Biol Rev Uv Abs Compounds

Biol. Rev. (1999), 74, pp. 311–345 Printed in the United Kingdom # Cambridge Philosophical Society 311

Ultraviolet radiation screening compounds

CHARLES S. COCKELL",* and JOHN KNOWLAND#

"Department of Plant Biology, Carnegie Institute of Washington, 290 Panama Street, Stanford, CA 94305-1297, U.S.A.#Department of Biochemistry, South Parks Road, University of Oxford, Oxford, OX1 3QU, U.K.

(Received 15 June 1998; revised 15 March 1999; accepted 25 March 1999)

ABSTRACT

Amongst the diversity of methods used by organisms to reduce damage caused by ultraviolet (UV) radiation,the synthesis of UV-screening compounds is almost ubiquitous. UV-screening compounds provide a passivemethod for the reduction of UV-induced damage and they are widely distributed across the microbial, plantand animal kingdoms. They share some common chemical features. It is likely that on early earth strongselection pressures existed for the evolution of UV-screening compounds. Many of these compounds probablyhad other physiological roles, later being selected for the efficacy of UV screening. The diversity inphysiological functions is one of the complications in studying UV-screening compounds and determiningthe true ecological importance of their UV-screening role. As well as providing protection against ambientUV radiation, species with effective screening may also be at an advantage during natural ozone depletionevents. In this review the characteristics of a wide diversity of UV-screening compounds are discussed andevolutionary questions are explored. As research into the range of UV-screening compounds represented inthe biosphere continues, so it is likely that the properties of many more compounds will be elucidated. Thesecompounds, as well as providing us with insights into natural responses to UV radiation, may also haveimplications for the development of artificial UV-screening methods to reduce human exposure to UVradiation.

Key words : UV radiation, evolution, screening, compounds.

CONTENTS

I. Introduction ............................................................................................................................ 312II. Advantages of passive UV screening ....................................................................................... 312

III. Chemical and absorbance characteristics of UV-screening compounds .................................. 314IV. Determining whether a compound has a screening role ......................................................... 318V. Scytonemin – a positively identified UV-screening compound................................................ 319

VI. Mycosporine-like amino acids and the ecological complications in studying UV-screeningcompounds............................................................................................................................... 320

VII. UV screening in plants............................................................................................................ 323VIII. UV screening and melanin...................................................................................................... 325

IX. Other candidate UV-screening compounds............................................................................. 326(1) Carotenoids ....................................................................................................................... 326(2) Other compounds ............................................................................................................. 327

X. UV-screening compounds and evolution................................................................................. 328(1) UV screening on prebiotic earth ...................................................................................... 328(2) UV screening and early life .............................................................................................. 331(3) Evolutionary relationships of UV-screening compounds................................................... 332(4) UV-screening compounds and the Berkner–Marshall hypothesis ..................................... 333(5) UV-screening compounds, ozone reductions and Phanerozoic palaeobiology .................. 334

* Present address : Charles Cockell, M}S 239-20, NASA Ames Research Center, Moffett Field, CA 94035-1000,USA.

312 Charles S. Cockell and John Knowland

XI. Artificial screening compounds and human UV protection .................................................... 335XII. Conclusions.............................................................................................................................. 338

XIII. Acknowledgements .................................................................................................................. 339XIV. References................................................................................................................................ 339

I. INTRODUCTION

For 3.8 billion years the evolution of methods toattenuate ultraviolet radiation has been a ubiquitousproblem for life and particularly for photosyntheticorganisms that depend on solar radiation for theirenergy requirements. For some organisms that livein subsurface regions of the earth, in the oceandepths, caves and other habitats that exclude solarradiation, the problem of UV radiation damage issolved. These organisms are not considered in thisreview.

For convenience ultraviolet (UV) radiation is splitinto four major bands. Vacuum UV is radiationwith a wavelength less than 200 nm. UV-C radiationoccupies the region between 200 and 280 nm.Neither vacuum UV nor UV-C reach the surface ofthe present-day earth because of atmosphericRayleigh scattering and ozone absorption, althoughthese regions are important parts of the extra-terrestrial spectrum (e.g. Nicolet, 1989). UV-Bradiation is energetically less damaging than UV-C.In the scientific literature, UV-B radiation is oftendefined as 280–320 nm. However, the legal definitionprovided by the International Commission onIllumination sets the UV-B radiation range as 280–315 nm. On earth, most of the UV-B radiationis attenuated by the ozone column that absorbsstrongly in the Hartley region (200–300 nm) andweakly in the Huggins Band (300–360 nm). Finally,UV-A radiation (315–400 nm) reaches the surface ofthe earth relatively unattenuated and is still lessenergetic than UV-B radiation.

UV radiation, and particularly the higher energywavelengths, has a range of effects. They includeDNA damage in most organisms (Harm, 1980;Karentz, Cleaver & Mitchell, 1991a ; Karentz et al.,1991b), inhibition of photosynthetic primary pro-ductivity in both micro-organisms (e.g. Smith et al.,1980, 1992; Ha$ der & Worrest, 1991; Cullen &Neale, 1994; Prezelin, Boucher & Schofield, 1994;Vincent & Roy, 1993) and higher plants (e.g. Tevini& Teramura, 1989; Tevini, 1993 and referencestherein), inhibition of nitrogenase activity (Sinha et

al., 1996), inhibition of heterocyst formation in somecyanobacteria (Sinha et al., 1996), reduction inmicrobial motility (e.g. Donkor, Amewowor &

Ha$ der, 1993a, b ; Donkor & Ha$ der, 1995), and adiversity of other responses that have been reviewedelsewhere (e.g. Tevini, 1993; Vincent & Roy, 1993;Vincent & Quesada, 1994).

It is accurate to view the overwhelming effect ofUV radiation as a damaging agent to a wide varietyof biological systems. However, it is wrong to painta picture of UV radiation as solely damaging. It hasmany beneficial roles in the biosphere. For example,UV-A reflected from petal anthocyanins (Flint &Caldwell, 1983) is essential for flower recognition bypollinating insects such as bees. UV-A is involved inbutterfly wing recognition during mating (Meyer-Rochow, 1991). Some spiders use the UV reflectanceof their webs to capture prey (Craig & Bernard,1990). Some fish that possess tetrachromatic visionvisualize UV-A at 360 nm and this may be essentialfor their visual acuity (e.g. Avery et al., 1983; Harosi& Hashimoto, 1983; Downing, Djamgoz &Bowmaker, 1986; Bowmaker & Kunz, 1987).Lizards and some other vertebrates also possess UV-A vision (Makino et al., 1995). Various growth andphotomorphogenetic effects in plants, mediatedthrough blue}UV-A receptors, involve UV-A radia-tion (Salisbury & Ross, 1992). As will becomeapparent in this review, some of the pigments andcompounds that absorb UV radiation have a role toplay in these processes.

II. ADVANTAGES OF PASSIVE UV SCREENING

A number of methods exist for coping with thepotential damage caused by UV radiation. They areillustrated in Fig. 1. An important response isdamage repair, particularly in DNA, which is one ofthe most susceptible biological targets (Jagger,1985). DNA photoreactivation, during which theblue light}UV-A inducible enzyme photolyasecleaves covalently linked thymine dimers in DNAgenerated by UV-B radiation is an importantresponse in most organisms (Sutherland, 1981). Thenon-light dependent removal of a section of DNAcontaining the damage lesion (DNA excision repairor ‘dark’ repair) has also evolved as an effectiverepair process (Hanawalt et al., 1979). Althoughthese mechanisms are effective, by definition they

313Ultraviolet radiation screening compounds

UVradiation

Avoidance

Protection

Repair

Phototaxis. Lifestyles that completelyavoid solar radiation

Physical substrates (rocks, iron, sulphur, etc.)Screening under organic compoundsQuenching of reactive oxygen species using carotenoidsSpecific UV-screening compounds:Mycosporine-like amino acids (many microorganisms),scytonemin (cyanobacteria),flavonoids (plants), melanin (animals)

Photoreactivation, excision repair, post-replication repair,de novo synthesis of proteins and lipids

Examples

Fig. 1. Strategies of ultraviolet (UV) radiation mitigation. UV-screening compounds provide a first line of defenceagainst UV radiation. Repair processes are subsequently used to deal with damage. The figure illustrates the diversityof responses that are available to an organism.

address damage that has already occurred. Becauserepair processes can never be 100% efficient, the lessdamage that occurs, the greater the potentialadvantage to the organism, regardless of the absolutelevels of repair that an organism may be able toelicit. This is particularly the case for single-celledorganisms, where even a single-point mutation canbe lethal. The synthesis of carotenoids that quenchoxygen free radicals generated by UV-inducedphotochemical reactions (Krinsky, 1979) is also animportant response in preventing UV-induced dam-age to a wide diversity of biological macromolecules.Again, however, its necessity stems from reactiveoxygen species formed by UV radiation that hasalready penetrated the cell.

Organisms may be able to avoid UV radiation byphototaxis, whereby they diurnally limit exposure toUV radiation by moving in response to the UV-Bgradient (e.g. Bebout & Garcia-Pichel, 1995).Phototaxis is less widely distributed than damagerepair processes and is likely to be energeticallydemanding for organisms regularly exposed to UVradiation.

For organisms exposed to UV radiation forsubstantial parts of their life-cycles and, like photo-trophic organisms, for their source of energy,mechanisms that passively screen UV radiation will

contribute to preventing UV-induced damage in thefirst place (see Fig. 1). UV screening may alsoprovide some energetic advantage by reducing theneed for constantly active avoidance and repairprocesses.

Two approaches exist to screen UV radiationpassively. There are a variety of physical substratesthat may be effective, such as a water columncontaining organic impurities. Dissolved organiccarbon is one of the primary factors in reducing UVpenetrability into freshwater systems (Booth &Morrow, 1997). Iron compounds can provide signifi-cant reductions in the UV region, particularlyoxidized ferric (Fe$+) iron whose absorptioncoefficient is an order of magnitude greater thanferrous (Fe#+) iron (e.g. Olsen & Pierson, 1986;Pierson, Mitchell & Ruff-Roberts, 1993). Iron-containing sediments could provide significant UVattenuation for benthic microbial communities(Garcia-Pichel, 1998). Elemental sulphur can alsopotentially provide specific UV absorption (Cockell,1998a). However, most physical substrates will non-specifically attenuate visible wavelengths either bydirect absorption or scattering, thus limiting ex-posure to photosynthetically active radiation forphototrophic organisms. For some organisms such aspolar cyanobacteria that dominate as a result of slow


persistent growth and tolerance of extreme environ-ments (Tang, Tremblay & Vincent, 1997), ratherthan fast optimized growth, this disadvantage maybe unimportant. The cryptoendolithic communitiesin the sandstone of the Ross Desert, Antarctica(Nienow, McKay & Friedmann, 1988) wheregrowth rates may be as low as one cell division peryear, are such an example. These communities livein the subsurface layers of rock, where UV isattenuated, but also visible light. In this case thegrowth in layers of rock offers the advantage of amore stable micro-climate. However, for manyorganisms in less extreme habitats, increased expo-sure to photosynthetically active radiation providedby UV-specific screening may be advantageous.

A second disadvantage of physical screeningmethods is that organisms are limited to the habitatswhere the physical substrate is available. Organismsthat can synthesize their own UV-protecting com-pounds have the opportunity to occupy a greaterdiversity of habitats, depending of course on limi-tations imposed by other environmental factors.

With these limitations in mind, it is unsurprisingthat the synthesis of passively screening compoundshas evolved as a ubiquitous first-line response to UVradiation.

III. CHEMICAL AND ABSORBANCE

CHARACTERISTICS OF UV-SCREENING

COMPOUNDS

The evolution of a successful UV-screening com-pound ultimately depends upon simple organicphotochemistry. The π-electron system is one of themost effective UV radiation absorbers. Π-electronsystems are primarily found in conjugated bondstructures that may be represented both in linearchained molecules with alternating single and doublebonds and in many aromatic and cyclic compoundscontaining electron resonance. The overlappingorbitals of π-electrons have absorption maxima inthe UV region that cause an energetic transition ofπ-electrons to anti-bonding π*-electron orbitals. Π-electron systems are a common chemical theme inthe function and characteristics of natural UV-screening molecules (Cockell, 1998b).

Alterations in the structure of a conjugatedmolecule change the absorbance characteristics andthus the region of the spectrum that is attenuated.Generally, the larger the molecule, the longer thewavelengths that are absorbed because, in the

CH2 CH CH CH CH CH2

CH2 CH CH CH2

(CH2 CH CH CH )2

OH

217

258

287

255

270

275

kmax (nm)

Fig. 2. Ultraviolet absorption maxima (λmax

) of someselected organic molecules demonstrating the batho-chromic shift that occurs when both the size of themolecule and the nature of the side groups is altered.

simplest terms, the wave-particle duality results inan increase in the wavelength of the electrons thatcan be accommodated in a larger molecular struc-ture. For example, in the conjugated molecule 1,3-butadiene, an important absorption maximum is at217 nm, whereas for 1,3,5-hexatriene the corre-sponding maximum is increased to 258 nm and for1,3,5,7-octatetraene it increases further to 287 nm inthe UV-B region (Fig. 2). Very long chain moleculessuch as the natural carotenoid, β-carotene (11ethylenic linkages) has an absorption in the visibleregion at 455 nm. The same shift in wavelength maybe achieved by increasing the number of conjugatedbonds and substituents in aromatic structures ratherthan in a linear chain. For example, benzene has anabsorbance peak at 255 nm, whereas naphthalene,that contains two rings, has the correspondingabsorbance at 275 nm (Fig. 2). Although manynatural linear carotenoids can absorb in the UVrange, their physiological importance in screening inmost known cases is secondary to aromaticderivatives. The role of carotenoids in screening andsome evolutionary aspects of the preferential selec-tion of aromatics as screening compounds arediscussed in greater detail in Section IX.

