Biol Rev Uv Abs Compounds

Biol. Rev. (1999), 74, pp.
311345 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 passive
method for the reduction of UV-induced damage and they are widely distributed across the microbial, plant
and animal kingdoms. They share some common chemical features. It is likely that on early earth strong
selection pressures existed for the evolution of UV-screening compounds. Many of these compounds probably
had other physiological roles, later being selected for the ecacy of UV screening. The diversity in
physiological functions is one of the complications in studying UV-screening compounds and determining
the true ecological importance of their UV-screening role. As well as providing protection against ambient
UV radiation, species with eective screening may also be at an advantage during natural ozone depletion
events. In this review the characteristics of a wide diversity of UV-screening compounds are discussed and
evolutionary questions are explored. As research into the range of UV-screening compounds represented in
the biosphere continues, so it is likely that the properties of many more compounds will be elucidated. These
compounds, as well as providing us with insights into natural responses to UV radiation, may also have
implications for the development of articial UV-screening methods to reduce human exposure to UV
radiation.
Key words : UV radiation, evolution, screening, compounds.
CONTENTS
I. Introduction ............................................................................................................................ 312
II. Advantages of passive UV screening....................................................................................... 312
III. Chemical and absorbance characteristics of UV-screening compounds .................................. 314
IV. Determining whether a compound has a screening role ......................................................... 318
V. Scytonemin a positively identied UV-screening compound................................................ 319
VI. Mycosporine-like amino acids and the ecological complications in studying UV-screening
compounds............................................................................................................................... 320
VII. UV screening in plants............................................................................................................ 323
VIII. 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 BerknerMarshall hypothesis ..................................... 333
(5) UV-screening compounds, ozone reductions and Phanerozoic palaeobiology.................. 334
* Present address : Charles Cockell, M\S 239-20, NASA Ames Research Center, Moett Field, CA 94035-1000,
USA.
312 Charles S. Cockell and John Knowland
XI. Articial screening compounds and human UV protection.................................................... 335
XII. Conclusions.............................................................................................................................. 338
XIII. Acknowledgements .................................................................................................................. 339
XIV. References................................................................................................................................ 339
I. INTRODUCTION
For 3.8 billion years the evolution of methods to
attenuate ultraviolet radiation has been a ubiquitous
problem for life and particularly for photosynthetic
organisms that depend on solar radiation for their
energy requirements. For some organisms that live
in subsurface regions of the earth, in the ocean
depths, caves and other habitats that exclude solar
radiation, the problem of UV radiation damage is
solved. These organisms are not considered in this
review.
For convenience ultraviolet (UV) radiation is split
into four major bands. Vacuum UV is radiation
with a wavelength less than 200 nm. UV-C radiation
occupies the region between 200 and 280 nm.
Neither vacuum UV nor UV-C reach the surface of
the present-day earth because of atmospheric
Rayleigh scattering and ozone absorption, although
these regions are important parts of the extra-
terrestrial spectrum (e.g. Nicolet, 1989). UV-B
radiation is energetically less damaging than UV-C.
In the scientic literature, UV-B radiation is often
dened as 280320 nm. However, the legal denition
provided by the International Commission on
Illumination sets the UV-B radiation range as 280
315 nm. On earth, most of the UV-B radiation
is attenuated by the ozone column that absorbs
strongly in the Hartley region (200300 nm) and
weakly in the Huggins Band (300360 nm). Finally,
UV-A radiation (315400 nm) reaches the surface of
the earth relatively unattenuated and is still less
energetic than UV-B radiation.
UV radiation, and particularly the higher energy
wavelengths, has a range of eects. They include
DNA 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 & Schoeld, 1994;
Vincent & Roy, 1993) and higher plants (e.g. Tevini
& Teramura, 1989; Tevini, 1993 and references
therein), inhibition of nitrogenase activity (Sinha et
al., 1996), inhibition of heterocyst formation in some
cyanobacteria (Sinha et al., 1996), reduction in
microbial motility (e.g. Donkor, Amewowor &
Ha$ der, 1993a, b; Donkor & Ha$ der, 1995), and a
diversity of other responses that have been reviewed
elsewhere (e.g. Tevini, 1993; Vincent & Roy, 1993;
Vincent & Quesada, 1994).
It is accurate to view the overwhelming eect of
UV radiation as a damaging agent to a wide variety
of biological systems. However, it is wrong to paint
a picture of UV radiation as solely damaging. It has
many benecial roles in the biosphere. For example,
UV-A reected from petal anthocyanins (Flint &
Caldwell, 1983) is essential for ower recognition by
pollinating insects such as bees. UV-A is involved in
buttery wing recognition during mating (Meyer-
Rochow, 1991). Some spiders use the UV reectance
of their webs to capture prey (Craig & Bernard,
1990). Some sh that possess tetrachromatic vision
visualize UV-A at 360 nm and this may be essential
for 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 and
photomorphogenetic eects in plants, mediated
through blue\UV-A receptors, involve UV-A radia-
tion (Salisbury & Ross, 1992). As will become
apparent in this review, some of the pigments and
compounds that absorb UV radiation have a role to
play in these processes.
II. ADVANTAGES OF PASSIVE UV SCREENING
A number of methods exist for coping with the
potential damage caused by UV radiation. They are
illustrated in Fig. 1. An important response is
damage repair, particularly in DNA, which is one of
the most susceptible biological targets (Jagger,
1985). DNA photoreactivation, during which the
blue light\UV-A inducible enzyme photolyase
cleaves covalently linked thymine dimers in DNA
generated by UV-B radiation is an important
response in most organisms (Sutherland, 1981). The
non-light dependent removal of a section of DNA
containing the damage lesion (DNA excision repair
or dark repair) has also evolved as an eective
repair process (Hanawalt et al., 1979). Although
these mechanisms are eective, by denition they
313 Ultraviolet radiation screening compounds
UV
radiation
Avoidance
Protection
Repair
Phototaxis. Lifestyles that completely
avoid solar radiation
Physical substrates (rocks, iron, sulphur, etc.)
Screening under organic compounds
Quenching of reactive oxygen species using carotenoids
Specific 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 rst line of defence
against UV radiation. Repair processes are subsequently used to deal with damage. The gure illustrates the diversity
of responses that are available to an organism.
address damage that has already occurred. Because
repair processes can never be 100% ecient, the less
damage that occurs, the greater the potential
advantage to the organism, regardless of the absolute
levels of repair that an organism may be able to
elicit. This is particularly the case for single-celled
organisms, where even a single-point mutation can
be lethal. The synthesis of carotenoids that quench
oxygen free radicals generated by UV-induced
photochemical reactions (Krinsky, 1979) is also an
important response in preventing UV-induced dam-
age to a wide diversity of biological macromolecules.
Again, however, its necessity stems from reactive
oxygen species formed by UV radiation that has
already penetrated the cell.
Organisms may be able to avoid UV radiation by
phototaxis, whereby they diurnally limit exposure to
UV radiation by moving in response to the UV-B
gradient (e.g. Bebout & Garcia-Pichel, 1995).
Phototaxis is less widely distributed than damage
repair processes and is likely to be energetically
demanding for organisms regularly exposed to UV
radiation.
For organisms exposed to UV radiation for
substantial 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 the
rst place (see Fig. 1). UV screening may also
provide some energetic advantage by reducing the
need for constantly active avoidance and repair
processes.
Two approaches exist to screen UV radiation
passively. There are a variety of physical substrates
that may be eective, such as a water column
containing organic impurities. Dissolved organic
carbon is one of the primary factors in reducing UV
penetrability into freshwater systems (Booth &
Morrow, 1997). Iron compounds can provide signi-
cant reductions in the UV region, particularly
oxidized ferric (Fe$
+
) iron whose absorption
coecient is an order of magnitude greater than
ferrous (Fe#
+
) iron (e.g. Olsen & Pierson, 1986;
Pierson, Mitchell & Ru-Roberts, 1993). Iron-
containing sediments could provide signicant UV
attenuation for benthic microbial communities
(Garcia-Pichel, 1998). Elemental sulphur can also
potentially provide specic UV absorption (Cockell,
1998a). However, most physical substrates will non-
specically attenuate visible wavelengths either by
direct absorption or scattering, thus limiting ex-
posure to photosynthetically active radiation for
phototrophic organisms. For some organisms such as
polar cyanobacteria that dominate as a result of slow
persistent growth and tolerance of extreme environ-
ments (Tang, Tremblay & Vincent, 1997), rather
than fast optimized growth, this disadvantage may
be unimportant. The cryptoendolithic communities
in the sandstone of the Ross Desert, Antarctica
(Nienow, McKay & Friedmann, 1988) where
growth rates may be as low as one cell division per
year, are such an example. These communities live
in the subsurface layers of rock, where UV is
attenuated, but also visible light. In this case the
growth in layers of rock oers the advantage of a
more stable micro-climate. However, for many
organisms in less extreme habitats, increased expo-
sure to photosynthetically active radiation provided
by UV-specic screening may be advantageous.
A second disadvantage of physical screening
methods is that organisms are limited to the habitats
where the physical substrate is available. Organisms
that can synthesize their own UV-protecting com-
pounds have the opportunity to occupy a greater
diversity of habitats, depending of course on limi-
tations imposed by other environmental factors.
With these limitations in mind, it is unsurprising
that the synthesis of passively screening compounds
has evolved as a ubiquitous rst-line response to UV
radiation.
III. CHEMICAL AND ABSORBANCE
CHARACTERISTICS OF UV-SCREENING
COMPOUNDS
The evolution of a successful UV-screening com-
pound ultimately depends upon simple organic
photochemistry. The -electron system is one of the
most eective UV radiation absorbers. -electron
systems are primarily found in conjugated bond
structures that may be represented both in linear
chained molecules with alternating single and double
bonds and in many aromatic and cyclic compounds
containing electron resonance. The overlapping
orbitals of -electrons have absorption maxima in
the UV region that cause an energetic transition of
-electrons to anti-bonding *-electron orbitals. -
electron systems are a common chemical theme in
the function and characteristics of natural UV-
screening molecules (Cockell, 1998b).
Alterations in the structure of a conjugated
molecule change the absorbance characteristics and
thus the region of the spectrum that is attenuated.
Generally, the larger the molecule, the longer the
wavelengths that are absorbed because, in the
CH
2

CH CH CH CH CH
2
CH
2

CH CH CH
2
(CH
2

CH CH CH )
2
OH
217
258
287
255
270
275
k
max
(nm)
Fig. 2. Ultraviolet absorption maxima (
max
) of some
selected organic molecules demonstrating the batho-
chromic shift that occurs when both the size of the
molecule and the nature of the side groups is altered.
simplest terms, the wave-particle duality results in
an increase in the wavelength of the electrons that
can be accommodated in a larger molecular struc-
ture. For example, in the conjugated molecule 1,3-
butadiene, an important absorption maximum is at
217 nm, whereas for 1,3,5-hexatriene the corre-
sponding maximum is increased to 258 nm and for
1,3,5,7-octatetraene it increases further to 287 nm in
the UV-B region (Fig. 2). Very long chain molecules
such as the natural carotenoid, -carotene (11
ethylenic linkages) has an absorption in the visible
region at 455 nm. The same shift in wavelength may
be achieved by increasing the number of conjugated
bonds and substituents in aromatic structures rather
than in a linear chain. For example, benzene has an
absorbance peak at 255 nm, whereas naphthalene,
that contains two rings, has the corresponding
absorbance at 275 nm (Fig. 2). Although many
natural linear carotenoids can absorb in the UV
range, their physiological importance in screening in
most known cases is secondary to aromatic
derivatives. The role of carotenoids in screening and
some evolutionary aspects of the preferential selec-
tion of aromatics as screening compounds are
discussed in greater detail in Section IX.
