanocoumarins and UVA increases the risk of skin cancer (Stern et al., 1998). Furanocoumarins also produce ROS in UVA light (Cannistraro and van de Vorst, 1977) and interact with cellular membranes and proteins (Beijersbergen van Hegenouwen et al., 1989).

Furanocoumarins were the first plant-derived compounds with a demonstrated light-dependent toxicity in the larvae of a herbivorous insect (Berenbaum, 1978). The production of furanocoumarins is linked to the openness of the plant habitat in Apiaceae (Berenbaum, 1981), suggesting a role for furanocoumarins in plant defense.

Hypericin. Photosensitization of livestock grazing on St. John's wort (Hypericum) (Clare, 1955) is caused by the red, fluorescent pigment hypericin [8] (Horsley, 1934), located in special glands of the plant. Hypericin absorbs between 530 and 600 nm (Knox and Dodge, 1985c), and illumination in the presence of hypericin kills insect larvae (Sandberg and Berenbaum, 1989), causes pigment loss and membrane damage in leaf disks (Knox and Dodge, 1985c) and inhibits protein kinases (Agostinis et al., 1996). Hypericin is predominantly a Type II photosensitizer (Knox and Dodge, 1985c), but Type I reactions may also occur (Duran and Song, 1986). Insects specialized to feeding on Hypericum avoid the toxic effects either by avoiding the hypericin-containing glands or by avoiding the activation of the poison by light (Sandberg and Berenbaum, 1989; Fields et al., 1990). Hypericin is also found in the photosensory pigment of Stentor (Song, 1981), and the ciliate is sensitive to light because of the endogenous hypericin (Yang et al., 1986).

Polyacetylenes (polyines) and thiophenes. Polyines and the biosynthetically related thio-phenes form a family of more than 750 UVA-absorbing phytochemicals (Bohlmann et al., 1973), with all phototoxic members in Asteraceae (Marchant and Cooper, 1987). The most studied compounds are phenylheptatriyne (PHT) [10] and a-terthienyl (a-T) [11].

The UVA-dependent biological effects of a-T range from inactivation of enzymes in vitro (Bakker et al., 1979), photokilling of bacteria (Downum et al., 1982) and inactivation of viruses (Hudson, 1989) to phototoxicity to the green alga Chlorella (Arnason et al., 1981).

The UVA level in sunlight and the a-T content in plant tissues are high enough to control lepidopteran larvae (Champagne etal., 1984). In higher plants, a-T and UVA cause growth retardation (Campbell et al., 1982), inhibit photosynthesis (Brennan, 1994) and promote decarboxylation oflAA(Brennan, 1996).

a-T is a strictly oxygen-dependent phototoxin and the production of xO2 during illumination of a-T (Bakker et al., 1979; Kagan et al., 1984a) probably causes the UVA-dependent biological effects (Marchant and Cooper, 1987). The nematocidal activity of a-T in Tagetes roots (Uhlenbroek and Bijloo, 1958) may be caused by xO2 produced after chemical excitation of a-T (Gommers and Bakker, 1988).

The biological activities of phototoxic polyines resemble those of a-T (Downum et al., 1982; Kagan et al., 1984b; Gong et al., 1988). Low concentrations of O2 are sufficient for PHT-sensitized phototoxicity (Kagan and Tuveson, 1988). Phototoxic polyines and thiophenes probably restrict insect herbivory on plants containing these compounds by acting both as feeding deterrents and photosensitizers (Champagne et al., 1984). Insects feeding on phototoxic Asteraceae have both behavioral (Guillet et al., 1995, 1997) and metabolic adaptations (Aucoin etal., 1990; Berenbaum, 1994; Guillet etal., 1997).

Other plant secondary metabolites. Several other classes of plant-derived chemicals like j-carbolines have phototoxic properties (Downum, 1992). New phototoxins like citral [12] (Asthana et al., 1992) and curcumin [13] (Chignell et al., 1994) are also found.

C. Porphyrins and Related Compounds

Porphyrins, precursors and constituents of heme and chlorophyll, are visual light absorbing pigments mainly capable of Type II reactions. Metabolic accumulation of protoporphyrin IX [14] mediates phototoxic poisoning of bacteria (Zoladek et al., 1996) and plants (Kunert and Dodge, 1989), and causes the photosensitivity of skin in the erythropoietic proto-porphyria disease in human (Kappas et al., 1995). Phylloerythrin [15], a chlorophyll degradation product, is the primary photosensitizer in hepatogenous photosensitivity of ruminants (Ivie, 1982), and hematoporphyrin derivatives are used in photodynamic cancer therapy (Dougherty et al., 1998). Chlorophylls sensitize the formation of ROS, and chloroplasts have a remarkable arsenal of antioxidant defenses (Foyer et al., 1994; Bartosz, 1997). The light-harvesting chlorophyll-protein complex of Photosystem II (PS II) may structurally avoid contact between triplet chlorophyll a and oxygen (Siefermann-Harms and Angerhofer, 1998). Derivatives of bacteriochlorophyll are promising new candidates for photodynamic cancer therapy (Scherz et al., 1997).

D. Cercosporin

A plant pathogenic fungus Cercospora produces a photosensitizer, cercosporin [9], and uses it to attack its host plant (Kuyama and Tamura, 1957; Daub and Ehrenshaft, 1993). This red pigment absorbs at 400-600 nm and emits red fluorescence. Illumination of plant tissues in the presence of cercosporin causes leakage of electrolytes and other cell constituents (Macri and Vianello, 1979). Illuminated cercosporin produces both *O2 and O2- in vitro (Daub and Hangarter, 1983), and Type II photosensitization is probably the most important mechanism in its pathogenic action (Daub and Ehrenshaft, 1993). Cercospora tolerates its own phototoxin, and the resistance is at least partially based on transient reduction of cercosporin by the mycelia (Daub et al., 1992).

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