As well as the absorption maximum, the extinctioncoefficient is also altered by the side groups and themolecular structure. Generally, aromatic com-pounds have lower extinction coefficients, ε, than


NH2

OH OH NH

OCH3

CO2H

NHOH

OCH3

NH

CO2H

OH OH

NH

OCH3

NH

CO2H

OH OH

Palythine (kmax = 320 nm) Asterina (kmax = 330 nm) Palythene (kmax = 360 nm)

NH

OH OH NH

OCH3

CO2H

OH

CH3 CO2H

NH

OH OH NH

CO2H

OH

CO2H

NH

OH OH NH

CO2H

OH

Palythinol (kmax = 332 nm) Porphyra (kmax = 334 nm) Shinorine (kmax = 334 nm)

CO2H

NH

OH OH NH

CO2H

OCH3

O

OH OH NH

CO2H

CO2H

NH

OCH3

OH OH NH

CO2H

Mycosporine-glycine:valine(kmax = 335 nm)

Mycosporine-glycine(kmax = 310 nm)

Palythenic acid(kmax = 337 nm)

O

OH OH

HO OCH3

OH

OCH3

OH OH

R2

R1O–OOOC

NH

N+

HCOOH

O R2

R1

Gadusol(kmax = 294 nm)

Nostoc commune E335(kmax = 335 nm)

R1 = Gal, Xyl, GlcUR2 = Gal, Glc, GlcN

OCH3 OCH3

OCH3

Fig. 3. Structures of some of the principal mycosporine-like amino acids (MAAs) found in nature. Gadusol, which isstructurally related to MAAs and found in fish roe is also shown as is E335, the Nostoc commune polysaccharide-linkedmycosporine (see text for details). λ

maxis the ultraviolet absorption maximum.


unsaturated aliphatic compounds. For example, εfor the benzene maximum at 255 nm is 230 mol−" lcm−", whereas for 1,3,5-hexatriene ε at the cor-responding absorption maximum at 258 nm is 80000mol−" l cm−". However, substitution can increase theextinction coefficient substantially. For example, inthe phenolate anion, ε is increased to 2600 mol−" lcm−". Generally, the more substituents that areadded to a molecule the more intense the absorptionbecomes. These type of substituent effects on theabsorption coefficient are important in achieving theefficacy of screening required in natural UV-screening compounds.

Here, it is worthwhile to illustrate the conceptsoutlined above with some natural examples.

Many organisms produce mycosporine-like aminoacids (MAAs) as a natural UV screen. The eleganceof their UV-absorbance properties lies in themodulation of the peak absorbance of a basiccyclohexenone or cyclohexenimine core structure.Fig. 3 shows some of the major MAAs found innature. Hetero-atoms with non-bonding electrons,such as oxygen or nitrogen and the halogens, canabsorb UV radiation when the electrons make atransition from n orbitals to π* orbitals. However,this absorption is quite weak. A more importanteffect of these substituents is their ability to alter theabsorption properties of aromatic ring structures.The non-bonding electrons can take part in theresonance of the ring structure which causes abathochromic shift in the absorbance maximum(Gillam, 1970). Thus, although the absorbance peakof the benzene ring structure is 255 nm, in theMAA cyclohexanone core structure it is increased to280 nm.

The degree of the bathochromic shift caused bythe substituent is determined by it’s mesomeric effect(the ability of the non-bonding lone pair electrons totake part in the electron resonance of the ringstructure). Mycosporine-glycine, which has a cyclo-hexanone core structure, has an absorbance maxi-mum at 310 nm. Palythine, which has a nitrogen-containing cyclohexenimine core structure ratherthan the cyclohexanone structure, but with identicalside groups on all other positions has its absorptionmaximum shifted to 320 nm. Further changes in theabsorption maxima are caused by substituent effects.For example, the MAA palythene has a conjugatedtriene system attached to the nitrogen which shiftsthe absorbance to 360 nm, an additional 40 nmcompared to palythine. Most of the UV-B-screeningMAAs use a cyclohexanone structure, whereas theUV-A-screening compounds use a cyclohexenimine

OHH

N

O

O

H

N

OH

1.2

1.0

0.8

0.6

0.4

0.2

0.0250 300 350 400 450 500 550 600 650 700 750 800

Wavelength (nm)

Ab

sorb

ance

Fig. 4. Phenolic and indolic structure of the widelydistributed terrestrial cyanobacterial UV-screening pig-ment, scytonemin. The figure also shows the absorbancespectrum of the oxidized pigment in tetrahydrofuran(adapted from Proteau et al., 1993). Absorption is shownas arbitrary units.

core structure, presumably because the non-bondingnitrogen electrons cause a greater bathochromic shifttowards the UV-A region. The evolutionary aspectsof this are discussed in Section IX. By synthesizing arange of MAAs, organisms might be able to screenbroadly in the UV-A and B range. In a singleorganism, the breadth of UV absorbance can spanfrom 230 to 400 nm and the peak absorbance canrange from 310 to 360 nm depending on thecomplement of MAAs.

The absorbance properties of a UV-screeningcompound are further changed by increasing thecomplexity of π-electron systems, their relationshipto other moieties and their number. The UV-absorbing compound scytonemin is a lipid-solublephenolic and indolic derivative produced in thesheaths of cyanobacteria (Garcia-Pichel &Castenholz, 1991; Garcia-Pichel, Sherry &Castenholz, 1992). This compound is present inthese organisms in addition to MAAs. Fig. 4 showsthe structure of scytonemin. The complexity of thering structures generates a specific type of UV-absorbance pattern (Proteau et al., 1993). The size ofthe molecule is partly responsible for the long-


HO

OH

R1

R2

R3

R4OH

O

CA

2

B

1.0

0.8

0.6

0.4

0.2

0250 300 350 400 450

354 nm

Wavelength (nm)

Ab

sorb

ance

Fig. 5. Basic structure of higher plant flavonoid molecules(adapted from Stafford, 1991). Changes in the side groupsresult in different molecules. With stereochemistry at C

#:

flavonone (R$, H; R

%, CFO), 3-hydroxyflavanone

(dihydroflavonol) (R$, OH; R

%, CFO), flavan-3-ol (R

$,

OH; R%, H). Loss of stereochemistry at C

#(double bond

positions refer to C-ring) : flavone (C#FC

$; R

$, H; R

%,

CFO), flavonol (C#FC

$; R

$, OH; R

%, CFO), antho-

cyanidin (O"FC

#; C

$FC

%; positive change on C ring; R

%,

H or OH), isoflavone (aryl migration to C$; R

%, CFO; B-

ring – R", R

#, H, or OH).

A typical absorbance profile for total phenolicsextracted in 85% acidified methanol is also shown. Thisextract is from the epidermis of the desert cactus, Opuntia

phaeocantha (C. S. Cockell, unpublished data). Absorptionis shown as arbitrary units.

wavelength UV-A screening. The molecule screensmost strongly from 325 nm to 425 nm across theUV-A, but the shoulder of the peak extends into theUV-B. The molecule also has an absorbance peak inthe UV-C with a maximum at 250 nm, which resultsfrom intense short-wavelength π to π* transitionsfound in most conjugated molecules.

Since the Cambrian explosion, many multicellularorganisms have also evolved other uses of the π-electron system specifically to screen UV radiation.Flavonoids (Fig. 5), produced by plants, screen inthe UV-A and B (Caldwell, Robberecht & Flint,1983). Ring B attached to the two-ring A and C

0.8

0.6

0.4

0.2

0.0200 300 400 500 600

Wavelength (nm)

Ab

sorb

ance

Fig. 6. Typical absorption profile of melanins (adaptedfrom Ravishankar et al., 1995). The pigment shows a non-specifically increasing absorbance towards shorter wave-lengths. The absorbance profile results from the manycomplex conjugated structures in the polymeric molecule.

system provides the UV-A and UV-B absorbance.Ring A also contributes to the absorbance in theUV-C region at approx. 250 nm. Because of thecomplex ring structure, single flavonoids may pro-vide broad UV-A and B screening, unlike MAAs,where evolution from a simple cyclohexenone orcyclohexenimine core structure provides only adiscrete absorbance maximum.

Melanin, used for screening UV in humans andmany vertebrates, also has a complex polymericstructure (Kollias et al., 1991) containing aromaticsand indole derivatives to provide UV-A and UV-Bscreening. Its complex polymeric structure generatesa rather non-specific screening (Fig. 6) with in-creasing efficacy at lower wavelengths (Crippa,Cristofoletti & Romeo, 1978; Menon et al., 1983).

Finally, it is important to realize that π-electron-containing molecules are also the primary targets ofUV radiation damage such as in unsaturated lipids,nucleic acids and aromatic and indolic amino acid-containing polypeptides. Thus, and perhapsunsurprisingly, a common chemical theme lies at theheart of UV radiation damage and screening(Cockell, 1998b).

Many compounds that happen to containaromatic or conjugated groups may fortuitouslyabsorb UV radiation. Therefore, to be sure that acompound produced by a cell either in sheaths orintracellularly has a physiologically significant UV-screening function, something more concrete than apurely structural definition is required. How do weknow that a compound has either specifically evolvedto screen UV radiation, or at least provides some


constitutive physiologically significant UV screeningfor an organism?

IV. DETERMINING WHETHER A COMPOUND

HAS A SCREENING ROLE

In attempts to elucidate the true screening role ofcyanobacterial scytonemin and mycosporine-likeamino acids, F. Garcia-Pichel, R. W. Castenholzand their co-workers made extensive considerationsof what criteria constitute a specific UV-screeningcompound (e.g. Garcia-Pichel, Wingard &Castenholz, 1993). From these studies a series ofrules was suggested for identifying a compound witha UV-screening role. Some compounds that screenUV radiation may not have an evolutionarilydedicated screening role. However, their presence insufficient quantities in a cell may provide someconstitutive physiologically significant UVscreening. Considering this caveat, the basicapproaches for demonstrating the physiologicalsignificance of potential UV-screening compoundsare elaborated below.

(1) The compound must obviously screen UVradiation. In most cases, a UV absorbance peak isfirst demonstrated by spectrophotometric analysis ofa cell extract from the organism. Methanolicextractions (e.g. a 2 h extraction at 45 °C in 80%aqueous methanol in the case of microbes, or a20 min to 1 h extraction at room temperature in75:24:1 methanol :water :HCl for ground planttissues) typically are used to extract and study water-soluble compounds such as intracellular MAAs orplant flavonoids (e.g. Robberecht & Caldwell, 1978;Scherer, Chen & Boger, 1988; Tevini, Braun &Fieser, 1991; Garcia-Pichel et al., 1993). Acetone,tetrahydrofuran, ethyl acetate and similar solventscan be used to study lipid-soluble molecules such asthose found in the sheaths of cyanobacteria (e.g.Garcia-Pichel & Castenholz, 1991). Methanol andacetone extractions can initially be compared todecide if the compound is an intracellular water-soluble compound or whether it is synthesized inmembranes or sheaths. Once a potential compoundis found, it can be purified by a variety of techniques.High-performance liquid chromatography (HPLC),where the detector is set to the λ

maxof the UV

absorbance peak (e.g. Nakamur, Kobayashi &Hirata, 1982; Carreto et al., 1990; Karentz et al.,1991a ; Tevini et al., 1991; Vogt, Gulz & Reznik,1991; Braun & Tevini, 1993) or thin layer chromato-graphy (TLC) (e.g. Mohle & Wellmann, 1982;

Beggs, Stolzer-Jehle & Wellmann, 1985; Garcia-Pichel & Castenholz, 1991) may be the first step toisolating the compound from other cell compoundsand confirming the existence of discretecompound(s) with a maximal absorbance in the UVrange.

A number of techniques have been used todetermine the structure of compounds, but nuclearmagnetic resonance spectroscopy (NMR) has provento be a particularly useful technique (e.g. Guilfordet al., 1988; Proteau et al., 1993; Wilmesmeier,Steuernagel & Wiermann, 1993).

(2) Inducibility of the compound in the living cellby UV radiation provides strong evidence of aspecific UV-screening function. This criterion can betested by placing commercially available UV-A- or-B-emitting lamps at different distances from theorganism under culture (e.g. Beggs et al., 1985;Garcia-Pichel et al., 1993). Because some bulbs emita spike in the UV-C at 254 nm, care must be takenin accurately defining the spectral emission. Cellextracts are taken from these organisms after fixedtime periods when the organisms have adapted tothe new UV radiation regime. The absorbancevalue at the wavelength of maximum absorbance isthen measured. An increase in absorbance thatcorrelates with increasing UV radiation flux isindicative of UV-induction of the compound. Strongcorrelations such as the linear relationship betweenUV-B radiation and induction of some plantflavonoids (e.g. Wellmann, 1975) may provideparticularly strong evidence for a UV-screening role.As a further, more detailed test, the action spectrumfor induction (the relative induction at differentwavelengths that may be generated by filters ormonochromatic UV radiation sources) might besimilar to the compound’s absorbance profile. Wave-lengths of maximum absorbance of the compoundmay cause maximum production of the compound(e.g. Garcia-Pichel et al., 1993).

As alluded to before, although induction of acompound by UV radiation may suggest a UV-screening role, it is important to recognize thatabsence of evidence does not constitute evidence ofabsence. A constitutively expressed compound canprovide some physiological advantage. As discussedin greater detail below, sporopollenins areconstitutively produced in algal cell walls and sporewalls, but have been observed to confer increasedUV tolerance in some species of algae (Xiong et al.,1997), presumably caused by the aromatic structuresthat they contain. Oat seedlings constitutivelyproduce flavonoids that provide some advantage in


mitigating UV damage during early stages of growth(Braun, 1991). Many cinnamoyl esters screen UV-Bin rye seedlings, but apparently are not specificallyinduced by increasing UV-B radiation (Tevini et al.,1991). Melanin is produced constitutively in dark-skinned people and provides some physiologicalbenefit (van der Leun & de Gruijl, 1993). Thus, afurther test must be to prove that a compoundprovides some physiologically significant benefit,regardless of whether it is produced constitutively orinducibly.

(3) A number of approaches can be used toexamine the efficacy of a compound’s ability toprovide physiologically worthwhile screening. Cellsstained with 4«,6-diamidino-2-phenylindole (DAPI),which binds to DNA, will fluoresce in the blue regionat 461 nm when they are excited with UV light ofwavelength 326 nm. In the case of inducible com-pounds, the fluorescence can be compared to cellsthat have been acclimated in the absence of UVradiation or in low-light conditions and in whichcompound concentrations are lower. Comparisons ofDAPI-fluorescence values allow the intensity of UVradiation reaching the nucleus of the cell to bedetermined and thus the potential DNA damagethat is mitigated by the compound (e.g. Garcia-Pichel et al., 1993). Techniques to determine if thecompound can reduce damage to photosystems inphotosynthetic organisms include measuring carbonuptake rates with ["%C]bicarbonate or measuringchlorophyll photobleaching by fluorescence at675 nm (Garcia-Pichel et al., 1993).

