il II 1 - American Chemical Society

Environmental phototoxicity. Solar ultraviolet radiation affects the toxicity of natural and man-made chemicals. Richard A. Larson. May R. BeFenbaum. ...
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Environmental phototoxicity Solar ultraviolet radiation affects the toxicity of natural and man-made chemicals

Richard A. Larson May R. BeFenbaum University of Illinois at UrbanaChMlPaign Urbana, Ill. 61801 Every living organism that inhabits the Earth's surface must cope with the sun's radiation. Many, such as vascular plants, algae, and photosynthetic bacteria, have developed techniques for harnessing some of the energy released by the sun. They use it to promote metabolic reactions-such as the fixation of carbon dioxide-that are energetically unfavorable yet supply vital chemical energy Animals use solar energy in visual processes and in other habitat adaptations; for example, some zooplankton will adjust their positions in a in water column in mwnse to .changes light intensity. In addition to visible lieht and infrared (R I ) radiation, the su&mits ultraviolet (UV) radiation in the 290-400 nm region (Figure 1). Approximately 4% of the total energy contained in sunlight occurs in the UV band. Distinctions sometimes are made hetween W - A (320-400 nm) and the more energetic radiation that is more strongly absorbed by biomolecules, UV-B (290-320 nm). The intensity of UV irradiation at the Earth's surface varies greatly with season; time of day; latitude; thichess of the atmosphere and the ozone layer; altitude; and cloud cover. Because it containspotent chemical energy, W radiation has the potential for directly damaging the biochemically important molecules that absorb it. In addition to the direct absorption of solar energy by cellular constituents, II another mechanism for toxicity is an indirect effect that occurs because of the absorption of sunlight by xenobiotics (or by naturally occurring com- This parsnip plant defends itself with phototoxic compounds. but the parsnip webworm pounds outside the target cell); these larva has become resistant to these compounds.

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may be converted by light or by subsequent light-promoted reactions to toxic forms that induce cellular damage.

Dim3 phototoxicity Although the absorption of IR and visible photon energy can generate rotational and lower vibrational states of a molecule, these wavelengths are not nearly as damaging as are the higher energy UV quanta, which increase vibration and electronic excitation. Nucleic acids and proteins absorb solar W radiation and therefore are prone to damage. The action spectrum for toxicity to a wide variety of cells, ranging from bacteria to mammalian epidermis, corresponds to the absorption spectrum of these molecules. The UV absorption of proteins extends into the solar W - B region, principally k a u s e of the amino acids tyrosine (Iand ) tryptopban 0 (see box). The latter compound has a molar extinction mfficient of 4020 at 290 MI, meaning that it absorbs solar UV quite strongly (a millimolar solution of II would absorb 99.99% of the incident 2Wm light within 1 cm). Tryptophan is highly susceptible to photolytic destruction, which creates a complex mixture of compounds (I). Although nucleic acid constituents have much lower absorbencies in the

solar UV region, many products of UVpromoted reactions have been reported, including thymine dimers (m), dimers between thymine and other nucleic acid bases, and protein-DNA adducts (2). Unless the DNA is repaired, these products presumably impair the normal genetic functioning of the cell. Com-

pared with RNA and other cell constituents, DNA is present in small quantities, yet it controls most cellular functions. DNA is particularly important in determining the sensitivity of living cells to UV radiation. Ultraviolet light appears to be toxic to all forms of unpigmented living

Envimn. Scl. Technol.. M I . 22,NO.4, 198@ 355

cells, including bacteria, protozoa, nematodes, arthropods, fish, birds, and mammals. There are, however, several protective mechanisms that mitigate W toxicity. Many organisms are negatively phototropic (tend to move away from light) and can behaviorally avoid damaging wavelengths. Pigments in outer epidermal layers, as well as structural materials such as feathers, scales, and air spaces, filter out or scatter some of these wavelengths. Several biochemical mechanisms for protection against UV radiation also exist. These include photoreactivation, or enzymatic repair that reverses damage caused by UV-B through exposure to W - A or longer wavelength radiation. This phenomenon is seen in almost all life forms, with the curious exceptions of certain bacteria and connective tissue cells (mast cells) from placental mmals; marsupial mast cells do undergo p h o t o d v a t i o n (2). One other form of biochemical repair is excision repair, or dark repair (so called because it is done in the absence of light), in which enzymes selectively excise or remove dimers and replace the cleaved portion with DNA replicated from the complementary strand. The sensitivity of DNA to UV damage often manifests itself as mutations in cells. Changes in germ cells can 256 Environ. Sci. Twhnol.. Voi. 22,No. 4, Inn-