As well as the absorption maximum, the extinction
coecient is also altered by the side groups and the
molecular structure. Generally, aromatic com-
pounds have lower extinction coecients, , than
NH
2
OH OH
NH
OCH
3
CO
2
H
NH
OH
OCH
3
NH
CO
2
H
OH OH
NH
OCH
3
NH
CO
2
H
OH OH
Palythine (k
max
= 320 nm) Asterina (k
max
= 330 nm) Palythene (k
max
= 360 nm)
NH
OH OH
NH
OCH
3
CO
2
H
OH
CH
3
CO
2
H
NH
OH OH
NH
CO
2
H
OH
CO
2
H
NH
OH OH
NH
CO
2
H
OH
Palythinol (k
max
= 332 nm) Porphyra (k
max
= 334 nm) Shinorine (k
max
= 334 nm)
CO
2
H
NH
OH OH
NH
CO
2
H
OCH
3
O
OH OH
NH
CO
2
H
CO
2
H
NH
OCH
3
OH OH
NH
CO
2
H
Mycosporine-glycine:valine
(k
max
= 335 nm)
Mycosporine-glycine
(k
max
= 310 nm)
Palythenic acid
(k
max
= 337 nm)
O
OH OH
HO
OCH
3
OH
OCH
3
OH OH
R
2
R
1
O
OOOC
N
H
N
+
H
COOH
O R
2
R
1
Gadusol
(k
max
= 294 nm)
Nostoc commune E335
(k
max
= 335 nm)
R
1
= Gal, Xyl, GlcU
R
2
= Gal, Glc, GlcN
OCH
3
OCH
3
OCH
3
Fig. 3. Structures of some of the principal mycosporine-like amino acids (MAAs) found in nature. Gadusol, which is
structurally related to MAAs and found in sh roe is also shown as is E335, the Nostoc commune polysaccharide-linked
mycosporine (see text for details).
max
is the ultraviolet absorption maximum.
unsaturated aliphatic compounds. For example,
for the benzene maximum at 255 nm is 230 mol
" l
cm
", whereas for 1,3,5-hexatriene at the cor-
responding absorption maximum at 258 nm is 80000
mol
" l cm
". However, substitution can increase the
extinction coecient substantially. For example, in
the phenolate anion, is increased to 2600 mol
" l
cm
". Generally, the more substituents that are
added to a molecule the more intense the absorption
becomes. These type of substituent eects on the
absorption coecient are important in achieving the
ecacy of screening required in natural UV-
screening compounds.
Here, it is worthwhile to illustrate the concepts
outlined above with some natural examples.
Many organisms produce mycosporine-like amino
acids (MAAs) as a natural UV screen. The elegance
of their UV-absorbance properties lies in the
modulation of the peak absorbance of a basic
cyclohexenone or cyclohexenimine core structure.
Fig. 3 shows some of the major MAAs found in
nature. Hetero-atoms with non-bonding electrons,
such as oxygen or nitrogen and the halogens, can
absorb UV radiation when the electrons make a
transition from n orbitals to * orbitals. However,
this absorption is quite weak. A more important
eect of these substituents is their ability to alter the
absorption properties of aromatic ring structures.
The non-bonding electrons can take part in the
resonance of the ring structure which causes a
bathochromic shift in the absorbance maximum
(Gillam, 1970). Thus, although the absorbance peak
of the benzene ring structure is 255 nm, in the
MAA cyclohexanone core structure it is increased to
280 nm.
The degree of the bathochromic shift caused by
the substituent is determined by its mesomeric eect
(the ability of the non-bonding lone pair electrons to
take part in the electron resonance of the ring
structure). 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 rather
than the cyclohexanone structure, but with identical
side groups on all other positions has its absorption
maximum shifted to 320 nm. Further changes in the
absorption maxima are caused by substituent eects.
For example, the MAA palythene has a conjugated
triene system attached to the nitrogen which shifts
the absorbance to 360 nm, an additional 40 nm
compared to palythine. Most of the UV-B-screening
MAAs use a cyclohexanone structure, whereas the
UV-A-screening compounds use a cyclohexenimine
OH
H
N
O
O
H
N
OH
1.2
1.0
0.8
0.6
0.4
0.2
0.0
250 300 350 400 450 500 550 600 650 700 750 800
Wavelength (nm)
A
b
s
o
r
b
a
n
c
e
Fig. 4. Phenolic and indolic structure of the widely
distributed terrestrial cyanobacterial UV-screening pig-
ment, scytonemin. The gure also shows the absorbance
spectrum of the oxidized pigment in tetrahydrofuran
(adapted from Proteau et al., 1993). Absorption is shown
as arbitrary units.
core structure, presumably because the non-bonding
nitrogen electrons cause a greater bathochromic shift
towards the UV-A region. The evolutionary aspects
of this are discussed in Section IX. By synthesizing a
range of MAAs, organisms might be able to screen
broadly in the UV-A and B range. In a single
organism, the breadth of UV absorbance can span
from 230 to 400 nm and the peak absorbance can
range from 310 to 360 nm depending on the
complement of MAAs.
The absorbance properties of a UV-screening
compound are further changed by increasing the
complexity of -electron systems, their relationship
to other moieties and their number. The UV-
absorbing compound scytonemin is a lipid-soluble
phenolic and indolic derivative produced in the
sheaths of cyanobacteria (Garcia-Pichel &
Castenholz, 1991; Garcia-Pichel, Sherry &
Castenholz, 1992). This compound is present in
these organisms in addition to MAAs. Fig. 4 shows
the structure of scytonemin. The complexity of the
ring structures generates a specic type of UV-
absorbance pattern (Proteau et al., 1993). The size of
the molecule is partly responsible for the long-
HO
OH
R
1
R
2
R
3
R
4
OH
O
C A
2
B
1.0
0.8
0.6
0.4
0.2
0
250 300 350 400 450
354 nm
Wavelength (nm)
A
b
s
o
r
b
a
n
c
e
Fig. 5. Basic structure of higher plant avonoid molecules
(adapted from Staord, 1991). Changes in the side groups
result in dierent molecules. With stereochemistry at C
#
:
avonone (R
$
, H; R
%
, C%O), 3-hydroxyavanone
(dihydroavonol) (R
$
, OH; R
%
, C%O), avan-3-ol (R
$
,
OH; R
%
, H). Loss of stereochemistry at C
#
(double bond
positions refer to C-ring): avone (C
#
%C
$
; R
$
, H; R
%
,
C%O), avonol (C
#
%C
$
; R
$
, OH; R
%
, C%O), antho-
cyanidin (O
"
%C
#
; C
$
%C
%
; positive change on C ring; R
%
,
H or OH), isoavone (aryl migration to C
$
; R
%
, C%O; B-
ring R
"
, R
#
, H, or OH).
A typical absorbance prole for total phenolics
extracted in 85% acidied methanol is also shown. This
extract is from the epidermis of the desert cactus, Opuntia
phaeocantha (C. S. Cockell, unpublished data). Absorption
is shown as arbitrary units.
wavelength UV-A screening. The molecule screens
most strongly from 325 nm to 425 nm across the
UV-A, but the shoulder of the peak extends into the
UV-B. The molecule also has an absorbance peak in
the UV-C with a maximum at 250 nm, which results
from intense short-wavelength to * transitions
found in most conjugated molecules.
Since the Cambrian explosion, many multicellular
organisms have also evolved other uses of the -
electron system specically to screen UV radiation.
Flavonoids (Fig. 5), produced by plants, screen in
the 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
0.0
200 300 400 500 600
Wavelength (nm)
A
b
s
o
r
b
a
n
c
e
Fig. 6. Typical absorption prole of melanins (adapted
from Ravishankar et al., 1995). The pigment shows a non-
specically increasing absorbance towards shorter wave-
lengths. The absorbance prole results from the many
complex conjugated structures in the polymeric molecule.
system provides the UV-A and UV-B absorbance.
Ring A also contributes to the absorbance in the
UV-C region at approx. 250 nm. Because of the
complex ring structure, single avonoids may pro-
vide broad UV-A and B screening, unlike MAAs,
where evolution from a simple cyclohexenone or
cyclohexenimine core structure provides only a
discrete absorbance maximum.
Melanin, used for screening UV in humans and
many vertebrates, also has a complex polymeric
structure (Kollias et al., 1991) containing aromatics
and indole derivatives to provide UV-A and UV-B
screening. Its complex polymeric structure generates
a rather non-specic screening (Fig. 6) with in-
creasing ecacy at lower wavelengths (Crippa,
Cristofoletti & Romeo, 1978; Menon et al., 1983).
Finally, it is important to realize that -electron-
containing molecules are also the primary targets of
UV radiation damage such as in unsaturated lipids,
nucleic acids and aromatic and indolic amino acid-
containing polypeptides. Thus, and perhaps
unsurprisingly, a common chemical theme lies at the
heart of UV radiation damage and screening
(Cockell, 1998b).
Many compounds that happen to contain
aromatic or conjugated groups may fortuitously
absorb UV radiation. Therefore, to be sure that a
compound produced by a cell either in sheaths or
intracellularly has a physiologically signicant UV-
screening function, something more concrete than a
purely structural denition is required. How do we
knowthat a compound has either specically evolved
to screen UV radiation, or at least provides some
constitutive physiologically signicant UV screening
for an organism?
IV. DETERMINING WHETHER A COMPOUND
HAS A SCREENING ROLE
In attempts to elucidate the true screening role of
cyanobacterial scytonemin and mycosporine-like
amino acids, F. Garcia-Pichel, R. W. Castenholz
and their co-workers made extensive considerations
of what criteria constitute a specic UV-screening
compound (e.g. Garcia-Pichel, Wingard &
Castenholz, 1993). From these studies a series of
rules was suggested for identifying a compound with
a UV-screening role. Some compounds that screen
UV radiation may not have an evolutionarily
dedicated screening role. However, their presence in
sucient quantities in a cell may provide some
constitutive physiologically signicant UV
screening. Considering this caveat, the basic
approaches for demonstrating the physiological
signicance of potential UV-screening compounds
are elaborated below.
(1) The compound must obviously screen UV
radiation. In most cases, a UV absorbance peak is
rst demonstrated by spectrophotometric analysis of
a cell extract from the organism. Methanolic
extractions (e.g. a 2 h extraction at 45 mC in 80%
aqueous methanol in the case of microbes, or a
20 min to 1 h extraction at room temperature in
75: 24: 1 methanol : water: HCl for ground plant
tissues) typically are used to extract and study water-
soluble compounds such as intracellular MAAs or
plant avonoids (e.g. Robberecht & Caldwell, 1978;
Scherer, Chen & Boger, 1988; Tevini, Braun &
Fieser, 1991; Garcia-Pichel et al., 1993). Acetone,
tetrahydrofuran, ethyl acetate and similar solvents
can be used to study lipid-soluble molecules such as
those found in the sheaths of cyanobacteria (e.g.
Garcia-Pichel & Castenholz, 1991). Methanol and
acetone extractions can initially be compared to
decide if the compound is an intracellular water-
soluble compound or whether it is synthesized in
membranes or sheaths. Once a potential compound
is found, it can be puried by a variety of techniques.
High-performance liquid chromatography (HPLC),
where the detector is set to the
max
of 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 rst step to
isolating the compound from other cell compounds
and conrming the existence of discrete
compound(s) with a maximal absorbance in the UV
range.
A number of techniques have been used to
determine the structure of compounds, but nuclear
magnetic resonance spectroscopy (NMR) has proven
to be a particularly useful technique (e.g. Guilford
et al., 1988; Proteau et al., 1993; Wilmesmeier,
Steuernagel & Wiermann, 1993).