In the case of a constitutively produced com-pound, natural inter-species variation in concen-tration might be used to examine the physiologicalsignificance of the compound (Xiong et al., 1996,1997). Care must be taken since in the functioningcell there may be other inter-species differences inrepair processes and synthesis of alternative UV-screening compounds. Optimally, mutants might begenerated for such studies in which the compound isexpressed at different levels. Given that many of thepathways of UV-screening compounds are nowbeing elucidated (such as MAA production by theshikimate acid pathway), the use of mutants to studythe physiological value of UV-screening compoundsis a promising area of development.

The screening ability of the compound is oftenexpressed as a ‘ screening factor ’. This value rangesfrom 0 (no screening) to 1 (complete screening) (e.g.Garcia-Pichel & Castenholz, 1993). Alternatively, itcan simply be expressed as a percentage absorbance.This number provides a convenient indication of the

value of the compound to the organism. In the caseof sheath compounds, the screening factor will simplybe the complementary value of the transmittance ofthe compound at a given wavelength. For intra-cellular compounds the screening factor calculationis more complex since the absorbance must beintegrated across the length of the cell. The endproximal to the radiation will have a value of zeroand the distal end will have a value that dependsupon the size of the cell and the absorbance andscattering of the compound at a given wavelength.The absorbance can be either measured or calcu-lated based on a given intracellular concentrationand a known extinction coefficient using the Beer–Lambert law, although approaches that also takeinto account scattering are likely to provide a moreaccurate appraisal. Theoretical models can be usedto calculate screening efficacy (Garcia-Pichel, 1994).

(4) Because screening compounds are a passivemethod of coping with UV radiation, proving thatenhanced survival under elevated UV radiation isdue to the compound may be complex. Induction ofother photoprotection mechanisms or physiologicalresponses including repair processes, may enhancesurvival. Thus, as a further test, the compoundshould provide increased resistance to UV radiationwhen other physiological processes are notfunctioning.

There are various methods for examining thephysiological value in cells lacking other functionalresponses. Desiccation is such a method. For micro-organisms, UV exposure during desiccation may befollowed by many of the techniques described underpoint (3) above. For multicellular organisms such asstarfish that may contain MAAs, this approach maybe problematic, since the desiccation will clearly killa whole organism. In such cases, the experimentermight concentrate on measurements of cellularprocesses that do not depend upon a fully functioningcell. The measurement of thymine dimer generationin DNA or measurements of enzyme damage by anappropriate assay may provide insights into theprotective efficacy of the compounds in isolated non-functional cells.

V. SCYTONEMIN – A POSITIVELY IDENTIFIED

UV-SCREENING COMPOUND

Na$ geli first described the brown colouration of somecyanobacteria and particularly cyanobacterial mats(Na$ geli & Schwenderer, 1877). This colouration is


now ascribed to scytonemin, the first compound toreceive rigorous application of the rules of screening.It is now presumed to have a dedicated screeningrole (e.g. Garcia-Pichel & Castenholz, 1991, 1993;Garcia-Pichel et al., 1992). The sheaths of a widerange of terrestrial cyanobacteria contain scyto-nemin. Planktonic forms apparently do not containthe compound, although a compound with similarabsorbance characteristics was reported from anIcelandic phytoplankton bloom (designated P380)(Llewellyn & Mantoura, 1997). It has been deter-mined by NMR to be a dimeric molecule ofmolecular weight 544 Da, made up of indolic andphenolic subunits and formed from the condensationof tryptophan and phenylpropanoid-derived sub-units. These provide the UV-A absorbance charac-teristics and its in vivo maximum at 370 nm(Proteau et al., 1993). Fig. 4 shows the structure ofscytonemin and the absorbance profile that providesthe first indication of a screening role. Scytonemin ismainly found in the green oxidized form, but it canbe reduced to a red form that in nature is found inthe lower reducing layers of cyanobacterial mats(Proteau et al., 1993).

The induction of scytonemin is proportional to theUV-A and visible light intensity. Light in the UV-Aregion of the spectrum is particularly effective ineliciting production (Garcia-Pichel & Castenholz,1991; Garcia-Pichel et al., 1992; Ehling-Schulz,Bilger & Scherer, 1997). Most cyanobacteria have aminimum photon fluence at which scytoneminbegins to be synthesized. It ranges from 99 µmol m−#

s−" in Diplocodon sp. up to 250 µmol m−# s−" inScytonema sp. ‘Scytonemin-free ’ cells are examinedwith low fluences (33 µmol m−# s−") and comparedto cells at higher irradiances in which scytonemin hasbeen induced. Natural light levels in the field havebeen found to be correlated with scytonemin levels inRivulari sp. colonies (Pentecost, 1993) although anegative correlation was found in Scytonema sp.populations. This negative correlation has beenexplained by the differing water availability in thetwo sites that were studied and the differing celldivision rates (Pentecost, 1993). Faster dividing cellsoften have less scytonemin because they are lessprone to damage that accumulates over longerperiods of time. When these factors were considered,Pentecost (1993) concluded that the field dataconcur with the laboratory-deduced UV-protectingrole of scytonemin.

Scytonemin can provide quite effective screeningthat ranges between 2 and 55% attenuation of lightat 320 nm at the single-cell level (Garcia-Pichel &

Castenholz, 1993). For cells under layers of mats orinside colonies of cells, the attenuation of UV-Aradiation may be even more effective. The physio-logical value of the compound has also been shown(Garcia-Pichel & Castenholz, 1991; Garcia-Pichel et

al., 1992). Scytonemin can effectively reduce photo-synthesis inhibition by UV-A radiation (measuredby oxygen evolution) and it can reduce photo-bleaching of chlorophyll a. In a Chlorogloeopsis species,fluorescence of chlorophyll at 680 nm shows a strongreduction when excited with UV-A wavelengths inscytonemin-containing cells in comparison to thoselacking it (Garcia-Pichel et al., 1992). In cells withscytonemin, the compound also allows for initiallyfaster growth rates when the cells are grown underUV illumination. The screening role of scytonemin isalso effective during physiological inactivity such asdesiccation, proving that the passive process of UVblocking is physiologically effective (Garcia-Pichel et

al., 1992).

VI. MYCOSPORINE-LIKE AMINO ACIDS AND

THE ECOLOGICAL COMPLICATIONS IN

STUDYING UV-SCREENING COMPOUNDS

Mycosporines were first identified in fungi as havinga role in UV-induced sporulation (Leach, 1965;Favre-Bonvin, Arpin & Brevard, 1976; Brook, 1981;Young & Patterson, 1982). Their relatives, themycosporine-like amino acids, have since been foundin a wide variety of organisms as diverse ascyanobacteria (Shibata, 1969; Sivalingam et al.,1974b ; Haxo et al., 1987; Karentz et al., 1991b ;Garcia-Pichel & Castenholz, 1993), red algae(Sivalingam, Ikawa & Nisizawa, 1974a ; Sivalingamet al., 1974b ; Takano et al., 1979; Tsujino et al., 1978;Tsujino, Yabe & Sekikawa, 1980; Sekikawa et al.,1986; Karentz et al., 1991b), dinoflagellates (Balch& Haxo, 1984; Carreto, DeMarco & Lutz, 1989;Carreto et al., 1990), lichens (Karentz et al., 1991b ;Budel, Karsten & Garcia-Pichel, 1997), corals andtheir associated biota (Shibata, 1969; Ito & Harata,1977; Takano, Uemura & Hirata, 1978a, b ; Dunlap& Chalker, 1986; Shick et al., 1992; Gleason, 1993;Dunlap & Shick, 1998), as well as many marineinvertebrates such as sea anemones (Scelfo, 1988;Shick, Lesser & Stochaj, 1991; Stochaj, Dunlap &Shick, 1994), limpets (Karentz, Bosch & Dunlap,1992), sponges (Nakamura et al., 1982), brine shrimp(Grant et al., 1985), sea urchins (Chioccara, Zeuli &Novellino, 1986; Adams, Carroll & Shick, 1996;


Carroll & Shick, 1996; Karentz, Dunlap & Bosch,1997), mussels (Chioccara et al., 1979), starfish(Nakamura, Kobayashi & Hirata, 1981, 1982), krill(Nakamura et al., 1982), and vertebrates including aspecies of Antarctic fish (Karentz et al., 1991b) andfish eggs (Chioccara et al., 1980).

There are approx. 19 MAAs found in marineorganisms. Some are shown in Fig. 3. Theirubiquitous presence across a large taxonomic rangeand geographical range is evidence not only of theirearly phylogenetic innovation, but potentially also oftheir importance as natural UV-screening com-pounds.

The basic cyclohexanone or cyclohexeniminechromophore responsible for UV absorbance isderived from the early stages of the shikimatepathway (Favre-Bonvin et al., 1987). The subsequentincorporation of the various amino acidic or imino-alcohol groups results in the diversity of MAAsfound in nature. HPLC has proven to be a powerfulmethod for MAA analysis (e.g. Nakamura et al.,1982). In a comprehensive study of Antarcticorganisms using HPLC, MAAs were found in a greatdiversity of invertebrates and also a species ofOsteichthyes ice fish (Karentz et al., 1991b). Inantarctic organisms, a complement of up to eightMAAs was found that might provide broad UVscreening. Many of the MAAs found (e.g. palythine,porphyra-334; shinorine, mycosporine-glycine andothers) in these organisms are identical to thosefound in tropical and temperate marine species.

An increase in concentration of MAAs associatedwith increases in UV flux has been observed directlyin organisms in the field. For example, the GreatBarrier Reef corals (Acropora spp.) at 20 m depthshowed significantly lower concentrations ofmycosporine-glycine and palythine than those inshallower waters (! 10 m) (Dunlap, Chalker &Oliver, 1986). In the Antarctic, surface planktonand invertebrates including krill (Euphausia superba)were found to have twice the MAA concentrationcompared to other non-surface species studied(Karentz et al., 1991b). These data as well as thedepth data acquired by others (e.g. Gleason, 1993;Shick et al., 1995; Dunlap & Shick, 1998 andreferences therein) that broadly correlate depth ofUV penetration into water with MAA concentrationhave been taken as evidence for a proposed ecologicalrole in UV protection.

Taking a similar approach to the study ofscytonemin, Garcia-Pichel & Castenholz (1993)studied the screening role of cyanobacterial MAAsthat they found in 13 out of 20 strains. They found

that in the cyanobacterium Gloeocapsa sp. thecompounds met the requirements described inSection IV for a UV-screening compound (Garcia-Pichel et al., 1993). MAAs were inducible by addingsupplemental UV radiation at between 2 and 6µmol m−# s−", causing levels of compound to increasebetween five- and tenfold (Garcia-Pichel et al., 1993),similar to the observed induction in the 13 othercyanobacterial strains (Garcia-Pichel & Castenholz,1993). The action spectrum for eliciting compoundsin Gloeocapsa sp. showed a peak at 320 nm, similar tothe absorbance profile for the compounds in thisspecies. Photobleaching of chlorophyll a measured asthe reduction of absorbance at 675 nm and photo-synthesis inhibition measured as the difference in["%C]bicarbonate uptake showed that in thedesiccated state MAAs do provide a physiologicallysignificant benefit to the cells. Furthermore, re-duction of blue-fluorescence of DAPI-labelled cellsnear 320 nm suggests that MAAs may also providesignificant screening to DNA. Attenuation of be-tween 10 and 26% of light at 320 nm has beenrecorded across 20 strains of cyanobacteria (Garcia-Pichel & Castenholz, 1993). For terrestrial cyano-bacteria, the attenuation in the UV-A region may becombined with that provided by scytonemin, toprovide a total screening up to 60% at 320 nm forsingle cells.

Induction of MAAs has also been observed indinoflagellates, including the production of up toseven MAAs in Alexandrium excavatum (e.g. Carreto et

al., 1990) in response to high light intensity, and theinduction of MAAs in Heterocapsa triquetra in responseto UV-B radiation (Wangberg, Persson & Karlson,1997).

An unusual context for MAAs is found in Nostoc

commune (Scherer et al., 1988; Bohm et al., 1995;Ehling-Schulz et al., 1997). The cyanobacteriumNostoc commune inhabits extreme arid and polarregions and grows as exposed mats on soils. Thewater-soluble mycosporines, that have absorptionmaxima at 312 and 335 nm (Bohm et al., 1985), areproduced extracellularly and are attached to thepolysaccharide matrix by different amino acids (Fig.3). They may account for between 7 and 10% of theorganism’s dry mass. This is considerably more thanintracellular MAAs, that typically range frombetween 0±16 and 0±84% of dry mass. Taking intoaccount the lower extinction coefficient (approx. 800mol−" l cm−") compared to most MAAs (approx.24000 mol−" l cm−"), Garcia-Pichel and Castenholz(1993) calculate that the screening efficacy of Nostoc

commune compound is very similar to MAAs. They


may provide a sunscreen factor of 0±7 for theseorganisms (Bohm et al., 1995). Like other com-pounds, the Nostoc commune MAAs are induced byUV-B radiation, as is the polysaccharide core itself(Ehling-Schulz et al., 1997). An artificial source ofUV-B radiation may increase compound concen-trations by five times or more. The compound doesnot seem to be produced by other cyanobacterialspecies and may be unique to Nostoc spp.

In many marine invertebrates and vertebrates,the de novo synthesis of MAAs is still in dispute and adietary origin from grazing on algae now seemsprobable (e.g. Shick, Dunlap & Larson, 1994;Stochaj et al., 1994; Carroll & Shick, 1996) withinternal modification of some of these compounds(Shick et al., 1992; Dunlap & Shick, 1998). Theshikimate acid pathway is not found in animals andso it is unlikely that it is biochemically possible forMAAs to be produced in animal tissues. Althoughsome accumulation of mycosporine-glycine:valinewas noted in intertidal Antarctic invertebratespecies, which was not found at lower trophic levels(Karentz et al., 1991b), it is possible that very lowconcentrations of this MAA may exist in some algalspecies consumed by invertebrates or that they aremodified after consumption. The picture may befurther complicated by the differential synthesis ofMAAs according to environmental changes. In thered-tide dinoflagellate Alexandrium excavatum, therelative proportions of different MAAs may alterrather quickly upon changes in light concentration(Carreto et al., 1990), which might potentially alterthe dietary intake by higher trophic levels.Continued studies of the synthesis, interconversionand structural modification of these types of com-pounds in selected ecosystems will undoubtedlyaddress many aspects of these questions.