result in outright mortality; changes in somatic cells may be responsible for certain forms of cancer, particularly in epidermal cells. Other UV effects on epidermal cells include erythema, or reddening. Erythemal activity peaks at 290-297 nm. Ultraviolet light is not entirely detrimental to animal life. Most vertebrates require exposure to sunlight in order to synthesize sterols such as vitamin D-3, or cholecalciferol. UV-B catalyzes the formation of vitamin D-3 from 7dehydrocholesterol. W and other shonwave radiation also may play a role in coordinating hormone changes corresponding to cycles of sexual activity and reproduction (3). There have been many reports on the deleterious physiological effects on plants exposed to high levels of UV-B, which may increase if stratospheric ozone concentrations decrease. Physiological and biochemical effects of W - B radiation include effects on enzymes; stomatal resistance; concentrations of chlorophylls, proteins, and lipids; reductions in leaf area; and damage to tissue (4).Some plants, however, a p pear to resist increased UV irradiation. The differential susceptibility of plants to W stress clearly is an important factor in their competitive relationships in terrestrial ecosystems. Experiments

with agriculturally important species pairs grown in pots have indicated that significant effects on biomass production took place when W-B was present either at ambient or artificially increased levels (5). Chemistry of phototoxic events The uptake of the electronic energy of a photon by a UV-absorbing molecule initiates photochemically damaging events in cells (the first law of photochemistry states that only light absorbed by a molecule has the potential to cause a chemical reaction). In the W band of the electromagnetic spectrum,the energy of a photon is sufficient to break covalent bonds; it is unusual, however, for this energy to be absorbed that efficiently. The absorption of a photon of UV light by a molecule normally results in its transformation from the low-energy ground state to a higher energy electronically excited state (Figure 2). Almost all stable molecules are singlets-that is, they have paired electrons in their ground states. The first excited state resulting from the acceptance of a photon also is a singlet; however, the energy transfer normally is so fast that spin inversion does not occur. Most excited states, especially singlets, are short-lived species. Excited singlet

products are lipid hydroperoxides: -CH=CH-CH2-CH=CH-CH-CH=CH-CH=CH-

(2,6-di-tert-butyl-4-methyl-

I

phenol, “butylated hydroxy-

OOH

Bilirubin and related bile pigments

I Cercosporin and related fungal

quinones Chlomphylls and degradation produt Furanocoumarins Hemes and related metalloporphy-

rins

Humic substances HVpericin

M&al oxide surfaces

I

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Naphthols Polyacelylenes and thiophenes Polycyclic aromatic hydrocarbons Protoporphyrins Ribofiavinand other Navins Svnthetii dves T&racydin& states lose their excess electronic energy in three principal ways: thermal reconversion to the ground state, with emission of heat; fluorescence (emission of a photon with lower energy than that of the exciting photon) and return to the singlet ground state; and internal conversion (intersystem crossing)-transformation of the singlet to a lower energy triplet excited state with unpaired electrons. Molecules in triplet excited states normally have much longer lifetimes than singlets and are much more liely to engage in chemical reactions. One of the most important fates of these molecules is energy transfer: The collision of the excited triplet (donor) with a different molecule in the ground state (acceptor) can, under certain conditions, result in the promotion of the acceptor to an excited state and the return of the donor molecule to the ground state. Compounds capable of the efficient transfer of their triplet energy to donor molecules are known as photosensitizers (see box). A potential route for the formation of a damaging species from a photochemically activated triplet state is the transfer of triplet energy to oxygen. The product of the energy-transfer reaction is singlet oxygen, IO2. Many classes of biological molecules are susceptible to attack by I&, including several protein amino acids (cysteine, methionine, tryptophan, and histidine), which react with it at rapid rates. Nucleic acid bases, with the exception of guanine, are not rapidly attacked (6). PolyunSaNrated fatty acids also react at slower rates than amino acids; the rate increases with the number of double bonds in the molecule (6),and the