(2) Inducibility of the compound in the living cell
by UV radiation provides strong evidence of a
specic UV-screening function. This criterion can be
tested by placing commercially available UV-A- or
-B-emitting lamps at dierent distances from the
organism under culture (e.g. Beggs et al., 1985;
Garcia-Pichel et al., 1993). Because some bulbs emit
a spike in the UV-C at 254 nm, care must be taken
in accurately dening the spectral emission. Cell
extracts are taken from these organisms after xed
time periods when the organisms have adapted to
the new UV radiation regime. The absorbance
value at the wavelength of maximum absorbance is
then measured. An increase in absorbance that
correlates with increasing UV radiation ux is
indicative of UV-induction of the compound. Strong
correlations such as the linear relationship between
UV-B radiation and induction of some plant
avonoids (e.g. Wellmann, 1975) may provide
particularly strong evidence for a UV-screening role.
As a further, more detailed test, the action spectrum
for induction (the relative induction at dierent
wavelengths that may be generated by lters or
monochromatic UV radiation sources) might be
similar to the compounds absorbance prole. Wave-
lengths of maximum absorbance of the compound
may cause maximum production of the compound
(e.g. Garcia-Pichel et al., 1993).
As alluded to before, although induction of a
compound by UV radiation may suggest a UV-
screening role, it is important to recognize that
absence of evidence does not constitute evidence of
absence. A constitutively expressed compound can
provide some physiological advantage. As discussed
in greater detail below, sporopollenins are
constitutively produced in algal cell walls and spore
walls, but have been observed to confer increased
UV tolerance in some species of algae (Xiong et al.,
1997), presumably caused by the aromatic structures
that they contain. Oat seedlings constitutively
produce avonoids that provide some advantage in
mitigating UV damage during early stages of growth
(Braun, 1991). Many cinnamoyl esters screen UV-B
in rye seedlings, but apparently are not specically
induced by increasing UV-B radiation (Tevini et al.,
1991). Melanin is produced constitutively in dark-
skinned people and provides some physiological
benet (van der Leun & de Gruijl, 1993). Thus, a
further test must be to prove that a compound
provides some physiologically signicant benet,
regardless of whether it is produced constitutively or
inducibly.
(3) A number of approaches can be used to
examine the ecacy of a compounds ability to
provide physiologically worthwhile screening. Cells
stained with 4h,6-diamidino-2-phenylindole (DAPI),
which binds to DNA, will uoresce in the blue region
at 461 nm when they are excited with UV light of
wavelength 326 nm. In the case of inducible com-
pounds, the uorescence can be compared to cells
that have been acclimated in the absence of UV
radiation or in low-light conditions and in which
compound concentrations are lower. Comparisons of
DAPI-uorescence values allow the intensity of UV
radiation reaching the nucleus of the cell to be
determined and thus the potential DNA damage
that is mitigated by the compound (e.g. Garcia-
Pichel et al., 1993). Techniques to determine if the
compound can reduce damage to photosystems in
photosynthetic organisms include measuring carbon
uptake rates with ["%C]bicarbonate or measuring
chlorophyll photobleaching by uorescence at
675 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 physiological
signicance of the compound (Xiong et al., 1996,
1997). Care must be taken since in the functioning
cell there may be other inter-species dierences in
repair processes and synthesis of alternative UV-
screening compounds. Optimally, mutants might be
generated for such studies in which the compound is
expressed at dierent levels. Given that many of the
pathways of UV-screening compounds are now
being elucidated (such as MAA production by the
shikimate acid pathway), the use of mutants to study
the physiological value of UV-screening compounds
is a promising area of development.
The screening ability of the compound is often
expressed as a screening factor. This value ranges
from 0 (no screening) to 1 (complete screening) (e.g.
Garcia-Pichel & Castenholz, 1993). Alternatively, it
can simply be expressed as a percentage absorbance.
This number provides a convenient indication of the
value of the compound to the organism. In the case
of sheath compounds, the screening factor will simply
be the complementary value of the transmittance of
the compound at a given wavelength. For intra-
cellular compounds the screening factor calculation
is more complex since the absorbance must be
integrated across the length of the cell. The end
proximal to the radiation will have a value of zero
and the distal end will have a value that depends
upon the size of the cell and the absorbance and
scattering of the compound at a given wavelength.
The absorbance can be either measured or calcu-
lated based on a given intracellular concentration
and a known extinction coecient using the Beer
Lambert law, although approaches that also take
into account scattering are likely to provide a more
accurate appraisal. Theoretical models can be used
to calculate screening ecacy (Garcia-Pichel, 1994).
(4) Because screening compounds are a passive
method of coping with UV radiation, proving that
enhanced survival under elevated UV radiation is
due to the compound may be complex. Induction of
other photoprotection mechanisms or physiological
responses including repair processes, may enhance
survival. Thus, as a further test, the compound
should provide increased resistance to UV radiation
when other physiological processes are not
functioning.
There are various methods for examining the
physiological value in cells lacking other functional
responses. Desiccation is such a method. For micro-
organisms, UV exposure during desiccation may be
followed by many of the techniques described under
point (3) above. For multicellular organisms such as
starsh that may contain MAAs, this approach may
be problematic, since the desiccation will clearly kill
a whole organism. In such cases, the experimenter
might concentrate on measurements of cellular
processes that do not depend upon a fully functioning
cell. The measurement of thymine dimer generation
in DNA or measurements of enzyme damage by an
appropriate assay may provide insights into the
protective ecacy of the compounds in isolated non-
functional cells.
V. SCYTONEMIN A POSITIVELY IDENTIFIED
UV-SCREENING COMPOUND
Na$ geli rst described the brown colouration of some
cyanobacteria and particularly cyanobacterial mats
(Na$ geli & Schwenderer, 1877). This colouration is
now ascribed to scytonemin, the rst compound to
receive rigorous application of the rules of screening.
It is now presumed to have a dedicated screening
role (e.g. Garcia-Pichel & Castenholz, 1991, 1993;
Garcia-Pichel et al., 1992). The sheaths of a wide
range of terrestrial cyanobacteria contain scyto-
nemin. Planktonic forms apparently do not contain
the compound, although a compound with similar
absorbance characteristics was reported from an
Icelandic phytoplankton bloom (designated P380)
(Llewellyn & Mantoura, 1997). It has been deter-
mined by NMR to be a dimeric molecule of
molecular weight 544 Da, made up of indolic and
phenolic subunits and formed from the condensation
of 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 of
scytonemin and the absorbance prole that provides
the rst indication of a screening role. Scytonemin is
mainly found in the green oxidized form, but it can
be reduced to a red form that in nature is found in
the lower reducing layers of cyanobacterial mats
(Proteau et al., 1993).
The induction of scytonemin is proportional to the
UV-A and visible light intensity. Light in the UV-A
region of the spectrum is particularly eective in
eliciting production (Garcia-Pichel & Castenholz,
1991; Garcia-Pichel et al., 1992; Ehling-Schulz,
Bilger & Scherer, 1997). Most cyanobacteria have a
minimum photon uence at which scytonemin
begins to be synthesized. It ranges from 99 mol m
#
s
" in Diplocodon sp. up to 250 mol m
# s
" in
Scytonema sp. Scytonemin-free cells are examined
with low uences (33 mol m
# s
") and compared
to cells at higher irradiances in which scytonemin has
been induced. Natural light levels in the eld have
been found to be correlated with scytonemin levels in
Rivulari sp. colonies (Pentecost, 1993) although a
negative correlation was found in Scytonema sp.
populations. This negative correlation has been
explained by the diering water availability in the
two sites that were studied and the diering cell
division rates (Pentecost, 1993). Faster dividing cells
often have less scytonemin because they are less
prone to damage that accumulates over longer
periods of time. When these factors were considered,
Pentecost (1993) concluded that the eld data
concur with the laboratory-deduced UV-protecting
role of scytonemin.
Scytonemin can provide quite eective screening
that ranges between 2 and 55% attenuation of light
at 320 nm at the single-cell level (Garcia-Pichel &
Castenholz, 1993). For cells under layers of mats or
inside colonies of cells, the attenuation of UV-A
radiation may be even more eective. The physio-
logical value of the compound has also been shown
(Garcia-Pichel & Castenholz, 1991; Garcia-Pichel et
al., 1992). Scytonemin can eectively reduce photo-
synthesis inhibition by UV-A radiation (measured
by oxygen evolution) and it can reduce photo-
bleaching of chlorophyll a. In a Chlorogloeopsis species,
uorescence of chlorophyll at 680 nm shows a strong
reduction when excited with UV-A wavelengths in
scytonemin-containing cells in comparison to those
lacking it (Garcia-Pichel et al., 1992). In cells with
scytonemin, the compound also allows for initially
faster growth rates when the cells are grown under
UV illumination. The screening role of scytonemin is
also eective during physiological inactivity such as
desiccation, proving that the passive process of UV
blocking is physiologically eective (Garcia-Pichel et
al., 1992).
VI. MYCOSPORINE-LIKE AMINO ACIDS AND
THE ECOLOGICAL COMPLICATIONS IN
STUDYING UV-SCREENING COMPOUNDS
Mycosporines were rst identied in fungi as having
a role in UV-induced sporulation (Leach, 1965;
Favre-Bonvin, Arpin & Brevard, 1976; Brook, 1981;
Young & Patterson, 1982). Their relatives, the
mycosporine-like amino acids, have since been found
in a wide variety of organisms as diverse as
cyanobacteria (Shibata, 1969; Sivalingam et al.,
1974b; Haxo et al., 1987; Karentz et al., 1991b;
Garcia-Pichel & Castenholz, 1993), red algae
(Sivalingam, Ikawa & Nisizawa, 1974a; Sivalingam
et al., 1974b; Takano et al., 1979; Tsujino et al., 1978;
Tsujino, Yabe & Sekikawa, 1980; Sekikawa et al.,
1986; Karentz et al., 1991b), dinoagellates (Balch
& Haxo, 1984; Carreto, DeMarco & Lutz, 1989;
Carreto et al., 1990), lichens (Karentz et al., 1991b;
Budel, Karsten & Garcia-Pichel, 1997), corals and
their 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 marine
invertebrates 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), starsh
(Nakamura, Kobayashi & Hirata, 1981, 1982), krill
(Nakamura et al., 1982), and vertebrates including a
species of Antarctic sh (Karentz et al., 1991b) and
sh eggs (Chioccara et al., 1980).
There are approx. 19 MAAs found in marine
organisms. Some are shown in Fig. 3. Their
ubiquitous presence across a large taxonomic range
and geographical range is evidence not only of their
early phylogenetic innovation, but potentially also of
their importance as natural UV-screening com-
pounds.
The basic cyclohexanone or cyclohexenimine
chromophore responsible for UV absorbance is
derived from the early stages of the shikimate
pathway (Favre-Bonvin et al., 1987). The subsequent
incorporation of the various amino acidic or imino-
alcohol groups results in the diversity of MAAs
found in nature. HPLC has proven to be a powerful
method for MAA analysis (e.g. Nakamura et al.,
1982). In a comprehensive study of Antarctic
organisms using HPLC, MAAs were found in a great
diversity of invertebrates and also a species of
Osteichthyes ice sh (Karentz et al., 1991b). In
antarctic organisms, a complement of up to eight
MAAs was found that might provide broad UV
screening. Many of the MAAs found (e.g. palythine,
porphyra-334; shinorine, mycosporine-glycine and
others) in these organisms are identical to those
found in tropical and temperate marine species.
An increase in concentration of MAAs associated
with increases in UV ux has been observed directly
in organisms in the eld. For example, the Great
Barrier Reef corals (Acropora spp.) at 20 m depth
showed signicantly lower concentrations of
mycosporine-glycine and palythine than those in
shallower waters (10 m) (Dunlap, Chalker &
Oliver, 1986). In the Antarctic, surface plankton
and invertebrates including krill (Euphausia superba)
were found to have twice the MAA concentration
compared to other non-surface species studied
(Karentz et al., 1991b). These data as well as the
depth data acquired by others (e.g. Gleason, 1993;
Shick et al., 1995; Dunlap & Shick, 1998 and
references therein) that broadly correlate depth of
UV penetration into water with MAA concentration
have been taken as evidence for a proposed ecological
role in UV protection.