The evolutionary significance of trophic-levelaccumulation is uncertain. It is tempting to speculatethat the shikimate acid pathway was lost in theearliest multicellular animals during the Cambrianand that trophic-level accumulation of UV-screening MAAs from algae was the predominantmeans of enhancing UV screening. Organisms thathad improved metabolic processing and distributionof MAAs to important biological sites would havehad some selective advantage, particularly in clearwaters, although why the pathway should have beenlost in animals is uncertain.

Although there is good laboratory and fieldevidence that MAAs can provide screening, they arealso a good example of the complexities that canexist with examining the role of UV-screening

compounds in protecting natural communities. Theyespecially underline the need to study these com-pounds in relation to other UV-mitigation methodsas well as other physiological functions of thecompound itself. There exist two complications thatmay arise in examining the prevalence of compoundsin nature and their correlations with UV exposure.

Firstly, UV repair processes, the quenching ofreactive oxygen species by carotenoids and photo-taxis may provide the required UV radiationresponse that may supplement or even negate theneed for UV-screening compounds in some species.In Gloeocapsa sp., for example, cells were able toacclimate to artificial increases in UV radiationprior to the maximal build-up of MAAs (Garcia-Pichel et al., 1993). This suggests that other processesmay also be important in mediating the immediateUV acclimation response in this organism, perhapsrepair processes. In a survey of cyanobacteriaundertaken by the same authors, some Oscillatoria

species that display a phototactic response to highlight and UV radiation intensities had a conspicuousabsence of MAAs. They may live in UV-exposedhabitats where non-phototactic cyanobacteria arefound such as Gloeocapsa spp. or Calothrix spp. that dopossess MAAs (Garcia-Pichel & Castenholz, 1993).

Simple morphology of some organisms, particu-larly chained diatoms such as Thalassiosira spp. andChaetoceros spp., may cause an order of magnitudedifference in intracellular UV exposure and thusDNA damage (Karentz et al., 1991a). In a study ofa diversity of Antarctic diatoms including Proboscia

spp. and Nitzschia spp., screening by the cell materialincluding the frustrule was found to be greater thanthe protection provided by the UV-screening com-pounds (Davidson et al., 1994; Davidson &Marchant, 1994). In some of these species, althoughan MAA peak was observed, it was not found to beUV-B inducible. Furthermore, in the species studied,UV-B tolerance was actually found to be higherthan in the Antarctic spring-bloomingprymnesiophyte Phaeocystis pouchettii that does pro-duce high concentrations of UV-B-inducible com-pounds (Marchant, Davidson & Kelly, 1991).

This type of screening compound versus otherresponse data demonstrates the importance of eluci-dating the complete range of UV responses in agiven organism. Thus, although wide surveys of UV-screening compounds in nature can be of initialvalue, interpretation of the data in the ecologicalcontext must be undertaken with great care.

The second complication is that the compoundsmay have other physiological functions as well as


UV screening. An example are cyanobacteria insome high-salt environments, where MAAs, with aninternal cell concentration of 98 mmol l−", have beenfound to be produced in response to increasing saltconcentrations. In this case, they are presumed tohave a role in balancing the osmotic pressure insidethe cells to counteract water loss (Oren, 1997). Aswell as enhancing UV screening, plant flavonoidshave proposed roles in flower recognition by insects,pollen development and sexual reproduction, plant–microbe interactions including nitrogen fixation,interactions with pathogens and parasitic plants andan involvement in the formation of plant structuralcompounds in seeds and bark (Koes, Quattrocchio& Mol, 1994). A truly rigorous correlation of theexistence of UV-screening compounds to UV ex-posures and habitats requires that the physiologicalfunctions of a compound are understood and thatthe relative importance of these functions at differentdevelopmental stages and in different habitats andenvironments is known.

An excellent example of these problems is illus-trated by a study of the presence of MAAs in thelenses of fish eyes (Dunlap et al., 1989). The focus ofthe eye is accomplished by the cornea and lens,which transmit light through to the retina. Thesetissues therefore have a role in screening UVradiation before it damages the retina. UV-A-absorbing compounds have been identified in thelens tissues of marine fish (Zigman, Paxia &Waldron, 1985; Zigman, 1987). Believing these tobe MAAs, Dunlap et al. (1989) extracted thecompounds from a wide diversity of fish lenses. Adiversity of well-described MAAs were found, in-cluding palythine, palythene, asterina-330 andpalythinol, although some were noticeably absentsuch as mycosporine-glycine. The patterns andconcentrations of MAAs showed no specificbehavioural or taxonomic trends. Indeed, in twodiurnal surface feeders (Spanish mackerel,Scomberomerus commerson and mackerel tuna, Euthynnus

affinis), MAA concentrations were found to be morethan three orders of magnitude lower than in someother species such as coral trout, Plectropomus leopardus

and green job-fish, Aprion virescens that feed through-out the water column. In the Scaridae (Parrotfish),the concentrations of palythene are so high that theeyes have a yellow colour.

Although the authors suggest that a more robustlight gradient might be used in future studies toexamine these relationships further, they also alludeto several factors that may explain their results.Filtering out short-wavelength light may improve

the visual acuity of some species of fish by reducingchromatic aberration (Muntz, 1973) and this mayaccount for the high concentrations of some MAAs,such as palythene in parrotfish eyes (λ

max¯ 360 nm).

Some species of fish exhibit tetrachromatic colourvision and have a UV-A-sensitive cone with maxi-mum sensitivity at 360 nm (e.g. Avery et al., 1983;Harosi & Hashimoto, 1983; Downing et al., 1986;Bowmaker & Kunz, 1987). In such species of fish,UV-A is important for visual acuity. In these cases,there may be a requirement for lower concentrationsof MAAs.

As the authors concede at the end of theirdiscussion ‘an understanding of the functionalsignificance of these compounds in fish eyes willrequire a more complete understanding of thestructure and function of many, presumably inter-acting, factors affecting vision in fishes ’.

VII. UV SCREENING IN PLANTS

Like many phototrophic micro-organisms, higherplants require an exposure to solar radiation for theirway of life. Phenylpropanoids including flavones,flavonols, cinnamoyl esters and anthocyanins pro-vide a UV-A and B screen. The flavonoids are todaythe most widely represented phenolic derivatives inthe biosphere (Harborne, 1964). Flavonoids providean effective UV screen that can reduce transmittanceof UV radiation through the epidermis, but allowthrough visible radiation for photosynthesis (Tevini& Iwanzik, 1983; Tevini et al., 1991). This screen,as well as reducing DNA damage, prevents UV-B-induced damage of the photosynthetic apparatus,particularly photosystem II (Noorudeen & Kulan-daivelu, 1982; Renger et al., 1989). It is nowwell established that flavonoids are UV-B-inducible(Mohle & Wellmann, 1982; Flint, Jordan &Caldwell, 1985; Barnes et al., 1988; Tevini et al.,1991) and in some cases such as in parsley,Petroselinum hortense, a linear relationship betweenflavonoid concentration and UV-B flux has beenobserved (Wellmann, 1975). Flavonoids can also beUV-A inducible, as has been demonstrated in Cistus

laurifolius (Vogt et al., 1991). The flavonoids respon-sible for UV screening may vary from species tospecies and according to developmental stage andtissues. Although single flavonoids may screen inboth the UV-A and UV-B region, most plantssynthesize a range of compounds that may provide amore effective screen. For example, in rye, Secale


cereale, seedlings isovitexin arabinoside and isovitexingalactoside were the two principal compoundsinduced by UV-B radiation (Tevini et al., 1991).

Other enzymes and products of the flavonoidbiosynthesis pathway have also been observed to beinduced by changes in the incident UV flux (e.g.Hadwiger & Schwochau, 1971; Lyndon, Teramura& Coffman, 1987; Zangerl & Berenbaum, 1987;Braun & Tevini, 1993), thus demonstrating acomplex UV response that may be regulated atmultiple levels. In legumes, this response has alsobeen shown to be linked through isoflavonoids to therepair of UV-induced DNA damage, particularlythymine dimer formation (Beggs et al., 1985). Theincreasing complexity of the UV-flavonoid responsein phylogenetically more recent plants (Stafford,1991; Koes et al., 1994) suggests a common ancestralresponse to UV radiation that over time and as aresult of UV selection pressure, has resulted in avariety of biochemical innovations and links to otherphysiological responses.

Flavonoids are principally deposited in the epi-dermal layers of the leaf (Robberecht & Caldwell,1978; Flint et al., 1985), although in some cases theyare also found in the epicuticular waxes (Vogt et al.,1991) and in the deeper mesophyll layers of theleaves (Weissenbock, Plesser & Trinks, 1976;Robberecht & Caldwell, 1978). They may also befound in leaf hairs (Karabourniotis et al., 1992). Thepigments are usually localized to the vacuoles ofepidermal cells (McClure, 1975), although theyhave also been reported in the cell wall of epidermalcells in some conifers (see Day, 1993 and referencestherein). Although vacuole localization can providethe necessary screening, some inefficiency undoubt-edly occurs with light that passes between the cellwalls. The multicellular localization of flavonoidsillustrates the first important difference betweenplants and micro-organisms. As will be discussed inmore detail in Section IX, multicellular distributionof UV-screens greatly enhances the possibility ofattenuation. Whereas a single-celled cyanobacteriamay screen 60% of UV radiation at 320 nm with acombination of MAAs and scytonemin (Garcia-Pichel & Castenholz, 1993), plants generally reduceUV at this wavelength by more than 90% before itreaches the mesophyll (Robberecht, Caldwell &Billings, 1980).

Plants in equatorial and high-altitude regions ofthe earth, where UV-B flux is generally higher,demonstrate the capacity of increased UV-B tol-erance by inducible flavonoid production. Most low-latitude species exhibit a lower epidermal trans-

mittance (less than 2%) compared to more tem-perate and higher latitude species that generallyexceed 5% (Robberecht et al., 1980). The speciesfound at low latitudes also include temperate-latitude species that have been introduced into theseregions such as the pea, Pisum sativum, furtherdemonstrating the capacity for photobiological ad-aptation. Since the unweighted UV-B flux is an orderof magnitude greater in low-latitude regions com-pared to high-latitude regions, these ranges providesome evidence of the acclimation capacity of plantsand their ability to change flavonoid composition innew UV regimes.

In an analogous way to the non-specific at-tenuation of UV-B radiation in diatoms by theirmorphological features, the morphological charac-teristics of plants or the physical properties of theirleaves may provide a significant contribution to UVreduction. Approximately 5% of the UV-B region ofthe spectrum is non-specifically reflected away bythe epidermal layer. However, in some plants withdense pubescence (Robberecht et al., 1980) or aglaucous surface (Mulroy, 1979), UV reflectancemay be as high as 20–70% (Caldwell et al., 1983).The epidermal cell structure can in some casesprovide reductions in UV by non-specific scattering.Robberecht and Caldwell (1978) reported that in 24plant species, a number of them had significantattenuation due to epidermal structure alone. In 16species studied, they reported that attenuation byUV-screening pigments was between 20 and 57% ofthe total UV attenuation, the rest being accountedfor by epidermal structure. These results are slightlyopen to question since the epidermis’ were extractedin acidified methanol for only 15 min. Using similarprocedures on desert cacti of the genus Opuntia,epidermal transmittance was found to be increasedby only 10–50% (C. S. Cockell, unpublished ob-servations). However, overnight extraction of theepidermis’, with four separate extractions, wasfound to reduce the concentration of intracellularflavonoids such that epidermal transmittance wasincreased by between 80 and 90%, suggestingthat the contribution of epidermal structure toUV-B attenuation may be greatly overestimated ifsufficient extraction periods are not allowed.

Anthocyanins are less efficient absorbers of UVradiation since their absorbance maximum is gener-ally near 520 nm, but the tail of their absorbanceprofile may extend into the UV-A region (Strack &Wray, 1989). As well as screening in leaves, they cancontribute to the absorbance of flower corollas(petals), being responsible for flower colour and


flower recognition by pollinators. They may con-tribute to the protection of pollen by helping tomitigate UV-A penetration into the anthers.Together with the absorbance provided byflavonoids, incident UV-A and B radiation withinanthers is reduced by 98% (Flint & Caldwell, 1983).Anthocyanins are induced by UV radiation below350 nm (Wellmann, Hrazdina & Grisebach, 1976;Beggs & Wellmann, 1985) and their induction mayalso depend on a complex interaction with thephytochrome system (Beggs & Wellmann, 1985).

As well as flavonoids, other aromatic-containinghigher plant pigments such as alkaloids absorb in theUV range. There is a negative correlation betweenalkaloid-bearing plants and latitude (Levin, 1976)and although alkaloids possess other functions suchas deterrents against grazing, it is also plausible theymay provide additional UV protection in somespecies.

VIII. UV SCREENING AND MELANIN

The animal melanins or ‘eumelanins ’ as they aremore correctly defined are found in eyes, feathers,insect cuticles, reptile skin, and the skin of a widevariety of mammals. They are found in particularlyhigh concentration in the inksac of the cuttlefishSepia officianalis (Prota, 1988) and this melanin hasbeen used as a standard for melanin studies. Thefocus on melanins and their role in photoprotectionin humans has brought them particular attentionsince the inverse correlation between skin cancer andmelanin production has been suggested (Scotto &Fraummeni, 1982). Thus, the literature on humanmelanin, the pigmentary system and its induction isvery substantial (e.g. Nordlund & Boissy, 1998).Here, we focus only on the screening properties ofthese pigments and their evolutionary significance.

Melanins are produced by the enzymatic oxi-dation of tyrosine by tyrosinase and then thesubsequent conversion of dopa to 5,6-dihydroxy-indole. This is a phenolic and indolic compound andthe basic building block of the eumelanin polymericstructure. This structure acts as the UV-absorbingchromophore.

Because of the complexity of eumelanins, and thusthe diversity of absorbance maxima of the π to π*transitions represented in the molecules, there is nospecific absorbance profile or maximum. However,they absorb increasingly strongly into the UV-Bregion of the spectrum (Fig. 6) (Crippa et al., 1978;Menon et al., 1983; Ravishankar, Muruganandam

& Suryanarayanan, 1995). Their dark colour resultsfrom some absorbance in visible wavelengths. Formany melanins, the plot of log[absorbance] againstwavelength gives a linear relationship (Spiegel-Adolf, 1937). The erythemal dose response orsuntanning effect induced by UV radiation shows anincrease in the UV-B portion of the spectrum, that isconsistent with the screening function of melanin.