Much evidence indicates that these peroxides contribute to the damage and dysfunction of cells and organelle membranes (7). Molecules in triplet state also may take part in photochemically promoted electron-transferreactions (photooxidations and photoreductions)that form reactive and potentially toxic intermediates. Particularly important in biological systems is the formation of by the superoxide radical anion, 02-, electron transfer from a photoexcited molecule such as ribdavin, tryptophan (8, or tyrosine (9) to molecular oxygen. In water, 02-is partly protonated to its conjugate acid, HOO. (pk= 4.8). Although 02-is a powerful nucleophile that may be able to displace the ester groups of some membrane lipids (IO), its normal fate in the cell is likely to be disproportionation to oxygen and hydrogen peroxide ( H 2 0 2 ) : HOO. + 0 2 - i H+ € I 2 0 2 9 This reaction occurs rapidly except at pH > 10 because the concentration of HOO. is negligible at a high pH (11). Although potentially damaging to some cellular constituents, the H202 produced by this reaction probably has a more important fate: reaction with trace metals such as reduced or complexed iron to form the exceedingly reactive hydroxyl radical (HO.): H202 iFeZ+ -t HO. HO- Fe3+ The hydroxyl radical is the most powerful oxidizing free-radical species known. It reacts very rapidly with most organic compounds (1-100% of the diffision-controlled rate) and also with many inorganic ions (12). For this reason, it must be considered as a potentially damaging agent if it is generated in the vicinity of biologically important molecules. Moreover, the product of the reaction of HO. and an organic molecule is an organic free radicalanother reactive species that may combine rapidly with nearby organic molecules or with oxygen to produce potentially harmful substances. +

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Light absorption by xenobiotics The first report of the insecticidal properties of photodynamic dyes dates back to 1928 (13). Since then, most research in this area has involved either the thiazine dye methylene blue (rv) or the halogenated fluorescein family of xanthene dyes, including rose bengal

(V). All of these dyes require visiblelight wavelengths for activation. A survey of the literature shows that photodynamic dyes are toxic to at least 20 species of insects in five orders and that all life stages, from egg to pupa, are vulnerable (14, 15). Most studies have shown toxicity is directly related to dye concentration and light intensity. The proposed mechanism for the toxicity of these dyes involves the generation of singlet oxygen, because their toxicity is observed to be generally correlated with phosphorescence quantum yield or the inverse of the fluorescence quantum yield (14).From both of these yields the activity of the first excited triplet state can be estimated. The few structure-activity studies that have been made demonstratethat toxicity for the xanthene dyes is a direct function of the number and weight of the halogen substitutes on the fluorescein ring (14). The primary motivation behind research in the efficacy of photodynamic dyes against insects is to develop new chemical control techniques. Photodynamic dyes have several attributes that make them promising candidates, including low toxicity to nontarget organisms, nonmutagenicity, and environmental photodegradability. Light-activated synthetic herbicides. Rebeiz and co-workers have developed potentially herbicidal compounds that interfere with the biosynthesis of chlorophylls (16). The basic component of the treatment mixture is the naturally occurring amino acid 6-aminolewlite (VI), an early intermediate on the biosynthetic pathway to chlorophyll. In essence, the treatment induces the accumulation in leaf tissue of relatively massive amounts of chlorophyll precursors (magnesium tetrapyrroles), which are extremely active photosensitizers. Singlet oxygen produced when this tissue is illuminated reacts with important biomolecules in the cells and eventually kills the plant. Surprisingly, some monocotyledonous crop plants such as corn and barley are quite resistant to the treatment, whereas some common weeds are highly susceptible (16). Phototoxiceffects of petroleum and its constitnents. Petroleum distillates such as kerosene have long been known to contain biocidal components that are produced by exposure of the material to sunlight (17). The photooxidation of No. 2 fuel oil also forms water-soluble compounds that are highly toxic to marine invertebrates, fish (18,algae, and yeasts (19). Payne et al. (20) have demonstrated that W-irradiated automobile crankcase oil is an extremely active mutagen in the Ames Salmonella reversion test, as is used crankcase oil; but UV-irradiated crankcase oil and several %vim. Sci. Technal.. Vol. 22. No. 4, 1988 3 x 7