Taking a similar approach to the study of
scytonemin, Garcia-Pichel & Castenholz (1993)
studied the screening role of cyanobacterial MAAs
that they found in 13 out of 20 strains. They found
that in the cyanobacterium Gloeocapsa sp. the
compounds met the requirements described in
Section IV for a UV-screening compound (Garcia-
Pichel et al., 1993). MAAs were inducible by adding
supplemental UV radiation at between 2 and 6
mol m
# s
", causing levels of compound to increase
between ve- and tenfold (Garcia-Pichel et al., 1993),
similar to the observed induction in the 13 other
cyanobacterial strains (Garcia-Pichel & Castenholz,
1993). The action spectrum for eliciting compounds
in Gloeocapsa sp. showed a peak at 320 nm, similar to
the absorbance prole for the compounds in this
species. Photobleaching of chlorophyll a measured as
the reduction of absorbance at 675 nm and photo-
synthesis inhibition measured as the dierence in
["%C]bicarbonate uptake showed that in the
desiccated state MAAs do provide a physiologically
signicant benet to the cells. Furthermore, re-
duction of blue-uorescence of DAPI-labelled cells
near 320 nm suggests that MAAs may also provide
signicant screening to DNA. Attenuation of be-
tween 10 and 26% of light at 320 nm has been
recorded across 20 strains of cyanobacteria (Garcia-
Pichel & Castenholz, 1993). For terrestrial cyano-
bacteria, the attenuation in the UV-A region may be
combined with that provided by scytonemin, to
provide a total screening up to 60% at 320 nm for
single cells.
Induction of MAAs has also been observed in
dinoagellates, including the production of up to
seven MAAs in Alexandrium excavatum (e.g. Carreto et
al., 1990) in response to high light intensity, and the
induction of MAAs in Heterocapsa triquetra in response
to 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 cyanobacterium
Nostoc commune inhabits extreme arid and polar
regions and grows as exposed mats on soils. The
water-soluble mycosporines, that have absorption
maxima at 312 and 335 nm (Bohm et al., 1985), are
produced extracellularly and are attached to the
polysaccharide matrix by dierent amino acids (Fig.
3). They may account for between 7 and 10% of the
organisms dry mass. This is considerably more than
intracellular MAAs, that typically range from
between 0n16 and 0n84% of dry mass. Taking into
account the lower extinction coecient (approx. 800
mol
" l cm
") compared to most MAAs (approx.
24000 mol
" l cm
"), Garcia-Pichel and Castenholz
(1993) calculate that the screening ecacy of Nostoc
commune compound is very similar to MAAs. They
may provide a sunscreen factor of 0n7 for these
organisms (Bohm et al., 1995). Like other com-
pounds, the Nostoc commune MAAs are induced by
UV-B radiation, as is the polysaccharide core itself
(Ehling-Schulz et al., 1997). An articial source of
UV-B radiation may increase compound concen-
trations by ve times or more. The compound does
not seem to be produced by other cyanobacterial
species and may be unique to Nostoc spp.
In many marine invertebrates and vertebrates,
the de novo synthesis of MAAs is still in dispute and a
dietary origin from grazing on algae now seems
probable (e.g. Shick, Dunlap & Larson, 1994;
Stochaj et al., 1994; Carroll & Shick, 1996) with
internal modication of some of these compounds
(Shick et al., 1992; Dunlap & Shick, 1998). The
shikimate acid pathway is not found in animals and
so it is unlikely that it is biochemically possible for
MAAs to be produced in animal tissues. Although
some accumulation of mycosporine-glycine: valine
was noted in intertidal Antarctic invertebrate
species, which was not found at lower trophic levels
(Karentz et al., 1991b), it is possible that very low
concentrations of this MAA may exist in some algal
species consumed by invertebrates or that they are
modied after consumption. The picture may be
further complicated by the dierential synthesis of
MAAs according to environmental changes. In the
red-tide dinoagellate Alexandrium excavatum, the
relative proportions of dierent MAAs may alter
rather quickly upon changes in light concentration
(Carreto et al., 1990), which might potentially alter
the dietary intake by higher trophic levels.
Continued studies of the synthesis, interconversion
and structural modication of these types of com-
pounds in selected ecosystems will undoubtedly
address many aspects of these questions.
The evolutionary signicance of trophic-level
accumulation is uncertain. It is tempting to speculate
that the shikimate acid pathway was lost in the
earliest multicellular animals during the Cambrian
and that trophic-level accumulation of UV-
screening MAAs from algae was the predominant
means of enhancing UV screening. Organisms that
had improved metabolic processing and distribution
of MAAs to important biological sites would have
had some selective advantage, particularly in clear
waters, although why the pathway should have been
lost in animals is uncertain.
Although there is good laboratory and eld
evidence that MAAs can provide screening, they are
also a good example of the complexities that can
exist with examining the role of UV-screening
compounds in protecting natural communities. They
especially underline the need to study these com-
pounds in relation to other UV-mitigation methods
as well as other physiological functions of the
compound itself. There exist two complications that
may arise in examining the prevalence of compounds
in nature and their correlations with UV exposure.
Firstly, UV repair processes, the quenching of
reactive oxygen species by carotenoids and photo-
taxis may provide the required UV radiation
response that may supplement or even negate the
need for UV-screening compounds in some species.
In Gloeocapsa sp., for example, cells were able to
acclimate to articial increases in UV radiation
prior to the maximal build-up of MAAs (Garcia-
Pichel et al., 1993). This suggests that other processes
may also be important in mediating the immediate
UV acclimation response in this organism, perhaps
repair processes. In a survey of cyanobacteria
undertaken by the same authors, some Oscillatoria
species that display a phototactic response to high
light and UV radiation intensities had a conspicuous
absence of MAAs. They may live in UV-exposed
habitats where non-phototactic cyanobacteria are
found such as Gloeocapsa spp. or Calothrix spp. that do
possess MAAs (Garcia-Pichel & Castenholz, 1993).
Simple morphology of some organisms, particu-
larly chained diatoms such as Thalassiosira spp. and
Chaetoceros spp., may cause an order of magnitude
dierence in intracellular UV exposure and thus
DNA damage (Karentz et al., 1991a). In a study of
a diversity of Antarctic diatoms including Proboscia
spp. and Nitzschia spp., screening by the cell material
including the frustrule was found to be greater than
the protection provided by the UV-screening com-
pounds (Davidson et al., 1994; Davidson &
Marchant, 1994). In some of these species, although
an MAA peak was observed, it was not found to be
UV-B inducible. Furthermore, in the species studied,
UV-B tolerance was actually found to be higher
than in the Antarctic spring-blooming
prymnesiophyte Phaeocystis pouchettii that does pro-
duce high concentrations of UV-B-inducible com-
pounds (Marchant, Davidson & Kelly, 1991).
This type of screening compound versus other
response data demonstrates the importance of eluci-
dating the complete range of UV responses in a
given organism. Thus, although wide surveys of UV-
screening compounds in nature can be of initial
value, interpretation of the data in the ecological
context must be undertaken with great care.
The second complication is that the compounds
may have other physiological functions as well as
UV screening. An example are cyanobacteria in
some high-salt environments, where MAAs, with an
internal cell concentration of 98 mmol l
", have been
found to be produced in response to increasing salt
concentrations. In this case, they are presumed to
have a role in balancing the osmotic pressure inside
the cells to counteract water loss (Oren, 1997). As
well as enhancing UV screening, plant avonoids
have proposed roles in ower recognition by insects,
pollen development and sexual reproduction, plant
microbe interactions including nitrogen xation,
interactions with pathogens and parasitic plants and
an involvement in the formation of plant structural
compounds in seeds and bark (Koes, Quattrocchio
& Mol, 1994). A truly rigorous correlation of the
existence of UV-screening compounds to UV ex-
posures and habitats requires that the physiological
functions of a compound are understood and that
the relative importance of these functions at dierent
developmental stages and in dierent habitats and
environments is known.
An excellent example of these problems is illus-
trated by a study of the presence of MAAs in the
lenses of sh eyes (Dunlap et al., 1989). The focus of
the eye is accomplished by the cornea and lens,
which transmit light through to the retina. These
tissues therefore have a role in screening UV
radiation before it damages the retina. UV-A-
absorbing compounds have been identied in the
lens tissues of marine sh (Zigman, Paxia &
Waldron, 1985; Zigman, 1987). Believing these to
be MAAs, Dunlap et al. (1989) extracted the
compounds from a wide diversity of sh lenses. A
diversity of well-described MAAs were found, in-
cluding palythine, palythene, asterina-330 and
palythinol, although some were noticeably absent
such as mycosporine-glycine. The patterns and
concentrations of MAAs showed no specic
behavioural or taxonomic trends. Indeed, in two
diurnal surface feeders (Spanish mackerel,
Scomberomerus commerson and mackerel tuna, Euthynnus
anis), MAA concentrations were found to be more
than three orders of magnitude lower than in some
other species such as coral trout, Plectropomus leopardus
and green job-sh, Aprion virescens that feed through-
out the water column. In the Scaridae (Parrotsh),
the concentrations of palythene are so high that the
eyes have a yellow colour.
Although the authors suggest that a more robust
light gradient might be used in future studies to
examine these relationships further, they also allude
to several factors that may explain their results.
Filtering out short-wavelength light may improve
the visual acuity of some species of sh by reducing
chromatic aberration (Muntz, 1973) and this may
account for the high concentrations of some MAAs,
such as palythene in parrotsh eyes (
max
l360 nm).
Some species of sh exhibit tetrachromatic colour
vision 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 sh,
UV-A is important for visual acuity. In these cases,
there may be a requirement for lower concentrations
of MAAs.
As the authors concede at the end of their
discussion an understanding of the functional
signicance of these compounds in sh eyes will
require a more complete understanding of the
structure and function of many, presumably inter-
acting, factors aecting vision in shes .
VII. UV SCREENING IN PLANTS
Like many phototrophic micro-organisms, higher
plants require an exposure to solar radiation for their
way of life. Phenylpropanoids including avones,
avonols, cinnamoyl esters and anthocyanins pro-
vide a UV-A and B screen. The avonoids are today
the most widely represented phenolic derivatives in
the biosphere (Harborne, 1964). Flavonoids provide
an eective UVscreen that can reduce transmittance
of UV radiation through the epidermis, but allow
through 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 now
well established that avonoids 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 between
avonoid concentration and UV-B ux has been
observed (Wellmann, 1975). Flavonoids can also be
UV-A inducible, as has been demonstrated in Cistus
laurifolius (Vogt et al., 1991). The avonoids respon-
sible for UV screening may vary from species to
species and according to developmental stage and
tissues. Although single avonoids may screen in
both the UV-A and UV-B region, most plants
synthesize a range of compounds that may provide a
more eective screen. For example, in rye, Secale
cereale, seedlings isovitexin arabinoside and isovitexin
galactoside were the two principal compounds
induced by UV-B radiation (Tevini et al., 1991).
Other enzymes and products of the avonoid
biosynthesis pathway have also been observed to be
induced by changes in the incident UV ux (e.g.
Hadwiger & Schwochau, 1971; Lyndon, Teramura
& Coman, 1987; Zangerl & Berenbaum, 1987;
Braun & Tevini, 1993), thus demonstrating a
complex UV response that may be regulated at
multiple levels. In legumes, this response has also
been shown to be linked through isoavonoids to the
repair of UV-induced DNA damage, particularly
thymine dimer formation (Beggs et al., 1985). The
increasing complexity of the UV-avonoid response
in phylogenetically more recent plants (Staord,
1991; Koes et al., 1994) suggests a common ancestral
response to UV radiation that over time and as a
result of UV selection pressure, has resulted in a
variety of biochemical innovations and links to other
physiological responses.