Melanins are formed in melanocytes in specificorganelles termed melanosomes which in humanshave a melanin content of between 17±9 and 72±3%(Duchon, Borovansky & Hach, 1973). The melaninoften forms in ‘caps ’ above the nucleus thatpresumably provide the most efficient UV protection(Gilchrest et al., 1996). Melanocytes are foundbetween the epidermis and dermis in the skin’s basalcell layer, the principal site of UV-induced pig-mentation (e.g. Friedmann & Gilchrest, 1987).Here, melanin is visualized as agglomerates (whichare essentially melanosomes). Agglomerates arecollections of melanin aggregates, themselves formedfrom melanin ‘particles ’ that are 30 nm sizedparticles of melanin polymers. Melanin particles arefound in the stratum corneum near the surface of theskin and in the epidermis and are also known as‘melanin dust ’ (Holbrook & Wulff, 1987). Theaccumulated effect of the melanin to be found indifferent skin layers (total depth approx. 250 µm)means that incident radiation is quite quicklyattenuated through the stratum corneum and epi-dermis. White skin transmits approx. 20–50% ofincident radiation at 300 nm through the epidermisand black skin approx. 2–10% (Everett et al., 1966;Kaidbey et al., 1979).

Melanin provides a fine example of the physio-logical value of both inducible and constitutivecompounds. In white populations melanin inductionis an important response to UV irradiation. How-ever, in many Asian and African populations, blackskin represents a screen that is predominatelyprovided by constitutive production of melanin. Inthe latter case, although the melanin response is notnecessarily correlated with UV fluence, the screen iscertainly of physiological importance during UVexposure (see below).

The response to UV irradiation is complex. Aswell as de novo melanin production, which appears tobe mediated through the existing tyrosinase pool,responses that may occur over the longer term mayinclude increases in the numbers of melanocytes andmelanosome formation. Thickening of the stratumcorneum and epidermis may also occur. Thisincreases skin folding and increases UV attenuation


by enhancing scattering, reflection and the surfacearea of absorption. It is part of the longer termphotoageing response (Kollias et al., 1991). Althoughit is inducible, in some ways it is analogous to thereductions in UV-penetration achieved by morpho-logical and scattering effects in plants and micro-organisms, independent of the concentration ofscreening compounds. The photoageing responsemeans that each successive UV exposure must behigher in order to induce the same tanning effect(Kollias et al., 1991).

The evolutionary importance of melanins is lessclear. UV-screening compounds in micro-organismshave a clear role since they may reduce photo-synthesis inhibition and other detrimental effects ofUV radiation such as DNA damage. For a single-celled organism this can be important, since damageto the cell may be lethal. It has previously beensuggested that sunburn is probably not an evolu-tionary selection pressure (Blum, 1961). However,for any animals that live in exposed regions forextended periods, particularly for hunting andfeeding, photoprotection in the skin layers may beimportant. The probability of skin cancer or otherlonger term negative photoageing effects occurringbefore breeding age may be increased without someform of photoprotection. Photoprotection may par-ticularly have been important in moving from forestregions into savannas and other open spaces duringperiods of drought or reduced food availability.Burnt skin may also act as a site of microbialinfection, a place for flies and other winged insects tolay eggs and as a site of irritation which mayexacerbate the infection risk. Thus, it is possible toenvisage that melanin does provide fitness value. Inhumans, the incidence of non-melanoma skin canceris 10–100 times higher in non-pigmented humanpopulations compared to pigmented human popu-lations (van er Leun & de Gruijl, 1993). Thesunburn effect in white Caucasians over short periodsof exposure before the melanin adaptive responseoccurs is ample evidence of the potential damagethat UV radiation might cause in any exposed skinon an animal without an effective photoprotectivestrategy.

IX. OTHER CANDIDATE UV-SCREENING

COMPOUNDS

Our understanding of the range and characteristicsof UV-screening compounds represented in thebiosphere is still in its infancy. The wide distributionof some compounds such as MAAs in marine

invertebrates and many micro-organisms, flavonoidsin higher plants, and melanins in many animalssuggests that a large number of compounds willprobably be found to share common characteristics,falling broadly into well-defined groups. However,some may be represented in only one or a few speciesif they are more recent innovations. Here, someother compounds are discussed.

(1) Carotenoids

The role of carotenoids in UV screening is stillsomewhat controversial. Their role in oxygen free-radical quenching and thus indirect photoprotectionfrom both UV and high visible light induced damagehas been established for some time (Krinsky, 1979;Demmig-Adams and Adams, 1992) and it is knownthat in many cases they are UV-inducible. Forexample, in Nostoc commune, concentrations of theouter-membrane carotenoids echinenone and myxo-xanthophyll were increased by approx. 40–50%after 5 h exposure to 1 Wm−# UV-B compared tocontrols (Ehling-Sculz et al., 1997).

In an analogous way to the higher trophic levelaccumulation of the UV-screening MAAs in marineorganisms, carotenoids are synthesized in algae,bacteria and plants de novo and acquired in the dietby higher trophic levels such as insects (Britton &Goodwin, 1981).

A diversity of experiments have not demonstratedsignificant screening advantages conferred by caro-tenoids. Under UV-B irradiation, no differences incompetitiveness were found in strains of the fungusPhycomyces blakesleeanus, regardless of carotenoidconcentrations or differences in carotenoid type(Cerda-Olmedo, Martin-Rojas & Cubero, 1996).Similar results were reported for Neurospora crassa

(Blanc, Tuveson & Sargent, 1976). Halophilic bac-teria are well known for carotenoid production, sinceit is these compounds that result in the highlycoloured (red or pink) natural communities. A rolein photoreactivation was ascribed for the caro-tenoids, but no passive UV screening could beobserved in pigmented versus unpigmented strains ofthe halophiles Halobacterium cutirubrum and Halo-bacterium salinarium (Sharma, Hepburn & Fitt, 1984).In a melanin-deficient mutant of the fungusWangiella dermatitidus, the carotenoids torulene andtorularhodin did provide some UV protection (Geis& Szaniszlo, 1984), but in the wild-type cells,melanin was found to provide the most importantUV protection.


The controversial nature of carotenoid involve-ment in passive UV screening is an interestingresearch problem since, as alluded to in Section II,from a chemical point of view they should makeeffective screens. It is unsurprising that many of thelonger carotenoids important in photosynthesis andphotochemical quenching are not UV screens sincethe minimum number of carbon double bondsrequired to provide quenching of singlet oxygenappears to be nine (Krinsky, 1971). Molecules withthis number of bonds and more tend to screen in thevisible region of the spectrum. However, linearconjugated molecules generally have higher ab-sorption coefficients than aromatics. It is surprisingthat compounds such as phytoene, that screens at340 nm and has three conjugated double bonds, aswell as shorter molecules that could screen at lowerwavelengths have not become more important. Theability to synthesize such compounds at sufficientscreening concentrations is possible, since manylong-chain carotenoids are synthesized at concen-trations of up to 2000 µg g−" dry mass (e.g.Cerda-Olmedo et al., 1996) which is similar, and insome cases greater, than the intracellular concen-trations associated with say, MAAs, which aresynthesized at intracellular concentrations rangingfrom 4 to 2903 µg g−" dry mass (Karentz et al.,1991b).

The original role of carotenoids is not known.Their ability to transfer energy means they areimportant in photosynthetic light reactions and theymay initially have had a primary role in photo-synthesis. Their role in quenching reactive oxygenspecies could have evolved during the increase inatmospheric oxygen partial pressures in the earlyProterozoic (Krinsky, 1971) or perhaps earlier inphototrophic organisms producing oxygen in theirmicro-environments. Thus, they may have taken ona photoprotective role as an additional function inthe earliest oxygenic photosynthesizers. The meta-bolic products of carotenoids have been postulatedto be involved in the synthesis of sporopollenin andother macromolecules (Krinsky, 1971). It is possiblethat in the earliest stages they may have beenintermediates in other biosynthetic pathways, laterbecoming used in light-related reactions. Thus,providing some phylogenetic reason for the lack ofthe use of carotenoids as passive screens is difficult.Furthermore, at later stages in evolution, when theycould have been selected again, aromatics werepreferentially selected as UV screens, for exampleflavonoids in plants and melanins in animals.

The relative unreactiveness of ringed aromatics

compared to linear, more energetically accessiblemolecules may be a reason why aromatics areusually found as UV-screens. A passive UV screenshould preferably have minimum interference withother cell biochemistry, particularly if it is constantlyproduced intracellularly. The role of other linearconjugated structures in passive or constitutive UVscreening in cells merits further analysis.

(2) Other compounds

The Antarctic spring-blooming Phaeocystis pouchetii,which blooms following the break-up of the sea-ice,produces colourless water-soluble compounds thatabsorb strongly in the UV-B region (Marchant et al.,1991). The colonial form is one of two life stages ofthis organism whereby the cells are not freeswimming, but float in the water column, embeddedin mucilage. UV protection might be expected to beimportant. The compounds produced absorbstrongly at 323 nm, with two other peaks at 271 and211 nm, the latter two being physiologically ir-relevant since they are in the UV-C. Their levels ofproduction correlate with total UV-B irradiance.With these compounds Antarctic colonial Phaeocystis

pouchetii may tolerate levels of UV-B up to0±32 Wm−#. In the motile life stage (or in the colonialform of East Australian Current strains of Phaeocystis

pouchetii, that do not produce as many compoundsand cannot increase concentrations in response toUV-B), loss of viability was significantly faster. Thisprovides circumstantial evidence of the fitness valueof these compounds, although better repair processescannot be ruled out (some Antarctic diatoms, whichmay have more efficacious repair processes, may notrequire UV-screening compounds to reach higherlevels of UV tolerance). The compounds areextruded into the water column and are rapidlydegraded by bacteria, suggesting they may be quiteshort-lived. Their presence in the water column mayprovide some fortuitous protection to otherorganisms in the vicinity of the blooms. It is possiblethat these compounds are MAAs or at least relatedto MAAs. Other UV-absorbing compounds havealso been found in the water column near phyto-plankton blooms. P380 was found in the surfacewaters near an Icelandic phytoplankton bloom(Llewellyn & Mantoura, 1997). It has a broadabsorbance from 300 to 470 nm and screens maxi-mally between 380 and 383 nm.

Many lichens also have a diversity of polyphenoliccompounds including usnic acid and parietine that


absorb strongly in the UV region and may beinvolved in photoprotection (Hawksworth & Hill,1984; Adams, Demmig-Adams & Lange, 1993;Solhaug & Gauslaa, 1996; Bachereau & Asta, 1997).In the case of lichens containing cyanobacteria,scytonemin itself may be the predominant screeningcompound (Budel et al., 1997).

As well as novel compounds in single-celled micro-organisms, many multicellular organisms may havenovel screening compounds. Gadusol and its relatedcompounds are structurally related to the myco-sporines (Fig. 3) and have been found in the roes ofcod Gadus morhua (Grant and Plack, 1980; Plack et

al., 1981) as well as in the eggs of Mediterranean fish(Chioccara et al., 1980) and brine shrimp (Grant et

al., 1985). A role in UV protection has yet to bedetermined. The high concentration of gadusolwhich may be about 0±3% of average dry mass inexposed Scottish cod roes ; (Plack et al., 1981) makesthe role of these compounds in UV protection likely.

Sporopollenin is a compound found in many algaeand also in the cell walls of pollen and spores(Atkinson, Gunning & John, 1972; Guilford et al.,1988; Delwiche, Graham & Thomson, 1989; Xionget al., 1996, 1997). It is an acetolysis-resistantbiopolymer made up of a diversity of aromatic andaliphatic residues (Meuter-Gerhards, Schwerdtfeger& Steuernagel, 1995). It does not appear to beinduced specifically by UV radiation and may havea function in antimicrobial activity. However, tol-erance of some algae such as Scenedesmus spp. and anEnallax sp. to UV-B radiation was correlated withcell wall concentration of sporopollenin (Xiong et

al., 1997). The compound does not have a specificabsorbance peak. Like melanin, its complex poly-meric structure generates a rather non-specificallyincreasing UV absorbance towards shorter wave-lengths. It may therefore act as a constitutive UVscreen, a screen that may be further enhanced byUV-induced synthesis of MAAs (Xiong et al., 1997).Sporopollenin is a good example of the fact that anyaromatic or conjugated bond-containing structurespresent in the cell wall or tissues of an organism maypotentially provide some UV screening, exacer-bating the problem in identifying those moleculesthat have evolved specifically to screen UV radi-ation. It is possible that some constitutive UVscreening might occur for other cytoskeleton-typestructures that contain aromatic amino-acids andsurround the cell wall, for example the chaperoninsthat have been proposed as an archeal cytoskeleton(e.g. Trent et al., 1997).

X. UV-SCREENING COMPOUNDS AND

EVOLUTION

Our knowledge of the specific biochemical andphysiological details of a diversity of UV-screeningcompounds, which has been addressed in previoussections, can provide us with a basis to consider someevolutionary questions. The role of UV radiation inevolution can be split broadly into four divisions.Firstly, UV radiation on pre-biotic earth. Secondly,UV radiation on Archean earth prior to theformation of the ozone shield, but when microbialcommunities existed. Thirdly, UV radiation duringthe early Proterozoic period when atmosphericoxygen partial pressures rose as a result of microbialphotosynthetic activity and geologic changes, thusresulting in the formation of the ozone column andfourthly, changes in UV radiation flux caused bynatural ozone depletion events since that time. Inthis section, the link between UV-screening com-pounds and evolution is explored in more detail.

(1) UV screening on prebiotic earth

Prior to the formation of the ozone layer thatoccurred when atmospheric oxygen concentrationsincreased during the early Proterozoic approx.2±1 Ga, the UV-C and UV-B radiation flux mayhave been much higher on the surface of the earththan it is today. This would have corresponded tothe time between the formation of the earth 4±8 Gaand approx. 2±0 Ga (Kasting & Donahue, 1980;Kasting, 1987, 1993; Francois & Gerard, 1988).Without such screening, the biologically effectiveirradiance to free-DNA would have been approxi-mately three orders of magnitude higher thanexperienced on earth today (Cockell, 1998a). Thesefluxes would have been significant on prebiotic earthsince they may have photolytically destroyed com-plex organic structures. However, there are manyways in which UV radiation may have beenattenuated.