that oxidized sulfur compounds are far less toxic than oxidized hydrocarbons. Water extracts of a No. 2 fuel oil are toxic to attached freshwater algal communities (attached to a solid substrate and not floating in the water column); the toxicity is greatly increased by light. Bleaching of the photosynthetic pigments of the algae is evident (30). Several oxidized hydrocarbon derivatives such as quinones and hydroperoxides are toxic to the green alga DUMliella biocuhta at low concentrations (0.01-0.7 ppm) (31). Individual aromatic hydrocarbons representative of types found in petroleum also are phototoxic under certain conditions. Anthracene (W) irradiated with UV-A in the presence of primate epithelial cells in tissue culture is bound to the DNA of the cells hy covalent linkages. It was postulated that the cation radical of the hydrocarbon attacks other petroleum products showed no the DNA bases in this reaction (32). significant mutagenic activity. Anthracene and other polycyclic aroSelby et al. (21) have shown that if matic hydrocarbons have been shown light of a wavelength > 360 nm is to be phototoxic to many aquatic orgaused, several natural and synthetic fuels nism including algae, zooplankton, (crude petroleum, shale oil, and coal- and fish (33-35). In addition, the polyderived oils) give rise to Ames-positive cyclic hydrocarbon fluoranthene inmutagens. Coal tar distillates exposed duces severe foliar injury in the presto UV become highly toxic to the fun- ence of UV when it is sprayed onto the gus CMdida albicanr and to several leaves of plants (36). It was suggested other bacteria and fungi (22).The pho- that a IO2 mechanism for toxicity is tochemical weathering of petroleum is most consistent with the results. a complex and poorly understood suhPhototoxic effects of naturally ocject that only recently has been re- curring chemicals from plants. Many viewed (23). Both singlet oxygen and organism, particularly higher plants, free.-radical pathways appear to con- use sunlight to enhance the toxicity of tribute to photooxidation(24).The geu- some of their metabolites; the resulting eral classes of compounds produced photoactive molecules constitute a line when complex petroleum products are of defense against predators such as inexposed to solar W include carboxylic sects. Table 1 lists the plant-derived acids, carbonyl compounds, alcohols, phototoxic molecules identified so far. Phototoxic plant products act through phenols, and hydroperoxides (19, 25, 26). AU of these substances are poten- a variety of mechanism. The most tially toxic, but the hydroperoxide frac- studied compounds, furanocoumarins tion of a photooxidized No. 2 fuel oil is 0 , typically are found in plants hy far the most toxic to microorganisms from the families Umbelliferae (carrot) (19). Moreover, the treatment of the and Rutaceae (rue). Interest in these photooxidized fuel oil with a mild re- chemicals has been stimulated by their ducing agent, which converts hydro- use in therapy of skin disorders and hy peroxides to alcohols, has resulted in their demonstrated mutagenicity. the significant reduction or elimination Furanocoumarins ahsorh W at about of toxic responses (26). The hydropr- 330 nm and are efficiently convected to oxide fraction also has been found to triplet states, which react with DNA contain mutagenic constituents when constituents to form 2 2 cyclotested in a Saccharomycesgene conver- adducts and with some amino acids to form photoadducts of uncertain strucsion test (27). Crude petroleum products have ture (37, 38). It also is possible that widely varying responses to light. In they react with the double bonds of some cases,the irradiated crude oil be- polyunsaturated fatty acids, although comes more toxic, but in other in- no reports of such a reaction have been stances there is little effect (28). The published. Nevertheless, a cycloaddidifference may be correlated with con- tion readon between 8-methoxypsoratent of free-radical and singlet oxygen len and the synthetic olefin, tetraquenchers such as sulfur compounds in methylethylene, has been ohserved to the crude oil, which have been shown occur efficiently (39). Furanmumarin to be readily photooxidized to sulfox- triplets also produce '02and &- (40, ides and sulfones (29). It is probable 41).