Flavonoids are principally deposited in the epi-
dermal layers of the leaf (Robberecht & Caldwell,
1978; Flint et al., 1985), although in some cases they
are also found in the epicuticular waxes (Vogt et al.,
1991) and in the deeper mesophyll layers of the
leaves (Weissenbock, Plesser & Trinks, 1976;
Robberecht & Caldwell, 1978). They may also be
found in leaf hairs (Karabourniotis et al., 1992). The
pigments are usually localized to the vacuoles of
epidermal cells (McClure, 1975), although they
have also been reported in the cell wall of epidermal
cells in some conifers (see Day, 1993 and references
therein). Although vacuole localization can provide
the necessary screening, some ineciency undoubt-
edly occurs with light that passes between the cell
walls. The multicellular localization of avonoids
illustrates the rst important dierence between
plants and micro-organisms. As will be discussed in
more detail in Section IX, multicellular distribution
of UV-screens greatly enhances the possibility of
attenuation. Whereas a single-celled cyanobacteria
may screen 60% of UV radiation at 320 nm with a
combination of MAAs and scytonemin (Garcia-
Pichel & Castenholz, 1993), plants generally reduce
UV at this wavelength by more than 90% before it
reaches the mesophyll (Robberecht, Caldwell &
Billings, 1980).
Plants in equatorial and high-altitude regions of
the earth, where UV-B ux is generally higher,
demonstrate the capacity of increased UV-B tol-
erance by inducible avonoid production. Most low-
latitude species exhibit a lower epidermal trans-
mittance (less than 2%) compared to more tem-
perate and higher latitude species that generally
exceed 5% (Robberecht et al., 1980). The species
found at low latitudes also include temperate-
latitude species that have been introduced into these
regions such as the pea, Pisum sativum, further
demonstrating the capacity for photobiological ad-
aptation. Since the unweighted UV-B ux is an order
of magnitude greater in low-latitude regions com-
pared to high-latitude regions, these ranges provide
some evidence of the acclimation capacity of plants
and their ability to change avonoid composition in
new UV regimes.
In an analogous way to the non-specic at-
tenuation of UV-B radiation in diatoms by their
morphological features, the morphological charac-
teristics of plants or the physical properties of their
leaves may provide a signicant contribution to UV
reduction. Approximately 5% of the UV-B region of
the spectrum is non-specically reected away by
the epidermal layer. However, in some plants with
dense pubescence (Robberecht et al., 1980) or a
glaucous surface (Mulroy, 1979), UV reectance
may be as high as 2070% (Caldwell et al., 1983).
The epidermal cell structure can in some cases
provide reductions in UV by non-specic scattering.
Robberecht and Caldwell (1978) reported that in 24
plant species, a number of them had signicant
attenuation due to epidermal structure alone. In 16
species studied, they reported that attenuation by
UV-screening pigments was between 20 and 57% of
the total UV attenuation, the rest being accounted
for by epidermal structure. These results are slightly
open to question since the epidermis were extracted
in acidied methanol for only 15 min. Using similar
procedures on desert cacti of the genus Opuntia,
epidermal transmittance was found to be increased
by only 1050% (C. S. Cockell, unpublished ob-
servations). However, overnight extraction of the
epidermis, with four separate extractions, was
found to reduce the concentration of intracellular
avonoids such that epidermal transmittance was
increased by between 80 and 90%, suggesting
that the contribution of epidermal structure to
UV-B attenuation may be greatly overestimated if
sucient extraction periods are not allowed.
Anthocyanins are less ecient absorbers of UV
radiation since their absorbance maximum is gener-
ally near 520 nm, but the tail of their absorbance
prole may extend into the UV-A region (Strack &
Wray, 1989). As well as screening in leaves, they can
contribute to the absorbance of ower corollas
(petals), being responsible for ower colour and
ower recognition by pollinators. They may con-
tribute to the protection of pollen by helping to
mitigate UV-A penetration into the anthers.
Together with the absorbance provided by
avonoids, incident UV-A and B radiation within
anthers is reduced by 98% (Flint & Caldwell, 1983).
Anthocyanins are induced by UV radiation below
350 nm (Wellmann, Hrazdina & Grisebach, 1976;
Beggs & Wellmann, 1985) and their induction may
also depend on a complex interaction with the
phytochrome system (Beggs & Wellmann, 1985).
As well as avonoids, other aromatic-containing
higher plant pigments such as alkaloids absorb in the
UV range. There is a negative correlation between
alkaloid-bearing plants and latitude (Levin, 1976)
and although alkaloids possess other functions such
as deterrents against grazing, it is also plausible they
may provide additional UV protection in some
species.
VIII. UV SCREENING AND MELANIN
The animal melanins or eumelanins as they are
more correctly dened are found in eyes, feathers,
insect cuticles, reptile skin, and the skin of a wide
variety of mammals. They are found in particularly
high concentration in the inksac of the cuttlesh
Sepia ocianalis (Prota, 1988) and this melanin has
been used as a standard for melanin studies. The
focus on melanins and their role in photoprotection
in humans has brought them particular attention
since the inverse correlation between skin cancer and
melanin production has been suggested (Scotto &
Fraummeni, 1982). Thus, the literature on human
melanin, the pigmentary system and its induction is
very substantial (e.g. Nordlund & Boissy, 1998).
Here, we focus only on the screening properties of
these pigments and their evolutionary signicance.
Melanins are produced by the enzymatic oxi-
dation of tyrosine by tyrosinase and then the
subsequent conversion of dopa to 5,6-dihydroxy-
indole. This is a phenolic and indolic compound and
the basic building block of the eumelanin polymeric
structure. This structure acts as the UV-absorbing
chromophore.
Because of the complexity of eumelanins, and thus
the diversity of absorbance maxima of the to *
transitions represented in the molecules, there is no
specic absorbance prole or maximum. However,
they absorb increasingly strongly into the UV-B
region of the spectrum (Fig. 6) (Crippa et al., 1978;
Menon et al., 1983; Ravishankar, Muruganandam
& Suryanarayanan, 1995). Their dark colour results
from some absorbance in visible wavelengths. For
many melanins, the plot of log[absorbance] against
wavelength gives a linear relationship (Spiegel-
Adolf, 1937). The erythemal dose response or
suntanning eect induced by UV radiation shows an
increase in the UV-B portion of the spectrum, that is
consistent with the screening function of melanin.
Melanins are formed in melanocytes in specic
organelles termed melanosomes which in humans
have a melanin content of between 17n9 and 72n3%
(Duchon, Borovansky & Hach, 1973). The melanin
often forms in caps above the nucleus that
presumably provide the most ecient UV protection
(Gilchrest et al., 1996). Melanocytes are found
between the epidermis and dermis in the skins basal
cell layer, the principal site of UV-induced pig-
mentation (e.g. Friedmann & Gilchrest, 1987).
Here, melanin is visualized as agglomerates (which
are essentially melanosomes). Agglomerates are
collections of melanin aggregates, themselves formed
from melanin particles that are 30 nm sized
particles of melanin polymers. Melanin particles are
found in the stratum corneum near the surface of the
skin and in the epidermis and are also known as
melanin dust (Holbrook & Wul, 1987). The
accumulated eect of the melanin to be found in
dierent skin layers (total depth approx. 250 m)
means that incident radiation is quite quickly
attenuated through the stratum corneum and epi-
dermis. White skin transmits approx. 2050% of
incident radiation at 300 nm through the epidermis
and black skin approx. 210% (Everett et al., 1966;
Kaidbey et al., 1979).
Melanin provides a ne example of the physio-
logical value of both inducible and constitutive
compounds. In white populations melanin induction
is an important response to UV irradiation. How-
ever, in many Asian and African populations, black
skin represents a screen that is predominately
provided by constitutive production of melanin. In
the latter case, although the melanin response is not
necessarily correlated with UV uence, the screen is
certainly of physiological importance during UV
exposure (see below).
The response to UV irradiation is complex. As
well as de novo melanin production, which appears to
be mediated through the existing tyrosinase pool,
responses that may occur over the longer term may
include increases in the numbers of melanocytes and
melanosome formation. Thickening of the stratum
corneum and epidermis may also occur. This
increases skin folding and increases UV attenuation
by enhancing scattering, reection and the surface
area of absorption. It is part of the longer term
photoageing response (Kollias et al., 1991). Although
it is inducible, in some ways it is analogous to the
reductions in UV-penetration achieved by morpho-
logical and scattering eects in plants and micro-
organisms, independent of the concentration of
screening compounds. The photoageing response
means that each successive UV exposure must be
higher in order to induce the same tanning eect
(Kollias et al., 1991).
The evolutionary importance of melanins is less
clear. UV-screening compounds in micro-organisms
have a clear role since they may reduce photo-
synthesis inhibition and other detrimental eects of
UV radiation such as DNA damage. For a single-
celled organism this can be important, since damage
to the cell may be lethal. It has previously been
suggested that sunburn is probably not an evolu-
tionary selection pressure (Blum, 1961). However,
for any animals that live in exposed regions for
extended periods, particularly for hunting and
feeding, photoprotection in the skin layers may be
important. The probability of skin cancer or other
longer term negative photoageing eects occurring
before breeding age may be increased without some
form of photoprotection. Photoprotection may par-
ticularly have been important in moving from forest
regions into savannas and other open spaces during
periods of drought or reduced food availability.
Burnt skin may also act as a site of microbial
infection, a place for ies and other winged insects to
lay eggs and as a site of irritation which may
exacerbate the infection risk. Thus, it is possible to
envisage that melanin does provide tness value. In
humans, the incidence of non-melanoma skin cancer
is 10100 times higher in non-pigmented human
populations compared to pigmented human popu-
lations (van er Leun & de Gruijl, 1993). The
sunburn eect in white Caucasians over short periods
of exposure before the melanin adaptive response
occurs is ample evidence of the potential damage
that UV radiation might cause in any exposed skin
on an animal without an eective photoprotective
strategy.
IX. OTHER CANDIDATE UV-SCREENING
COMPOUNDS
Our understanding of the range and characteristics
of UV-screening compounds represented in the
biosphere is still in its infancy. The wide distribution
of some compounds such as MAAs in marine
invertebrates and many micro-organisms, avonoids
in higher plants, and melanins in many animals
suggests that a large number of compounds will
probably be found to share common characteristics,
falling broadly into well-dened groups. However,
some may be represented in only one or a few species
if they are more recent innovations. Here, some
other compounds are discussed.
(1) Carotenoids
The role of carotenoids in UV screening is still
somewhat controversial. Their role in oxygen free-
radical quenching and thus indirect photoprotection
fromboth UV and high visible light induced damage
has been established for some time (Krinsky, 1979;
Demmig-Adams and Adams, 1992) and it is known
that in many cases they are UV-inducible. For
example, in Nostoc commune, concentrations of the
outer-membrane carotenoids echinenone and myxo-
xanthophyll were increased by approx. 4050%
after 5 h exposure to 1 Wm
# UV-B compared to
controls (Ehling-Sculz et al., 1997).
In an analogous way to the higher trophic level
accumulation of the UV-screening MAAs in marine
organisms, carotenoids are synthesized in algae,
bacteria and plants de novo and acquired in the diet
by higher trophic levels such as insects (Britton &
Goodwin, 1981).
A diversity of experiments have not demonstrated
signicant screening advantages conferred by caro-
tenoids. Under UV-B irradiation, no dierences in
competitiveness were found in strains of the fungus
Phycomyces blakesleeanus, regardless of carotenoid
concentrations or dierences 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, since
it is these compounds that result in the highly
coloured (red or pink) natural communities. A role
in photoreactivation was ascribed for the caro-
tenoids, but no passive UV screening could be
observed in pigmented versus unpigmented strains of
the halophiles Halobacterium cutirubrum and Halo-
bacterium salinarium (Sharma, Hepburn & Fitt, 1984).