Other atmospheric attenuators may have existedin the early atmosphere. Atmospheric attenuation ofUV radiation by high concentrations of sulphur orH

#S has previously been suggested (Sagan, 1973;

Kasting et al., 1989). Prebiotic evolution in the deepocean or subsurface earth may also have allowed lifeto evolve well before it radiated onto the surface of aUV-exposed earth, by which time it may havedeveloped damage responses.

However, if UV flux was much higher than todayand some pre-biological reactions occurred in po-


tentially exposed regions such as in water bodies ofearly cratons and land-masses or in the oceans, thenscreening may have been important to reduce UV-induced destruction of complex organic molecules(Cleaves & Miller, 1998). Shallow water is not avery good attenuator of UV radiation and even insea water, the salts that produce UV-attenuatingbromide or nitrate can only produce highly effectiveabsorption of UV radiation below 220 nm in surfacelayers (Ogura & Hanya, 1966). UV-B radiationmay penetrate tens of metres into clear water (Smithet al., 1992). The possibility that organic moleculesmay have acted as a UV screen in the aqueousenvironments of early earth was first considered bySagan (1973). Many cyclic or conjugated organicmolecules, could potentially have been important,although without land plants, dissolved organiccarbon (today provided predominantly by humicsubstances) would have been at much lower con-centrations. Significant concentrations of hydrogencyanide (HCN) polymer may have existed in theoceans of prebiotic earth, formed from cyanidepolymerizations. HCN polymer may have providedattenuation of wavelengths below 260 nm (Cleaves& Miller, 1998). In combination with other UVabsorbers such as HS− or reduced iron, UVpenetration into earth’s early oceans could havebeen substantially reduced. Significant delivery ofaromatic compounds and organics by a higherimpact flux on early earth and by interstellar dustparticles (Chyba & Sagan, 1992) may also haveprovided some UV-screening organics. Specifically,polycyclic aromatic hydrocarbons (PAHs) may haveprovided some type of UV screen as they will absorbin the UV region (M. Bernstein and T. Halasinski,personal communication). In carbonaceous chond-ritic meteorites they represent 90% of the organicmaterial (Deamer, Mahon & Bosco, 1994 andreferences therein). Thus, they may have been animportant part of the prebiotic organic inventory.

Although finding specific UV absorbers amongstthis diversity of prebiotic possibilities is an interestingavenue of research, it must be remembered that theπ to π* electron transitions in many small conjugatedorganic structures will screen in the UV-C regionand in many, where the molecules are long enoughor substituent groups of the molecule of the ap-propriate structure, UV-B absorbance may occur aswell (see Section III). Any surfaces or small waterbodies in which a build-up of even a thin layer oforganically enriched material containing conjugatedaliphatic or aromatic compounds occurred couldpotentially have provided UV screening for pre-

biotic aqueous reactions in exposed regions of theearth (Cockell, 1998b). If we assume a molecularweight of approx. 160 Da for a typical small UV-Cor UV-B absorber (the typical molecular weight of aUV-absorbing linear molecule or a three-ringedaromatic structure which is typical for many PAHsfound in carbonaceous meteorites) and an averageextinction coefficient of 10000 mol−" l cm−" acrossthe UV range (which is at the lower end for a smalllinear conjugated molecule, but the upper end forsome aromatics), then it is apparent that even ata concentration of 16±5 µg cm−" ; such compoundscould provide a 90% reduction of incident UVradiation.

A particular problem for prebiotic earth is thehigh UV absorbance of the nucleic acids. In theearly period of the Archean, it is supposed that thesurface of earth was host to an ‘RNA world’ inwhich RNA catalysis, probably enveloped by earlyamphiphilic membranes, presented the first type ofcell-like structures (Lazcano, 1994). During thisstage the high UV-C and UV-B absorbance ofnucleic acids might have made reproducibility ofRNA replication a biochemical challenge unlessthese molecules were shielded. Kolb, Dworkin &Miller (1994), in attempting to reconcile the need fora pre-RNA molecular order, suggest that candidateearly precursors to RNA (the five-membered urazoleand guanazole) would also have been resistant toUV damage since they are transparent above220 nm. Whilst this is certainly true in the earlieststages of the RNA world, the ozone shield did notform until approx. 2 billion years ago. It does notnegate the requirement for nucleic acid shielding onprebiotic earth once the RNA world did come intoexistence. Unsaturated amphiphilic membranemolecules may have provided non-specific screeningand scattering of UV-C and UV-B radiation throughconjugation, thus reducing the biologically effectiveirradiances to early encapsulated nucleic acids andhelping to increase the reproducibility of the geneticmachinery. One might imagine a selection pressurefor more UV-absorbing unsaturated phospholipidsin early membrane structures. However, until single-celled organisms evolved that had effective repairprocesses or could enhance UV screening by thespecific production of UV-screening compounds,then it is likely that much of importance in theprebiotic world occurred in regions that wereshielded or at least under the fortuitous build-up oforganic molecules or inorganic compounds thatprovided protection to reactions occurring under-neath.


Table 1. A selection of ultraviolet (UV)-screening methods found on present-day earth which may have been relevant on

early earth (adapted from Cockell, 1998a).

UV-screening strategies Example Comments

Physical methodsIron compounds Many mat-forming organisms

(Pierson et al., 1993)Provides specific UV absorption

Sulphur Thermophilic mats and organismsnear hydrothermal regions (Cockell,1998b)

Specific UV absorption

Solid NaCl Evaporites}halophiles (Rothschild,1990)

Specific UV absorption

Water column Oceanic and freshwater communities(Sagan, 1973; Margulis et al., 1976)

Not very effective attenuationwithout impurities such as iron ordissolved organics. Trade-offproblem between UV penetrationand euphotic zone

Rock}sand}soil}snow (particularlywith impurities)}calciumcarbonate} gypsum (calciumsulphate)

Endolithic communities, desertcrusts, snow algae etc. (Nienow &Friedmann, 1993; Garcia-Pichel &Belnap, 1996)

Many of these physical substrates arenon-specific and will attenuatevisible wavelengths, although theyhave the advantage of generating alocal microenvironment for theorganisms, particularly in extremeenvironments

Sediments Benthic habitats (Garcia-Pichel &Bebout, 1996)

Attenuation of UV radiation under amillimetre or more of sediment.Important in benthic andcontinental shelf areas

Organics Any π-electron-containingchromophore (Sagan, 1973;Cockell, 1998b)

May have been important onprebiotic earth. Small compoundsprovide non-specific UV-Cabsorption

Biological methodsMatting Many cyanobacterial mats (Margulis

et al., 1976)Dead or living upper layers protectlower layers. Can be specific to UVif upper layers contain UV-screening compounds

UV-screening compounds Scytonemin in cyanobacteria,mycosporine-like amino acids, etc.(e.g. Garcia-Pichel et al., 1993;Vincent & Quesada, 1994)

Specific biological screening of UVradiation

Fortuitous production of organicsthat screen UV

Mycosporine-like amino acids inresponse to osmotic effects (Oren,1997)

May occur when other physiologicalprocesses produce compounds thathappen to screen in the UV

It is also important to note that for some prebioticreactions the UV flux may have been advantageous.In the laboratory, UV radiation has been shown toprovide the energy for the formation of complexorganics, some of which may have had prebiologicalsignificance (Groth & Weyssenhoff, 1960; Sagan &

Khare, 1971). In some of the earliest stages oforganic complexification, UV flux may have beenadvantageous.

As our knowledge of the chemical reactions thatultimately led to the first self-replicating nucleicacids and thence to the first unicellular-like


organisms emerges, so surely our understanding ofthe positive and negative roles of UV radiation onprebiotic earth will improve and with it ourunderstanding of the importance of UV screening atcritical stages along this path.

(2) UV screening and early life

Many micro-organisms today suffer photosynthesisinhibition and DNA damage caused by UV radi-ation. It is likely therefore, that if early exposed lifedid exist under a greatly elevated UV-B and a UV-C regime, it faced a much greater problem with UVdamage (Sagan, 1973; Margulis, Walker & Ram-bler, 1976; Rambler & Margulis, 1980; Cockell,1998a ; Garcia-Pichel, 1998). Repair processes suchas photolyase repair of thymine dimers may wellhave been more effective, but the screening of UVradiation was probably also very important. Inmany situations physical substrates may have pro-vided sufficient screening. As described in Section II,layers of UV-B- and UV-C-absorbing reduced ironcompounds (Olsen & Pierson, 1986; Pierson et al.,1993) may act as an effective UV screen. Organismsunder layers of sulphur (Cockell, 1998a) andorganisms in deep waters may have gained thebenefit of UV radiation attenuation. Endolithiccommunities that live in the subsurface layers ofrocks (Nienow & Friedmann, 1993) may receivesome UV-screening benefit. Table 1 shows a di-versity of potential screening strategies that mayhave been available to early life on earth (adaptedfrom Cockell, 1998a). As discussed above, there aredisadvantages with physical substrates with respectto their non-specific attenuation of visible radiation.In some organisms this would have created aselection pressure for UV-screening compounds,providing the possibility for higher visible lightexposure.

An additional and strong evolutionary selectionpressure for UV-screening compounds must haveexisted on early earth. Three and a half billion yearsago, the sun, that is a typical main sequence star, ispresumed to have been 25% less luminous (Kasting,1993). Thus, the photosynthetically active radiationat the surface of the earth would have been less,which may have caused the euphotic zone (the zoneat which photosynthesis equals respiration) to havebeen slightly higher in the early oceans. The higherUV penetration into early waters caused by the lackof an ozone shield and possibly the proportionallygreater UV flux emitted by the sun as a residual of

its early T-Tauri stage (Canuto et al., 1982) wouldhave resulted in a more significant problem in thetrade-off between photosynthesis and UV radiationexposure in earth’s early oceans. The selectionpressure may have been intense for organisms thatcould exploit the high photosynthetically activeradiation in upper regions of the photic zone whilstmitigating UV radiation damage (Cockell, 1998a).UV-screening compounds would have been one ofthe principal solutions. This trade-off and selectionpressure would also have applied to terrestrialorganisms. UV-screening compounds would haveprovided the flexibility for life to inhabit moreexposed habitats.

The nature of the first specific UV-screeningcompounds on Archean earth is unknown, althoughan interesting hypothesis is that early aromatic-containing reaction centres were some of the earliestUV-screens that over time altered from a non-productive dissipative UV-screen to a light-harvesting role in photosynthesis (Mulkidjanian &Junge, 1997 and references therein).

Many compounds represented on present-dayearth and that are presumed to have been repre-sented on early earth may have provided substantialprotection for cyanobacteria (Garcia-Pichel, 1998).Both scytonemin and MAAs, which have screeningfactors equivalent to screening of between 2 and55% and 10 and 26% respectively in many speciesof cyanobacteria at the single cell level (at 320 nm),could have provided excellent screening from UV-Band later, as oxygen partial pressures increased andphotooxidative effects became more prominent, UV-A as well (Garcia-Pichel, 1998). Screening wouldhave been greatly enhanced when these compoundswere produced in layers of mats or in colonial growthforms. This would have helped reduce DNA damagerates from early earth values to values experiencedby exposed organisms today (Cockell, 1998a).

As alluded to in Section III, screening of UV-Cradiation on early earth could have been non-specifically accomplished by a wide variety ofmolecules, since as well as the more effective non-specific scattering of short-wavelength radiation,many small organic compounds will absorb thisregion of the spectrum. Scytonemin has a UV-Cpeak (Fig. 4) and when produced extracellularly itmay have been quite effective in providing a first-line reduction of UV-C flux penetrating the cell.Note that these UV-C peaks do not exist as a resultof evolutionary selection, unlike the UV-B and UV-A peaks in MAAs and scytonemin. They result fromUV-C-absorbing π to π* transitions found in most


conjugated organic structures. The non-specificnature of UV-C screening is demonstrated nicely bythe UV-C peaks in many phylogenetically morerecent organisms such as Antarctic Phaeocystis

pouchetti (Hariot) Lagerheim (Marchant et al., 1991)and many higher plant flavonoids (e.g. Shimazaki,Igarashi & Kondo, 1988; Markham, 1989). Theseorganisms did not evolve under a UV-C flux. Usedin combination with other strategies, UV-screeningcompounds would have been effective across thewhole UV range.

(3) Evolutionary relationships of UV-screening compounds

The evolutionary origins of many UV-screeningcompounds are still unknown. It is likely that manyevolved from other physiological roles to later fulfil aUV-screening function. Because potentially anycompound with aromatic or conjugated systemsmay provide passive UV radiation screening, wewould expect that the production of compoundsfor other physiological roles that fortuitously pro-vided UV screening would subsequently be selectedfor in organisms, if they provided some physiologicaland competitive advantage. Such an evolutionaryorigin for the UV-screening role of flavonoids inhigher plants has been proposed. They are presumedin their earliest stages to have been chemicalmessengers or physiological regulators (Stafford,1991) or a chemical defence against early pathogensand herbivores (Jorgensen, 1993) that would havebeen effective at low concentrations (Stafford, 1991).Later evolution in the pathways would have allowedboth structural improvements in UV screening andincreases in concentrations. MAAs have a role asosmotic regulators in some cyanobacteria (1997)and such alternative roles may have given rise to thefirst UV-screening MAAs (Cockell, 1998a). Asdiscussed before, the evolutionary origin of UV-screening compounds in other physiological rolesis today one of the primary complications in attri-buting compounds to a UV-screening role.

The evolution of the MAAs as specific UV-screensmay represent an early innovation in dealing withArchean UV-B flux. Many of the simpler MAAssuch as mycosporine-glycine specifically absorb inthe UV-B. It has been suggested that later, asoxygen levels increased, UV-A-screening MAAsbecame important since many of the effects of UV-A are mediated through oxygen free radicals(Garcia-Pichel, 1998) and thus the contribution ofUV-A as a damaging agent in the biosphere would

have increased. UV-A protection may also havebeen important well before the rise in oxygen partialpressures since many photosynthetic microbes mayhave been producing oxygen in their microenviron-ments in the late Archean (Garcia-Pichel, 1998). Inthese compounds, the nitrogen atom replacing theketone function has a greater mesomeric effect onthe benzene ring and absorbance is shifted into theUV-A. It is plausible that a mutation in the earliestUV-B-screening compounds resulted in a UV-Ascreen which became physiologically advantageous.Because MAAs are found in many eukaryotic algae,it is likely that they were passed to the eukaryoticalgae by cyanobacteria in the plastidic line.