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Furanocoumarins have been demonstrated to be phototoxic to a wide variety of organism, ranging from viruses and bacteria through mammals (mcluding humans). Manifestations of toxicity in multicellular eukaryotes generally involve adverse effects on epidermal tissues; human reactions to furanocoumarins include erythema and blistering. In plants, furanocoumarins generally are localized in ducts and channels; they have been reported to be present in cuticles, however, and they also can be found in almost all aboveground plant parts (42). Naturally occurring thiophenes, such as a-terthienyl (IX),display a broad range of phototoxic effects toward a variety of organisms (43). Similarly, the biosynthetidy related aromatic polyacetylenes are broadly biocidal. Phenylhexatriyne 0,for example, is toxic to bacteria, fungi, protozoa, nematodes, insects, and fish, as well as to human fibroblast cells (44). Light and oxygen--especially '9appear to play critical roles in the toxicity of polythiophenes and aromatic polyacetylenes. More specifically, there is evidence that photoexcited aterthienyl is a photosensitizer (45). Downum et al. have suggested that a '&-mediated attack on membrane proteins is responsible for the membrane photcdestmction ohserved in E. coli treated with a-terthienyl (46). Many plants, especially in the families Guttiferae and F'olygonaceae, contain complex, photochemicdy and photobiolog i d y active polycyclic quinones (47). Livestock poisoning by the ingestion of Hypericum (St. John's wort or Klamath weed) species has long been known. The active principle, hypericin (XI), occurs in glands on the leaves, petals, sepals, and stems of the plant. It is a dark red, fluorescent substance that is rather soluble in alkaline or alcoholic aqueous solution. It displays phototoxicity only when it is irradiated with visible light; near-UV wavelengths, by contrast, are virtually ineffective. Oxygen is required for the toxic effects to animals, which include erythrocyte hemolysis. Hypericin promotes the photooxidation of tyramine by an apparent IO2 process (48). Several classes of tricyclic alkaloids also are phototoxic to a variety of organisms. For instance, furanoquinoline and @-carbolinealkaloids generally are phototoxic. The toxicity of the former is believed to he associated with the formation of a monofunctional covalent bond formation with pyrimidine bases in DNA, as are the structurally similar hut biosynthetically distinct furanocoumarins. Nuclear DNA also is thought to be the primary target site for the @-carbolinealkaloids (49).

Defense mechanisms Enzymatic defenses. Organisms have evolved a variety of enzymes that detoxify photochemical or photochemically generated toxicants (Figure 3). These include catalase, an enzyme that converts H24 to water and oxygen; superoxide dismutase, found in all aerobic organisms, which catalyzes the destruction of 02-;and glutathione peroxidase, an important enzyme found in animals (but not in plants) that catalyzes the reduction of hydroperoxides

(94.

One proposed system for cellular defense against reactive oxygen species consists of a sequence of enzymes in cytoplasm, peroxisomes, and mitochondria (51). In this system, superoxide dismutase removes superoxide anion radicals but generates hydrogen peroxide in the process. Catalase promotes the decomposition of hydrogen peroxide to water and oxygen; glutathione peroxidase converts lipid peroxides that result from the reaction with hydroxyl radicals to lipid alcohols and water. This last reaction is associated with one catalyzed by glutathione reductase, in which oxidized glutathione receives reducing equivalents from reduced nicotinamide-adenine dinucleotide phosphate (NADPH)to regenerate

reduced glutathione. In addition to biochemid mechanisms for decomposing excited oxygen species, other detoxification mechanisms alter the strumre of phototoxic compounds to render them nonphototoxic. The polysubstrate monooxygenases or mixed function oxidases (MFOs), a complex of membranebound enzymes that catalyze a variety of oxidative reactions,have been implicated in the detoxification of furanocoumarins in at least two species of insects ( 5 2 ) . The carboxylic acid metabolites resulting from MFO metabolism are incapable of photobinding to DNA. Small molecules. Larson (53)has reviewed the occurrence of potential quenchers and inhibitors of phototoxic plant chemicals that are known to occur in insects. These compounds include potential '02quencbers such as tertiary amines, furans, carotenoids, and tetrasubstituted oletins. They also comprise free-radical quenchers and peroxide deE, stroyers such as vitamin C, vi& hindered secondary amines, and sulfur compounds. Little or no experimental work, however, has been carried out to test the hypothesis that these chemicals actually function as defensive substances in the insect.

Acknowledgments We thank K.Marley, I. Nitao, A. Zangerl, and R. G. Zepp for helpful discussions; the National Research Council for providing a =Nor associateship to Richard A. Larson; and the National Science Foundation for an award (#BSR 835 1407) to May R. Berenhaum. This article has been reviewed for suitability as an ES&T feature by Wendell L. Dilling, Midland, Mich. 48640. References (1) Piled, M . 2 ; Santus, R.; Land, E. 1.

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