In a melanin-decient mutant of the fungus
Wangiella dermatitidus, the carotenoids torulene and
torularhodin did provide some UV protection (Geis
& Szaniszlo, 1984), but in the wild-type cells,
melanin was found to provide the most important
UV protection.
The controversial nature of carotenoid involve-
ment in passive UV screening is an interesting
research problem since, as alluded to in Section II,
from a chemical point of view they should make
eective screens. It is unsurprising that many of the
longer carotenoids important in photosynthesis and
photochemical quenching are not UV screens since
the minimum number of carbon double bonds
required to provide quenching of singlet oxygen
appears to be nine (Krinsky, 1971). Molecules with
this number of bonds and more tend to screen in the
visible region of the spectrum. However, linear
conjugated molecules generally have higher ab-
sorption coecients than aromatics. It is surprising
that compounds such as phytoene, that screens at
340 nm and has three conjugated double bonds, as
well as shorter molecules that could screen at lower
wavelengths have not become more important. The
ability to synthesize such compounds at sucient
screening concentrations is possible, since many
long-chain carotenoids are synthesized at concen-
trations of up to 2000 g g
" dry mass (e.g.
Cerda-Olmedo et al., 1996) which is similar, and in
some cases greater, than the intracellular concen-
trations associated with say, MAAs, which are
synthesized at intracellular concentrations ranging
from 4 to 2903 g g
" dry mass (Karentz et al.,
1991b).
The original role of carotenoids is not known.
Their ability to transfer energy means they are
important in photosynthetic light reactions and they
may initially have had a primary role in photo-
synthesis. Their role in quenching reactive oxygen
species could have evolved during the increase in
atmospheric oxygen partial pressures in the early
Proterozoic (Krinsky, 1971) or perhaps earlier in
phototrophic organisms producing oxygen in their
micro-environments. Thus, they may have taken on
a photoprotective role as an additional function in
the earliest oxygenic photosynthesizers. The meta-
bolic products of carotenoids have been postulated
to be involved in the synthesis of sporopollenin and
other macromolecules (Krinsky, 1971). It is possible
that in the earliest stages they may have been
intermediates in other biosynthetic pathways, later
becoming used in light-related reactions. Thus,
providing some phylogenetic reason for the lack of
the use of carotenoids as passive screens is dicult.
Furthermore, at later stages in evolution, when they
could have been selected again, aromatics were
preferentially selected as UV screens, for example
avonoids in plants and melanins in animals.
The relative unreactiveness of ringed aromatics
compared to linear, more energetically accessible
molecules may be a reason why aromatics are
usually found as UV-screens. A passive UV screen
should preferably have minimum interference with
other cell biochemistry, particularly if it is constantly
produced intracellularly. The role of other linear
conjugated structures in passive or constitutive UV
screening 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 that
absorb strongly in the UV-B region (Marchant et al.,
1991). The colonial form is one of two life stages of
this organism whereby the cells are not free
swimming, but oat in the water column, embedded
in mucilage. UV protection might be expected to be
important. The compounds produced absorb
strongly at 323 nm, with two other peaks at 271 and
211 nm, the latter two being physiologically ir-
relevant since they are in the UV-C. Their levels of
production correlate with total UV-B irradiance.
With these compounds Antarctic colonial Phaeocystis
pouchetii may tolerate levels of UV-B up to
0n32 Wm
#. In the motile life stage (or in the colonial
form of East Australian Current strains of Phaeocystis
pouchetii, that do not produce as many compounds
and cannot increase concentrations in response to
UV-B), loss of viability was signicantly faster. This
provides circumstantial evidence of the tness value
of these compounds, although better repair processes
cannot be ruled out (some Antarctic diatoms, which
may have more ecacious repair processes, may not
require UV-screening compounds to reach higher
levels of UV tolerance). The compounds are
extruded into the water column and are rapidly
degraded by bacteria, suggesting they may be quite
short-lived. Their presence in the water column may
provide some fortuitous protection to other
organisms in the vicinity of the blooms. It is possible
that these compounds are MAAs or at least related
to MAAs. Other UV-absorbing compounds have
also been found in the water column near phyto-
plankton blooms. P380 was found in the surface
waters near an Icelandic phytoplankton bloom
(Llewellyn & Mantoura, 1997). It has a broad
absorbance from 300 to 470 nm and screens maxi-
mally between 380 and 383 nm.
Many lichens also have a diversity of polyphenolic
compounds including usnic acid and parietine that
absorb strongly in the UV region and may be
involved 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 screening
compound (Budel et al., 1997).
As well as novel compounds in single-celled micro-
organisms, many multicellular organisms may have
novel screening compounds. Gadusol and its related
compounds are structurally related to the myco-
sporines (Fig. 3) and have been found in the roes of
cod Gadus morhua (Grant and Plack, 1980; Plack et
al., 1981) as well as in the eggs of Mediterranean sh
(Chioccara et al., 1980) and brine shrimp (Grant et
al., 1985). A role in UV protection has yet to be
determined. The high concentration of gadusol
which may be about 0n3% of average dry mass in
exposed Scottish cod roes ; (Plack et al., 1981) makes
the role of these compounds in UV protection likely.
Sporopollenin is a compound found in many algae
and also in the cell walls of pollen and spores
(Atkinson, Gunning & John, 1972; Guilford et al.,
1988; Delwiche, Graham & Thomson, 1989; Xiong
et al., 1996, 1997). It is an acetolysis-resistant
biopolymer made up of a diversity of aromatic and
aliphatic residues (Meuter-Gerhards, Schwerdtfeger
& Steuernagel, 1995). It does not appear to be
induced specically by UV radiation and may have
a function in antimicrobial activity. However, tol-
erance of some algae such as Scenedesmus spp. and an
Enallax sp. to UV-B radiation was correlated with
cell wall concentration of sporopollenin (Xiong et
al., 1997). The compound does not have a specic
absorbance peak. Like melanin, its complex poly-
meric structure generates a rather non-specically
increasing UV absorbance towards shorter wave-
lengths. It may therefore act as a constitutive UV
screen, a screen that may be further enhanced by
UV-induced synthesis of MAAs (Xiong et al., 1997).
Sporopollenin is a good example of the fact that any
aromatic or conjugated bond-containing structures
present in the cell wall or tissues of an organism may
potentially provide some UV screening, exacer-
bating the problem in identifying those molecules
that have evolved specically to screen UV radi-
ation. It is possible that some constitutive UV
screening might occur for other cytoskeleton-type
structures that contain aromatic amino-acids and
surround the cell wall, for example the chaperonins
that have been proposed as an archeal cytoskeleton
(e.g. Trent et al., 1997).
X. UV-SCREENING COMPOUNDS AND
EVOLUTION
Our knowledge of the specic biochemical and
physiological details of a diversity of UV-screening
compounds, which has been addressed in previous
sections, can provide us with a basis to consider some
evolutionary questions. The role of UV radiation in
evolution can be split broadly into four divisions.
Firstly, UV radiation on pre-biotic earth. Secondly,
UV radiation on Archean earth prior to the
formation of the ozone shield, but when microbial
communities existed. Thirdly, UV radiation during
the early Proterozoic period when atmospheric
oxygen partial pressures rose as a result of microbial
photosynthetic activity and geologic changes, thus
resulting in the formation of the ozone column and
fourthly, changes in UV radiation ux caused by
natural ozone depletion events since that time. In
this 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 that
occurred when atmospheric oxygen concentrations
increased during the early Proterozoic approx.
2n1 Ga, the UV-C and UV-B radiation ux may
have been much higher on the surface of the earth
than it is today. This would have corresponded to
the time between the formation of the earth 4n8 Ga
and approx. 2n0 Ga (Kasting & Donahue, 1980;
Kasting, 1987, 1993; Francois & Gerard, 1988).
Without such screening, the biologically eective
irradiance to free-DNA would have been approxi-
mately three orders of magnitude higher than
experienced on earth today (Cockell, 1998a). These
uxes would have been signicant on prebiotic earth
since they may have photolytically destroyed com-
plex organic structures. However, there are many
ways in which UV radiation may have been
attenuated.
Other atmospheric attenuators may have existed
in the early atmosphere. Atmospheric attenuation of
UV radiation by high concentrations of sulphur or
H
#
S has previously been suggested (Sagan, 1973;
Kasting et al., 1989). Prebiotic evolution in the deep
ocean or subsurface earth may also have allowed life
to evolve well before it radiated onto the surface of a
UV-exposed earth, by which time it may have
developed damage responses.
However, if UV ux was much higher than today
and some pre-biological reactions occurred in po-
tentially exposed regions such as in water bodies of
early cratons and land-masses or in the oceans, then
screening may have been important to reduce UV-
induced destruction of complex organic molecules
(Cleaves & Miller, 1998). Shallow water is not a
very good attenuator of UV radiation and even in
sea water, the salts that produce UV-attenuating
bromide or nitrate can only produce highly eective
absorption of UV radiation below 220 nm in surface
layers (Ogura & Hanya, 1966). UV-B radiation
may penetrate tens of metres into clear water (Smith
et al., 1992). The possibility that organic molecules
may have acted as a UV screen in the aqueous
environments of early earth was rst considered by
Sagan (1973). Many cyclic or conjugated organic
molecules, could potentially have been important,
although without land plants, dissolved organic
carbon (today provided predominantly by humic
substances) would have been at much lower con-
centrations. Signicant concentrations of hydrogen
cyanide (HCN) polymer may have existed in the
oceans of prebiotic earth, formed from cyanide
polymerizations. HCN polymer may have provided
attenuation of wavelengths below 260 nm (Cleaves
& Miller, 1998). In combination with other UV
absorbers such as HS
or reduced iron, UV
penetration into earths early oceans could have
been substantially reduced. Signicant delivery of
aromatic compounds and organics by a higher
impact ux on early earth and by interstellar dust
particles (Chyba & Sagan, 1992) may also have
provided some UV-screening organics. Specically,
polycyclic aromatic hydrocarbons (PAHs) may have
provided some type of UV screen as they will absorb
in the UV region (M. Bernstein and T. Halasinski,
personal communication). In carbonaceous chond-
ritic meteorites they represent 90% of the organic
material (Deamer, Mahon & Bosco, 1994 and
references therein). Thus, they may have been an
important part of the prebiotic organic inventory.
Although nding specic UV absorbers amongst
this diversity of prebiotic possibilities is an interesting
avenue of research, it must be remembered that the
to * electron transitions in many small conjugated
organic structures will screen in the UV-C region
and in many, where the molecules are long enough
or substituent groups of the molecule of the ap-
propriate structure, UV-B absorbance may occur as
well (see Section III). Any surfaces or small water
bodies in which a build-up of even a thin layer of
organically enriched material containing conjugated
aliphatic or aromatic compounds occurred could
potentially have provided UV screening for pre-
biotic aqueous reactions in exposed regions of the
earth (Cockell, 1998b). If we assume a molecular
weight of approx. 160 Da for a typical small UV-C
or UV-B absorber (the typical molecular weight of a
UV-absorbing linear molecule or a three-ringed
aromatic structure which is typical for many PAHs
found in carbonaceous meteorites) and an average
extinction coecient of 10000 mol
" l cm
" across
the UV range (which is at the lower end for a small
linear conjugated molecule, but the upper end for
some aromatics), then it is apparent that even at
a concentration of 16n5 g cm
" ; such compounds
could provide a 90% reduction of incident UV
radiation.