The relationship between flavonoids and MAAs isless clear, but nevertheless intriguing. Plants aregenerally believed to have arisen from the greenalgae (Chlorophyta) (Niklas, 1976; Atsatt, 1988).One recent hypothesis suggests that land plants havea biphyletic origin as the product of an endocellularmutualism between a green alga (most probably acharophycean alga) and a fungus-like organism(Jorgensen, 1993) and it was suggested that thismutualism may even have been made possible by theUV-screening properties of flavonoids protecting thefungus. If this was the case then the synthesis offlavonoids as the principal UV screen would havearisen in the earliest charophycean algae. However,since the existence of flavonoids in present-day algaeis questionable (Markham, 1988), it is difficult tospeculate accurately on the nature of UV protectionin ancestral land plants. If green algae were the trueorigin of land plants and flavonoids are onlyrepresented in later plants, one might even speculatethat early land plants may initially have beendependent on MAAs or related compounds fromgreen algae instead of flavonoids. Some present-daygreen algae do contain MAAs (e.g. Karentz et al.,1991b). The present-day members of Nitella,Coleochaete and other genera of green algae arepostulated to be the ancestors of plants (e.g.Delwiche et al., 1989). Examination of the nature ofUV screening in these genera would be a worthwhilearea of evolutionary investigation. Later, flavonoidsmight have evolved from the phenylpropanoidbiosynthesis pathways as chemical messengers. Astheir absorbance properties both in the UV-A and Bsuperseded algal compounds, it is possible to imaginethat the MAA response to UV became redundant.Ultimately, the MAA pathway may have been lostaltogether as selection pressures for flavonoids andtheir associated diversity of biochemical usesincreased.


Other compounds have evolutionary origins thatare unknown. Cyanobacteria were some of theearliest oxygenic photosynthetic organisms on earth.Many of the terrestrial forms produce scytonemin,suggesting that it was a later innovation in thedeepest branches of many cyanobacteria, or waslater lost in marine species. The structure ofscytonemin suggests that it may be formed from thecondensation of tryptophan and phenylpropanoidderivatives (Proteau et al., 1993). Its reliance onoxygen for synthesis might support the contention ofa later evolutionary origin after the evolution ofoxygenic photosynthesis (Garcia-Pichel, 1998). Itspredominant screening peak in the UV-A ratherthan the UV-B also suggests some specific evolutionagainst the photooxidative effects of UV-A radia-tion.

The synthesis of animal eumelanins from thecondensation reactions with tyrosine mediatedthrough the action of tyrosinase is very differentfrom other pigment pathways and may suggest thatthe eumelanins have an independent evolutionaryorigin. As well as land animals, the presence ofmelanin in the ink sacs of cuttlefish (Prota, 1988)and many insects and molluscs as well as fungisuggests an early evolution.

Pictures are beginning to emerge of the evol-utionary relationships of UV-screening compoundsand their significance in the course of evolution. Inorder to understand the origins of UV screening andthe evolutionary relationships, a key area of investi-gation should be the study of UV screening in thedeepest branches of the phylogenetic tree. Moredetailed examination of photosynthetically deepbranches such as Chloroflexus spp. and the deepestbranches of the archaea, particularly exposedthermophilic genera, would also be of interest.

(4) UV-screening compounds and theBerkner–Marshall hypothesis

The evolution of UV screening is central to somefundamental questions in evolution. Berkner andMarshall (1965) originally proposed that the in-vasion of land 420 million years ago was dependentupon the build-up of atmospheric oxygen and theformation of the ozone shield that would havereduced UV flux. This hypothesis has subsequentlybeen supported by others in the context of landplants (e.g. Lowry, Lee & Hebant, 1980). Thehypothesis is an attractive one since it provides acongruence between a major innovation in evolutionand a major geophysical change in the earth’s

environment. However, aside from the major factthat a significant ozone shield was probably in placewell before the evolution of land plants and possiblyas long ago as 2 Ga (Kasting, 1987), it is highlyequivocal for a number of other physiological andpalaeobiological reasons.

Firstly, it is understood from the fossil recordthat stromatolitic microbial communities inhabitedintertidal regions some three billion years before theinvasion of land by plants (e.g. Knoll, 1992; Schopf& Klein, 1992). Apart from water containingimpurities, the water column, and particularlyshallow water, is not a particularly effective at-tenuator of UV radiation. Many microbial com-munities probably inhabited these exposed niches bya combination of UV-screening compound pro-duction and the use of the matting habit, wherebythe uppermost layers of the colony either die orprovide screening to the lower layers of the colony(Margulis et al., 1976). Other mechanisms such asphototaxis may also have been important. In a sense,the matting habit can be regarded as a method ofachieving the advantages of a multicellular UV-screening response by a unicellular organism. Fromthis perspective a multicellular algae or an earlyancestral leaf (that may have an epidermal layerprotecting photosynthetic regions below) can beregarded as an exquisite advance on the mattinghabit with the epidermis being a dedicated andevolved upper layer of the ‘mat’. UV-B penetrationinto the mesophyll of a leaf in modern-day plants canbe reduced to less than 2% of ambient. Colonies ofcyanobacteria, through the use of matting, canachieve similar reductions in UV radiation in the toplayers of the colony. From these arguments alone, itseems illogical that stromatolites evolved in inter-tidal and benthic continental-shelf regions of theworld 3±5 billion years ago and yet a multicellularalga (and a potential early ancestral plant), that hadfurther refined the UV protection of photosyntheticorganelles and DNA by specific multicellularadaptations, would have been constrained for somethree billion years more.

Secondly, there is now evidence for the presence ofexposed terrestrial microbial communities in thefossil record 1±2 billion years ago (Horodyski &Knauth, 1994). For the same reasons as describedpreviously, this evidence is in conflict with the delayin the development of early plants since if exposedmicro-organisms could survive, there should havebeen little constraint to early multicellular plants.

Thirdly, if we assume that Archean oceanic waterscontained little organic matter (since most UV-


absorbing organic matter in the present-day oceansis derived from land organisms), 1% penetration ofUV-B may have occurred to approx. 25–50 m inopen ocean water as for clear waters in the present-day oceans (Booth & Morrow, 1997). A single-celledisolated cyanobacterium at this depth might be ableto attenuate approx. 26% of the UV radiationit receives using MAAs which is the upper limitof MAA screening found in some cyanobacteria(Garcia-Pichel & Castenholz, 1993). They wouldreceive approx. 0±7% of incident UV radiation tointernal biological targets at such depths. Since aplant leaf can reduce incident flux to less than 2%,we can see that the differences in UV exposurebetween a suspended single-celled organism in thephotic zone and an early multicellular plant areprobably within the same order of magnitude.

Finally, the view of land plants (and indeed thecolonization of land generally) occurring as a resultof some major alteration in the UV radiationenvironment may be unnecessary from a generalevolutionary standpoint. If life was well establishedon land 1±2 billion years ago, then the colonizationof land by green algae, the evolution of uprightstructures and the eventual diversification of theseearly ancestral forms into bryophytes and tracheo-phytes by 420 million years ago may have been agradual evolutionary process that may well havebeen favoured by a gradually reducing UV flux, butmay not have required it.

The availability of atmospheric oxygen may wellhave been a factor for land plant colonization inother contexts since the lignin biosynthesis requiredfor structural support involves the use of oxygen(Lowry et al., 1980). It seems likely that within themilieu of significant evolutionary challenges andinnovations posed to land plants which includeprevention of desiccation, biosynthesis of supportstructures and accumulation of nutrients andminerals through root systems, an answer to thedelay of land plant colonization until the Silurianexists. However, it seems unlikely that UV radiationwould have been a critical constraint.

As a final factor in this discussion it is interestingto contemplate the link between UV radiation fluxand multicellularity in general terms. The evolutionof multicellular structures, at least in the Animalia,was probably linked to the availability of oxygen forrespiration (Berkner & Marshall, 1965). However,because of the screening advantage offered bymultiple cell layers and multiple localization ofscreening compounds, intense UV radiation shouldin a sense be a selection pressure for multicellularity.

Although the microbial matting habit is caused bymultiple ecological factors, the advantages in UVradiation attenuation afforded by the matting habitcan be seen as early evidence for the selectionpressure towards a multicellular UV-screeningstructure. Reductions in surficial UV radiation onthe Cambrian earth might have actually reduced therelative selection advantage for multicellular organ-isms compared to unicellular organisms, althoughclearly all organisms would gain an overall advan-tage under reduced UV flux.

(5) UV-screening compounds, ozonereductions and Phanerozoic palaeobiology

The last billion years of biological evolution hasoccurred under the protection of a UV-screeningozone column. Data from the total ozone monitoringsatellite (TOMS) show that natural variations inozone column abundance occur even over monthlyand annual periods. During the year, variations mayoccur of between 5 and 25%, that may be reflectedin the UV exposure of some biomes (e.g. Moorthy &Kathiresan, 1997). During the year, short-termvariations may also occur of up to 40% of totalozone column abundance over periods of even a fewdays to a month due to irregularities in ozoneconcentrations (TOMS data, NASA Goddard SpaceFlight Center). More longer term variations in UV-B reaching the surface of the earth may occur fromthe eccentricity of the earth’s orbit (which causes a7% variation in radiation incident on the top of theatmosphere between perihelion and aphelion).Variations in cloud cover for periods of days andweeks may cause significant variations in UV-Bradiation. Thus, organisms must possess a robustnessto deal with regular variations in surface UV-B overshort time periods. Passive UV-screening pigmentsmay provide some of this intrinsic robustness.

However, a number of natural events can depleteozone over more significant time periods (years) andat levels that are far more biologically significantthan most other natural variations. These eventsmay be evolutionarily significant (Cockell, 1999).Natural ozone-depleting events include large-scalevolcanism involving the injection of ozone-depletingmagmatic chlorine (Johnston, 1980; Vogelmann,Ackerman & Turco, 1992), asteroid and cometimpact events (e.g. Turco et al., 1982; Toon et al.,1997) and cosmic rays emanating from neutron starmergers or the supernova remnant shells of closesupernovae events (Ruderman, 1974; Aikin,Chandra & Stecher, 1980; Ellis & Schramm, 1995;


Thorsett, 1995). In the case of impact events, NO isgenerated that depletes ozone. Depending on scale itmay cause depletion of up to 85% (Turco et al.,1982). Impact events resulting in such levels of ozonedepletion may occur at a frequency of one every 10million years. Close cosmic events (neutron starmergers less than 1 kiloparsec and supernovae lessthan 10 pc) would produce high energy cosmicparticles that cause the ionization of atmosphericoxygen and nitrogen and the formation of NO.Depletion in these case may be up to 20% at theequator and 60% in Northern latitudes (Crutzen &Bruhl, 1996) and may occur at a frequency of oneevery 100 million years (Thorsett, 1975). Thefrequency of ozone depletion caused by volcanicactivity is less clear, but there have been at least ninemajor basaltic volcanic episodes over the last 250million years (Rampino, Self & Stothers, 1988).

The exact level of increase in UV-B radiation willdepend upon the latitude on earth and can becalculated by radiative transfer models. For thepurposes of the discussion in this review, however,the fact that some of these events may cause ozonedepletions much greater than 50% suffices toillustrate that significant ozone depletions that equal,and in many cases exceed, anthropogenically-causeddepletions occur over geological time-scales.

Over such time periods, exposed organisms witheffective UV protection and repair processes may beat an advantage compared to those with less effectivestrategies. Repair processes and phototaxis etc. mayprovide some level of intrinsic robustness to elevatedUV-B radiation. It is likely that compounds thatprovide passive screening against elevated UV-B willalso be important. The ability to tolerate relativelyrapid increases in UV flux will therefore dependupon these various processes combined and will bespecies specific. We would expect that for increasingseverity of ozone depleting events a greater pro-portion of exposed species may be stressed. As yetthere is little understanding of the ecological effectsof such events on natural populations and the degreeto which various UV protection methods mayprovide greater survival over evolutionary timeperiods, particularly in low latitude regions (Cockell,1999). Studies on UV screening in terrestrial andmarine realms of the antarctic and the efficacy ofthese processes in dealing with the effects ofanthropogenically-caused depletion may be of valuein assessing the effects of natural ozone depletionevents.

In studying recent changes in the UV radiationenvironment, UV-screening compounds have found

some use. The longevity of some compounds such asscytonemin and their potential preservation hasbeen suggested previously as an interesting target ofpaleobiological investigation (Garcia-Pichel &Castenholz, 1991). Preserved compounds have beeneffectively used to examine UV radiation regimes inlakes by measuring their concentrations in sedimentcores. These data have been used to provideinformation on the dissolved organic carbon com-position of lakes (and hence UV penetration to thebenthos) which in itself can be used as an indicatorof localized environmental changes (Leavitt et al.,1997). Because the concentration of UV-screeningcompounds depend on the changes in the UVpenetration of the water column, delineatingchanges in lake chemistry from natural changes instratospheric ozone may be difficult. Exposed cyano-bacterial stromatolites or organisms in clear-waterlakes that might better reflect changes in the ozonecolumn rather than local environmental conditionsmight be used for palaeo-analysis of changes indirect UV flux. Analysis of UV-screening com-pounds (particularly UV-B screening compounds)in cores taken from arctic or antarctic clear waterlakes might be used to search for direct evidence ofnatural ozone depleting events.

XI. ARTIFICIAL SCREENING COMPOUNDS

AND HUMAN UV PROTECTION

Mankind’s attempts to deal with unwanted effects ofsunlight are to an increasing extent based ondeveloping chemicals that absorb ultraviolet radi-ation. Some factors of importance are discussed heresince they provide an interesting comparison to theevolution of natural UV-screening compounds, andan insight into the motives that have driven peopleto attempt to develop artificial compounds thatallow them to tolerate increased exposure to UV-Bradiation. Furthermore, as the properties andcharacteristics of natural compounds are betterunderstood, a convergence of human needs andnatural responses is likely to become increasinglyimportant.