A particular problem for prebiotic earth is the
high UV absorbance of the nucleic acids. In the
early period of the Archean, it is supposed that the
surface of earth was host to an RNA world in
which RNA catalysis, probably enveloped by early
amphiphilic membranes, presented the rst type of
cell-like structures (Lazcano, 1994). During this
stage the high UV-C and UV-B absorbance of
nucleic acids might have made reproducibility of
RNA replication a biochemical challenge unless
these molecules were shielded. Kolb, Dworkin &
Miller (1994), in attempting to reconcile the need for
a pre-RNA molecular order, suggest that candidate
early precursors to RNA (the ve-membered urazole
and guanazole) would also have been resistant to
UV damage since they are transparent above
220 nm. Whilst this is certainly true in the earliest
stages of the RNA world, the ozone shield did not
form until approx. 2 billion years ago. It does not
negate the requirement for nucleic acid shielding on
prebiotic earth once the RNA world did come into
existence. Unsaturated amphiphilic membrane
molecules may have provided non-specic screening
and scattering of UV-Cand UV-B radiation through
conjugation, thus reducing the biologically eective
irradiances to early encapsulated nucleic acids and
helping to increase the reproducibility of the genetic
machinery. One might imagine a selection pressure
for more UV-absorbing unsaturated phospholipids
in early membrane structures. However, until single-
celled organisms evolved that had eective repair
processes or could enhance UV screening by the
specic production of UV-screening compounds,
then it is likely that much of importance in the
prebiotic world occurred in regions that were
shielded or at least under the fortuitous build-up of
organic molecules or inorganic compounds that
provided 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 methods
Iron compounds Many mat-forming organisms
(Pierson et al., 1993)
Provides specic UV absorption
Sulphur Thermophilic mats and organisms
near hydrothermal regions (Cockell,
1998b)
Specic UV absorption
Solid NaCl Evaporites\halophiles (Rothschild,
1990)
Specic UV absorption
Water column Oceanic and freshwater communities
(Sagan, 1973; Margulis et al., 1976)
Not very eective attenuation
without impurities such as iron or
dissolved organics. Trade-o
problem between UV penetration
and euphotic zone
Rock\sand\soil\snow (particularly
with impurities)\calcium
carbonate\ gypsum (calcium
sulphate)
Endolithic communities, desert
crusts, snow algae etc. (Nienow &
Friedmann, 1993; Garcia-Pichel &
Belnap, 1996)
Many of these physical substrates are
non-specic and will attenuate
visible wavelengths, although they
have the advantage of generating a
local microenvironment for the
organisms, particularly in extreme
environments
Sediments Benthic habitats (Garcia-Pichel &
Bebout, 1996)
Attenuation of UV radiation under a
millimetre or more of sediment.
Important in benthic and
continental shelf areas
Organics Any -electron-containing
chromophore (Sagan, 1973;
Cockell, 1998b)
May have been important on
prebiotic earth. Small compounds
provide non-specic UV-C
absorption
Biological methods
Matting Many cyanobacterial mats (Margulis
et al., 1976)
Dead or living upper layers protect
lower layers. Can be specic to UV
if 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)
Specic biological screening of UV
radiation
Fortuitous production of organics
that screen UV
Mycosporine-like amino acids in
response to osmotic eects (Oren,
1997)
May occur when other physiological
processes produce compounds that
happen to screen in the UV
It is also important to note that for some prebiotic
reactions the UV ux may have been advantageous.
In the laboratory, UV radiation has been shown to
provide the energy for the formation of complex
organics, some of which may have had prebiological
signicance (Groth & Weyssenho, 1960; Sagan &
Khare, 1971). In some of the earliest stages of
organic complexication, UV ux may have been
advantageous.
As our knowledge of the chemical reactions that
ultimately led to the rst self-replicating nucleic
acids and thence to the rst unicellular-like
organisms emerges, so surely our understanding of
the positive and negative roles of UV radiation on
prebiotic earth will improve and with it our
understanding of the importance of UV screening at
critical stages along this path.
(2) UV screening and early life
Many micro-organisms today suer photosynthesis
inhibition and DNA damage caused by UV radi-
ation. It is likely therefore, that if early exposed life
did exist under a greatly elevated UV-B and a UV-
C regime, it faced a much greater problem with UV
damage (Sagan, 1973; Margulis, Walker & Ram-
bler, 1976; Rambler & Margulis, 1980; Cockell,
1998a; Garcia-Pichel, 1998). Repair processes such
as photolyase repair of thymine dimers may well
have been more eective, but the screening of UV
radiation was probably also very important. In
many situations physical substrates may have pro-
vided sucient screening. As described in Section II,
layers of UV-B- and UV-C-absorbing reduced iron
compounds (Olsen & Pierson, 1986; Pierson et al.,
1993) may act as an eective UV screen. Organisms
under layers of sulphur (Cockell, 1998a) and
organisms in deep waters may have gained the
benet of UV radiation attenuation. Endolithic
communities that live in the subsurface layers of
rocks (Nienow & Friedmann, 1993) may receive
some UV-screening benet. Table 1 shows a di-
versity of potential screening strategies that may
have been available to early life on earth (adapted
from Cockell, 1998a). As discussed above, there are
disadvantages with physical substrates with respect
to their non-specic attenuation of visible radiation.
In some organisms this would have created a
selection pressure for UV-screening compounds,
providing the possibility for higher visible light
exposure.
An additional and strong evolutionary selection
pressure for UV-screening compounds must have
existed on early earth. Three and a half billion years
ago, the sun, that is a typical main sequence star, is
presumed to have been 25% less luminous (Kasting,
1993). Thus, the photosynthetically active radiation
at the surface of the earth would have been less,
which may have caused the euphotic zone (the zone
at which photosynthesis equals respiration) to have
been slightly higher in the early oceans. The higher
UV penetration into early waters caused by the lack
of an ozone shield and possibly the proportionally
greater UV ux emitted by the sun as a residual of
its early T-Tauri stage (Canuto et al., 1982) would
have resulted in a more signicant problem in the
trade-o between photosynthesis and UV radiation
exposure in earths early oceans. The selection
pressure may have been intense for organisms that
could exploit the high photosynthetically active
radiation in upper regions of the photic zone whilst
mitigating UV radiation damage (Cockell, 1998a).
UV-screening compounds would have been one of
the principal solutions. This trade-o and selection
pressure would also have applied to terrestrial
organisms. UV-screening compounds would have
provided the exibility for life to inhabit more
exposed habitats.
The nature of the rst specic UV-screening
compounds on Archean earth is unknown, although
an interesting hypothesis is that early aromatic-
containing reaction centres were some of the earliest
UV-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-day
earth and that are presumed to have been repre-
sented on early earth may have provided substantial
protection for cyanobacteria (Garcia-Pichel, 1998).
Both scytonemin and MAAs, which have screening
factors equivalent to screening of between 2 and
55% and 10 and 26% respectively in many species
of cyanobacteria at the single cell level (at 320 nm),
could have provided excellent screening from UV-B
and later, as oxygen partial pressures increased and
photooxidative eects became more prominent, UV-
A as well (Garcia-Pichel, 1998). Screening would
have been greatly enhanced when these compounds
were produced in layers of mats or in colonial growth
forms. This would have helped reduce DNA damage
rates from early earth values to values experienced
by exposed organisms today (Cockell, 1998a).
As alluded to in Section III, screening of UV-C
radiation on early earth could have been non-
specically accomplished by a wide variety of
molecules, since as well as the more eective non-
specic scattering of short-wavelength radiation,
many small organic compounds will absorb this
region of the spectrum. Scytonemin has a UV-C
peak (Fig. 4) and when produced extracellularly it
may have been quite eective in providing a rst-
line reduction of UV-C ux penetrating the cell.
Note that these UV-C peaks do not exist as a result
of evolutionary selection, unlike the UV-B and UV-
A peaks in MAAs and scytonemin. They result from
UV-C-absorbing to * transitions found in most
conjugated organic structures. The non-specic
nature of UV-C screening is demonstrated nicely by
the UV-C peaks in many phylogenetically more
recent organisms such as Antarctic Phaeocystis
pouchetti (Hariot) Lagerheim (Marchant et al., 1991)
and many higher plant avonoids (e.g. Shimazaki,
Igarashi & Kondo, 1988; Markham, 1989). These
organisms did not evolve under a UV-C ux. Used
in combination with other strategies, UV-screening
compounds would have been eective across the
whole UV range.
(3) Evolutionary relationships of UV-
screening compounds
The evolutionary origins of many UV-screening
compounds are still unknown. It is likely that many
evolved from other physiological roles to later full a
UV-screening function. Because potentially any
compound with aromatic or conjugated systems
may provide passive UV radiation screening, we
would expect that the production of compounds
for other physiological roles that fortuitously pro-
vided UV screening would subsequently be selected
for in organisms, if they provided some physiological
and competitive advantage. Such an evolutionary
origin for the UV-screening role of avonoids in
higher plants has been proposed. They are presumed
in their earliest stages to have been chemical
messengers or physiological regulators (Staord,
1991) or a chemical defence against early pathogens
and herbivores (Jorgensen, 1993) that would have
been eective at lowconcentrations (Staord, 1991).
Later evolution in the pathways would have allowed
both structural improvements in UV screening and
increases in concentrations. MAAs have a role as
osmotic regulators in some cyanobacteria (1997)
and such alternative roles may have given rise to the
rst UV-screening MAAs (Cockell, 1998a). As
discussed before, the evolutionary origin of UV-
screening compounds in other physiological roles
is today one of the primary complications in attri-
buting compounds to a UV-screening role.
The evolution of the MAAs as specic UV-screens
may represent an early innovation in dealing with
Archean UV-B ux. Many of the simpler MAAs
such as mycosporine-glycine specically absorb in
the UV-B. It has been suggested that later, as
oxygen levels increased, UV-A-screening MAAs
became important since many of the eects of UV-
A are mediated through oxygen free radicals
(Garcia-Pichel, 1998) and thus the contribution of
UV-A as a damaging agent in the biosphere would
have increased. UV-A protection may also have
been important well before the rise in oxygen partial
pressures since many photosynthetic microbes may
have been producing oxygen in their microenviron-
ments in the late Archean (Garcia-Pichel, 1998). In
these compounds, the nitrogen atom replacing the
ketone function has a greater mesomeric eect on
the benzene ring and absorbance is shifted into the
UV-A. It is plausible that a mutation in the earliest
UV-B-screening compounds resulted in a UV-A
screen which became physiologically advantageous.
Because MAAs are found in many eukaryotic algae,
it is likely that they were passed to the eukaryotic
algae by cyanobacteria in the plastidic line.
The relationship between avonoids and MAAs is
less clear, but nevertheless intriguing. Plants are
generally believed to have arisen from the green
algae (Chlorophyta) (Niklas, 1976; Atsatt, 1988).
One recent hypothesis suggests that land plants have
a biphyletic origin as the product of an endocellular
mutualism between a green alga (most probably a
charophycean alga) and a fungus-like organism
(Jorgensen, 1993) and it was suggested that this
mutualism may even have been made possible by the
UV-screening properties of avonoids protecting the
fungus. If this was the case then the synthesis of
avonoids as the principal UV screen would have
arisen in the earliest charophycean algae. However,
since the existence of avonoids in present-day algae
is questionable (Markham, 1988), it is dicult to
speculate accurately on the nature of UV protection
in ancestral land plants. If green algae were the true
origin of land plants and avonoids are only
represented in later plants, one might even speculate
that early land plants may initially have been
dependent on MAAs or related compounds from
green algae instead of avonoids. Some present-day
green algae do contain MAAs (e.g. Karentz et al.,
1991b). The present-day members of Nitella,
Coleochaete and other genera of green algae are
postulated to be the ancestors of plants (e.g.
Delwiche et al., 1989). Examination of the nature of
UV screening in these genera would be a worthwhile
area of evolutionary investigation. Later, avonoids
might have evolved from the phenylpropanoid
biosynthesis pathways as chemical messengers. As
their absorbance properties both in the UV-A and B
superseded algal compounds, it is possible to imagine
that the MAA response to UV became redundant.
Ultimately, the MAA pathway may have been lost
altogether as selection pressures for avonoids and
their associated diversity of biochemical uses
increased.
Other compounds have evolutionary origins that
are unknown. Cyanobacteria were some of the
earliest oxygenic photosynthetic organisms on earth.