The first compound to be used widely (Natow,1986; Patel, Highton & Moy, 1992) was para-aminobenzoic acid (PABA); superficially an ap-pealing choice because it is a natural product. Itabsorbs UV-B quite strongly and so protectsefficiently against sunburn, and has been used sincethe 1920s. Although it is a crystalline solid whichdissolves poorly in water, it penetrates the stratumcorneum. It does so even if it is suspended in an


emulsion at pH 4±2; when it is largely insoluble, andcan be recovered in the urine. When applied as asolution in ethanol it can accumulate in the skin(Algra & Knox, 1978), so that it can be a veryeffective photoprotective agent. But drawbacksgradually became evident. PABA can stain bothnatural (cotton) fabrics and synthetic ones such asnylon and polyesters, perhaps because it is photo-sensitive, and a number of people became sensitizedto PABA and developed dermatitis (Patel et al.,1992). These problems, and particularly the prac-tical ones associated with actual use by sunbathers,stimulated a search for alternatives well before thereports of potential problems with DNA damage,notably an ability to sensitize the formation ofthymine dimers in DNA in cultured cells, started toappear (Sutherland, 1982; Sutherland & Griffin,1984). The search resulted in, among other things,the introduction of esters of PABA derivatives. Onein particular is worth considering in some detail. Itis 2-ethylhexyl-4 dimethylaminobenzoate, in whichthe carboxyl group of PABA has been converted intoa branched chain ester, and the amino-group hasbeen converted into a dimethylamino group. Thismaterial, known commercially as Padimate-O,Octyl Dimethyl PABA, O-PABA, OD-PABA, Esca-lol 507 (and sometimes as Arlatone UV-B, Solar-chem O or UV-Absorb DMO) was particularlyattractive because it is a colourless, oily liquid that isvirtually insoluble in water and did not stainclothing. It became, for a while, one of the mostwidely used UV-screens, and offers very goodprotection against sunburn. As assayed by Ames testsusing yeast, it did not appear to have the potential todamage DNA, at least in the absence of deliberateillumination (Bonin et al., 1982), and so appeared tohave many advantages.

However, it is important to remember that,despite their name, UV-screens and sunblockscannot simply eliminate the light energy which fallson them. They have to do something with it, and thechemicals involved are really energy transducerswhich absorb UV energy and convert it to someother form. It is important to understand exactlywhat happens, because compounds such as PABAand Padimate-O can form excited states. Such statesare not easy to characterize, but can be intenselyreactive, raising the possibility that sunscreens couldhave unwanted side-effects, as indeed was shown forPABA (Sutherland, 1982; Sutherland & Griffin,1984).

PABA sensitizes the formation of thymine dimersin DNA. It does so because it forms a triplet state

which can react directly with DNA, forming thyminedimers. However, there are other possibilities. Trip-let PABA can react with oxygen to form singletoxygen (Allen, Gossett & Allen, 1996). It can alsogenerate solvated electrons, which react with oxygento make the superoxide radical anion O[−

#(Allen et

al., 1996; Martincigh, Allen & Allen, 1997). Proton-ation of the superoxide radical anion followed bydismutation generates O

#and hydrogen peroxide.

Hydrogen peroxide can react with the superoxideradical anion in the Haber–Weiss reaction, or withFe#+ (inevitably present in biological systems) in theFenton reaction to produce hydroxyl radicals, whichcan damage DNA in various ways. This means (a)that any mutagenic effects of PABA cannot neces-sarily be attributed to the formation of thyminedimers, and (b) that PABA could have undesirableside-effects, and it is possible that this is why naturalselection has not chosen PABA as a commonUV-screening compound.

What about Padimate-O? On a simple view, onemight expect it to behave in the same way, but infact it appears to behave rather differently. The firstclue came from a structural comparison with afunctionally identical compound, ethyl-4-dimethyl-aminobenzoate, which is simply the correspondingethyl ester. It is used as an industrial photoinitiatorof polymerization (Wayne, 1988), which it catalysesbecause it generates carbon-centred radicals underUV illumination (Forster & Hester, 1981). Ascarbon-centred radicals can, in the presence of waterand oxygen, react in various ways to form highlyreactive hydroxyl and peroxy radicals, one wouldexpect the sunscreen Padimate-O to have thepotential to inflict damage on a variety of bio-logically important molecules. Experiments usingyeast showed that it is killed by illuminatedPadimate-O, and suggested that DNA damage alsooccurs. It could contribute to the toxicity becausemutations were generated and rapidly-dividing cellsthat were deficient in DNA repair were moresensitive than non-dividing, repair-proficient cells(Knowland et al., 1993). This work raises thetheoretical possibility that the beneficial effects ofsunscreens in reducing UV exposure could bebalanced by potentially harmful effects caused bysunscreen penetrating the skin, which both PABAand Padimate-O are known to do (Arancibia et al.,1981; Blank et al., 1982). DNA damage, if it occurs,could be especially important. In order to investigatethis possibility it is essential first to define the kind ofdamage that any given sunscreen inflicts when it isilluminated. The simplest way to do this is to study,


in preliminary experiments, what happens withDNA in vitro. It emerges that, unlike PABA,Padimate-O does not sensitize the formation ofthymine dimers in DNA. Rather, it generates directstrand breaks and oxidative lesions, mainly but notexclusively at GC base pairs, that can be detected ina variety of ways (McHugh & Knowland, 1998).Thus, experiments intended to reveal the kind ofdamage inflicted on human cells by sunlight in thepresence of Padimate-O must take this into account.The same is true for other organic sunscreens, buttheir photochemistry in relation to potential DNAdamage is, in most cases, very poorly understood.Other aspects also merit further attention. Forexample, it appears that illumination can catalyzebreakdown of organic sunscreens (Roscher et al.,1994; Schwack & Rudolph, 1995), and it is not clearwhat the consequences could be.

As an alternative to organic sunscreens, physicalsunscreens have also been developed. They are basedon titanium dioxide and zinc oxide, which are bothwhite pigments. The former is widely used in paint.If the particle sizes are relatively large, creamscontaining these pigments simply appear white,which makes them cosmetically unattractive. Inorder to avoid this problem, manufacturers reducethe particle size to the range 20–50 nm, because inthis size range the interaction with light obeysRayleigh’s laws of light scattering, whereby theintensity of scattered light is inversely proportionalto the fourth power of the wavelength (Judin, 1993).This means that they scatter the short, UV wave-lengths far more efficiently than visible ones. To theextent that scattering is backwards rather thanforwards, it is protective. However, the oxideparticles also absorb UV light to a substantialextent, and, like organic sunscreens, they interactwith that energy. Both titanium dioxide and zincoxide are semiconductors. Interaction with UVpromotes electrons from the valence band to theconduction band, generating single electrons andelectron deficient states, known as ‘holes ’. Anywavelength shorter than approx. 385 nm willachieve this, so that these materials are excitable byboth UV-A and UV-B. The electrons and ‘holes ’either recombine or migrate rapidly (in less thanapprox. 0±01 ns) to the surface. In aqueous environ-ments, ‘holes ’ react with water or hydroxyl ions, andelectrons react with oxygen, forming hydroxylradicals and superoxide (Serpone, 1996):

h+OH−UOH[,e−O

#UO

#

[−,

Both hydroxyl radicals and superoxide are highlyreactive, so that ZnO and TiO

#are effective

photocatalysts of oxidation. Indeed, TiO#

has beenextensively investigated in connection with oxidationof environmental pollutants (Bahnemann et al.,1994). Although it is not clear whether TiO

#and

ZnO as used in UV-screens can penetrate humanskin or not, where the evidence is limited anduncertain (see Dunford et al., 1997 and referencestherein), it is clear that small particles of TiO

#can

enter human cells (Kubota et al., 1994) and thatlarger ones can too (Wamer, Yin & Wei, 1997).Consequently, it is important to investigate thepotential of both TiO

#and ZnO to attack DNA,

taking into account both the type of damage whichmight be inflicted and whether it actually occurs.Here, it is particularly important to study thepreparations actually used in UV-screens, becausethey are often coated with other materials which areintended to reduce or eliminate photoactivity,although whether such coatings are invariablysuccessful is hard to judge. It is also important toconsider the type of TiO

#used, because its activity

depends on crystal structure, the anatase form beingmore active than the rutile form. Anatase TiO

#with

a particle size of 450 nm clearly generates hydroxylradicals, as demonstrated using the spin trap 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), and itcatalyses oxidative damage to DNA and RNA,producing hydroxylated guanine residues (Wamer et

al., 1997).A survey of preparations found in a range of

conventional UV-screens found that there is a widerange of ability to catalyse photooxidation, but thatall were active to some extent (Dunford et al., 1997).The same survey also demonstrated DNA damage incultured human cells illuminated in the presence ofTiO

#with light intensities very similar to those

found under the stratum corneum. The damagefound was exactly that which would be predicted –direct strand breaks and oxidative lesions attribut-able to hydroxyl radicals. In that study, damage tonuclear DNA was found, in contrast to the work ofWamer et al. (1997), which, using a different assayand much larger particles of TiO

#, did not report

damage to nuclear DNA. Although it is clear thatmuch more work needs to be done, it would appearthat the potential of sunscreens to damage DNA isnot confined to organic sunscreens.

It is not the purpose of this review to consider inany detail the many studies that have been con-ducted using direct biological approaches, becausethat has been done many times before, but a few


comments may be relevant to the more chemical andmolecular work outlined. It has been shown thatcertain UV-screen preparations reduce the tumoursfound in control hairless mice, although whether thisshould be interpreted in terms of prevention oftumourgenesis (Kligman, Akin & Kligman, 1980) orsimply delay (Wulf et al., 1982) is not entirely clear.It is also clear that some can reduce the formation ofthymine dimers in mouse skin under illuminationwhich under-represents UV-A (Ananthaswamy et

al., 1997). It is hard to know whether suggestionsfrom work on a melanoma-susceptible hybrid fish(Setlow et al., 1993) showing that UV-A is par-ticularly important in that species apply to mice andmen, but if UV-A is indeed a relevant risk factor,then much of the earlier work, conducted beforegood solar simulators which adequately representnatural sunlight became available, may have to bere-assessed. One problem with these whole-animalexperiments is that they cannot easily distinguishbetween the effects of screens applied to skin and theeffects of that which diffuses into the skin, but on theother hand they do address an important biologicalend-point : the formation of a tumour. The overallconclusion has to be that as many approaches aspossible need to be applied to the question of the rolethat sunscreens play in protecting us from theunwanted effects of the sun.

Natural sunscreens may provide some solutions.An interesting parallel has occurred in research.As we have begun to understand the potentialphotoxidative problems associated with some of theartificial sunscreens, so we have also begun toelucidate the role that many natural UV-screeningcompounds have in anti-oxidant activity. Thisfunctional link may not be a coincidence. UV-screening flavonoids are important anti-oxidants inplants (Larson, 1988). In corals, the UV-screeningMAA, mycosporine-glycine, is a strong anti-oxidant(Dunlap & Yamamoto, 1995). Thus, it is possiblethat within the complement of natural UV-screeningcompounds, some are effective not just in passivescreening, but also at quenching reactive oxygenspecies. They may provide an additional line ofdefence against photooxidative damage as well asother compounds that are presumed to have adedicated anti-oxidant role, such as carotenoids.Compounds such as flavonoids and mycosporine-glycine are likely to be of great interest in thedevelopment of human sunscreens. Thus, a conver-gence of the study of natural UV-screens and thedevelopment of sunscreens for human requirementsseems inevitable.

Secondly, of course, research on human sunscreensmay provide directions for research on naturalUV-screening compounds. Having investigated thefate of energy produced by UV screening in humansuncreams the question arises as to how significantthis is in natural populations and what happens toenergy produced by natural UV-screening com-pounds. How necessary are the antioxidant proper-ties of some MAAs, flavonoids and some carotenoidsin mitigating UV-induced damage generated by theUV absorbance of these UV-screening compoundsin the first place? What is the nature of thisinteraction between antioxidant activity and UVscreening in natural compounds? Answers to thesequestions are likely to have many natural scienceand human commercial implications.

XII. CONCLUSIONS

(1) UV-screening compounds are represented inwell-defined groups (for example cyanobacterialscytonemin, MAAs, flavonoids and eumelanins)across a great diversity of organisms. This suggeststhat the specific screening of UV radiation has beena strategy for coping with its damaging effects for aconsiderable time. The screening compounds dis-covered to date use the π to π* transitions associatedwith conjugated bond structures, aromatic orindolic derivatives to screen UV radiation, providinga common chemical theme.

(2) Since a UV-screening compound is bydefinition a passive process, it is important to ensurethat any experimentally observed screening is indeedphysiologically relevant. A series of basic rules havedeveloped that allows the experimenter to check therole of a compound in UV screening. By thesecriteria both scytonemin, and in some cases MAAs,have been found to have a UV-screening role as wellas many plant flavonoids and animal eumelanins.Some compounds such as sporopollenin and melaninare examples of compounds that may provideconstitutive, but physiologically relevant UVscreening.

(3) Although compounds provide an effectiveUV-specific shield, particularly for photosyntheticorganisms, it is also clear that other UV mechanismssuch as phototaxis and particularly protein andnucleic acid repair processes may provide an effectiveUV response and in many cases may actually reduceor even negate the need for UV-screening com-pounds. Simple measures of concentrations andscreening characteristics of compounds do not


provide insights into an organism’s UV tolerance.More detailed studies of UV-screening compoundsand their screening abilities in relation to othermodes of UV response will be an important avenueof future research, particularly in attempting tounderstand possible changes in interspecies com-petitiveness in response to changes in UV radiation,caused by natural or anthropogenic ozone depletion.

(4) Another avenue of future research is tocontinue work to understand the representation ofdifferent types of UV-screening compounds in agreater diversity of organisms. This work will havegreat evolutionary interest. It can provide moredetailed insights into how ubiquitous and necessaryUV-screening compounds are in many relatedspecies and during the course of their evolutionarydevelopment. Furthermore, through such work wecan gain better insights into the role of UV-screeningcompounds on early earth. Studies on the deepestbranches of the phylogenetic tree will elucidate theorigins of UV screening.

UV radiation has been a ubiquitous feature of theexposed surface of earth since the origin of life itself.It is the one of the most damaging forms of radiationthat penetrates to the surface of the earth and istherefore a highly important regulator of organismsurvival and ecosystem balance. We can expect thework that has been completed to date to provide afoundation for increasing our understanding of therole of UV-screening compounds in survival underthe various UV regimes of the earth, past andpresent and future.

XIII. ACKNOWLEDGEMENTS

Acknowledgements are due to two anonymous reviewersfor thoughtful and helpful ideas and suggestions on themanuscript.

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