Many of the terrestrial forms produce scytonemin,
suggesting that it was a later innovation in the
deepest branches of many cyanobacteria, or was
later lost in marine species. The structure of
scytonemin suggests that it may be formed from the
condensation of tryptophan and phenylpropanoid
derivatives (Proteau et al., 1993). Its reliance on
oxygen for synthesis might support the contention of
a later evolutionary origin after the evolution of
oxygenic photosynthesis (Garcia-Pichel, 1998). Its
predominant screening peak in the UV-A rather
than the UV-B also suggests some specic evolution
against the photooxidative eects of UV-A radia-
tion.
The synthesis of animal eumelanins from the
condensation reactions with tyrosine mediated
through the action of tyrosinase is very dierent
from other pigment pathways and may suggest that
the eumelanins have an independent evolutionary
origin. As well as land animals, the presence of
melanin in the ink sacs of cuttlesh (Prota, 1988)
and many insects and molluscs as well as fungi
suggests an early evolution.
Pictures are beginning to emerge of the evol-
utionary relationships of UV-screening compounds
and their signicance in the course of evolution. In
order to understand the origins of UV screening and
the evolutionary relationships, a key area of investi-
gation should be the study of UV screening in the
deepest branches of the phylogenetic tree. More
detailed examination of photosynthetically deep
branches such as Chloroexus spp. and the deepest
branches of the archaea, particularly exposed
thermophilic genera, would also be of interest.
(4) UV-screening compounds and the
BerknerMarshall hypothesis
The evolution of UV screening is central to some
fundamental questions in evolution. Berkner and
Marshall (1965) originally proposed that the in-
vasion of land 420 million years ago was dependent
upon the build-up of atmospheric oxygen and the
formation of the ozone shield that would have
reduced UV ux. This hypothesis has subsequently
been supported by others in the context of land
plants (e.g. Lowry, Lee & Hebant, 1980). The
hypothesis is an attractive one since it provides a
congruence between a major innovation in evolution
and a major geophysical change in the earths
environment. However, aside from the major fact
that a signicant ozone shield was probably in place
well before the evolution of land plants and possibly
as long ago as 2 Ga (Kasting, 1987), it is highly
equivocal for a number of other physiological and
palaeobiological reasons.
Firstly, it is understood from the fossil record
that stromatolitic microbial communities inhabited
intertidal regions some three billion years before the
invasion of land by plants (e.g. Knoll, 1992; Schopf
& Klein, 1992). Apart from water containing
impurities, the water column, and particularly
shallow water, is not a particularly eective at-
tenuator of UV radiation. Many microbial com-
munities probably inhabited these exposed niches by
a combination of UV-screening compound pro-
duction and the use of the matting habit, whereby
the uppermost layers of the colony either die or
provide screening to the lower layers of the colony
(Margulis et al., 1976). Other mechanisms such as
phototaxis may also have been important. In a sense,
the matting habit can be regarded as a method of
achieving the advantages of a multicellular UV-
screening response by a unicellular organism. From
this perspective a multicellular algae or an early
ancestral leaf (that may have an epidermal layer
protecting photosynthetic regions below) can be
regarded as an exquisite advance on the matting
habit with the epidermis being a dedicated and
evolved upper layer of the mat . UV-B penetration
into the mesophyll of a leaf in modern-day plants can
be reduced to less than 2% of ambient. Colonies of
cyanobacteria, through the use of matting, can
achieve similar reductions in UV radiation in the top
layers of the colony. From these arguments alone, it
seems illogical that stromatolites evolved in inter-
tidal and benthic continental-shelf regions of the
world 3n5 billion years ago and yet a multicellular
alga (and a potential early ancestral plant), that had
further rened the UV protection of photosynthetic
organelles and DNA by specic multicellular
adaptations, would have been constrained for some
three billion years more.
Secondly, there is now evidence for the presence of
exposed terrestrial microbial communities in the
fossil record 1n2 billion years ago (Horodyski &
Knauth, 1994). For the same reasons as described
previously, this evidence is in conict with the delay
in the development of early plants since if exposed
micro-organisms could survive, there should have
been little constraint to early multicellular plants.
Thirdly, if we assume that Archean oceanic waters
contained little organic matter (since most UV-
absorbing organic matter in the present-day oceans
is derived from land organisms), 1% penetration of
UV-B may have occurred to approx. 2550 m in
open ocean water as for clear waters in the present-
day oceans (Booth & Morrow, 1997). A single-celled
isolated cyanobacterium at this depth might be able
to attenuate approx. 26% of the UV radiation
it receives using MAAs which is the upper limit
of MAA screening found in some cyanobacteria
(Garcia-Pichel & Castenholz, 1993). They would
receive approx. 0n7% of incident UV radiation to
internal biological targets at such depths. Since a
plant leaf can reduce incident ux to less than 2%,
we can see that the dierences in UV exposure
between a suspended single-celled organism in the
photic zone and an early multicellular plant are
probably within the same order of magnitude.
Finally, the view of land plants (and indeed the
colonization of land generally) occurring as a result
of some major alteration in the UV radiation
environment may be unnecessary from a general
evolutionary standpoint. If life was well established
on land 1n2 billion years ago, then the colonization
of land by green algae, the evolution of upright
structures and the eventual diversication of these
early ancestral forms into bryophytes and tracheo-
phytes by 420 million years ago may have been a
gradual evolutionary process that may well have
been favoured by a gradually reducing UV ux, but
may not have required it.
The availability of atmospheric oxygen may well
have been a factor for land plant colonization in
other contexts since the lignin biosynthesis required
for structural support involves the use of oxygen
(Lowry et al., 1980). It seems likely that within the
milieu of signicant evolutionary challenges and
innovations posed to land plants which include
prevention of desiccation, biosynthesis of support
structures and accumulation of nutrients and
minerals through root systems, an answer to the
delay of land plant colonization until the Silurian
exists. However, it seems unlikely that UV radiation
would have been a critical constraint.
As a nal factor in this discussion it is interesting
to contemplate the link between UV radiation ux
and multicellularity in general terms. The evolution
of multicellular structures, at least in the Animalia,
was probably linked to the availability of oxygen for
respiration (Berkner & Marshall, 1965). However,
because of the screening advantage oered by
multiple cell layers and multiple localization of
screening compounds, intense UV radiation should
in a sense be a selection pressure for multicellularity.
Although the microbial matting habit is caused by
multiple ecological factors, the advantages in UV
radiation attenuation aorded by the matting habit
can be seen as early evidence for the selection
pressure towards a multicellular UV-screening
structure. Reductions in surcial UV radiation on
the Cambrian earth might have actually reduced the
relative selection advantage for multicellular organ-
isms compared to unicellular organisms, although
clearly all organisms would gain an overall advan-
tage under reduced UV ux.
(5) UV-screening compounds, ozone
reductions and Phanerozoic palaeobiology
The last billion years of biological evolution has
occurred under the protection of a UV-screening
ozone column. Data from the total ozone monitoring
satellite (TOMS) show that natural variations in
ozone column abundance occur even over monthly
and annual periods. During the year, variations may
occur of between 5 and 25%, that may be reected
in the UV exposure of some biomes (e.g. Moorthy &
Kathiresan, 1997). During the year, short-term
variations may also occur of up to 40% of total
ozone column abundance over periods of even a few
days to a month due to irregularities in ozone
concentrations (TOMS data, NASA Goddard Space
Flight Center). More longer term variations in UV-
B reaching the surface of the earth may occur from
the eccentricity of the earths orbit (which causes a
7% variation in radiation incident on the top of the
atmosphere between perihelion and aphelion).
Variations in cloud cover for periods of days and
weeks may cause signicant variations in UV-B
radiation. Thus, organisms must possess a robustness
to deal with regular variations in surface UV-B over
short time periods. Passive UV-screening pigments
may provide some of this intrinsic robustness.
However, a number of natural events can deplete
ozone over more signicant time periods (years) and
at levels that are far more biologically signicant
than most other natural variations. These events
may be evolutionarily signicant (Cockell, 1999).
Natural ozone-depleting events include large-scale
volcanism involving the injection of ozone-depleting
magmatic chlorine (Johnston, 1980; Vogelmann,
Ackerman & Turco, 1992), asteroid and comet
impact events (e.g. Turco et al., 1982; Toon et al.,
1997) and cosmic rays emanating from neutron star
mergers or the supernova remnant shells of close
supernovae events (Ruderman, 1974; Aikin,
Chandra & Stecher, 1980; Ellis & Schramm, 1995;
Thorsett, 1995). In the case of impact events, NO is
generated that depletes ozone. Depending on scale it
may cause depletion of up to 85% (Turco et al.,
1982). Impact events resulting in such levels of ozone
depletion may occur at a frequency of one every 10
million years. Close cosmic events (neutron star
mergers less than 1 kiloparsec and supernovae less
than 10 pc) would produce high energy cosmic
particles that cause the ionization of atmospheric
oxygen and nitrogen and the formation of NO.
Depletion in these case may be up to 20% at the
equator and 60% in Northern latitudes (Crutzen &
Bruhl, 1996) and may occur at a frequency of one
every 100 million years (Thorsett, 1975). The
frequency of ozone depletion caused by volcanic
activity is less clear, but there have been at least nine
major basaltic volcanic episodes over the last 250
million years (Rampino, Self & Stothers, 1988).
The exact level of increase in UV-B radiation will
depend upon the latitude on earth and can be
calculated by radiative transfer models. For the
purposes of the discussion in this review, however,
the fact that some of these events may cause ozone
depletions much greater than 50% suces to
illustrate that signicant ozone depletions that equal,
and in many cases exceed, anthropogenically-caused
depletions occur over geological time-scales.
Over such time periods, exposed organisms with
eective UV protection and repair processes may be
at an advantage compared to those with less eective
strategies. Repair processes and phototaxis etc. may
provide some level of intrinsic robustness to elevated
UV-B radiation. It is likely that compounds that
provide passive screening against elevated UV-B will
also be important. The ability to tolerate relatively
rapid increases in UV ux will therefore depend
upon these various processes combined and will be
species specic. We would expect that for increasing
severity of ozone depleting events a greater pro-
portion of exposed species may be stressed. As yet
there is little understanding of the ecological eects
of such events on natural populations and the degree
to which various UV protection methods may
provide greater survival over evolutionary time
periods, particularly in low latitude regions (Cockell,
1999). Studies on UV screening in terrestrial and
marine realms of the antarctic and the ecacy of
these processes in dealing with the eects of
anthropogenically-caused depletion may be of value
in assessing the eects of natural ozone depletion
events.
In studying recent changes in the UV radiation
environment, UV-screening compounds have found
some use. The longevity of some compounds such as
scytonemin and their potential preservation has
been suggested previously as an interesting target of
paleobiological investigation (Garcia-Pichel &
Castenholz, 1991). Preserved compounds have been
eectively used to examine UV radiation regimes in
lakes by measuring their concentrations in sediment
cores. These data have been used to provide
information on the dissolved organic carbon com-
position of lakes (and hence UV penetration to the
benthos) which in itself can be used as an indicator
of localized environmental changes (Leavitt et al.,
1997). Because the concentration of UV-screening
compounds depend on the changes in the UV
penetration of the water column, delineating
changes in lake chemistry from natural changes in
stratospheric ozone may be dicult. Exposed cyano-
bacterial stromatolites or organisms in clear-water
lakes that might better reect changes in the ozone
column rather than local environmental conditions
might be used for palaeo-analysis of changes in
direct UV ux. Analysis of UV-screening com-
pounds (particularly UV-B screening compounds)
in cores taken from arctic or antarctic clear water
lakes might be used to search for direct evidence of
natural ozone depleting events.
XI. ARTIFICIAL SCREENING COMPOUNDS
AND HUMAN UV PROTECTION
Mankinds attempts to deal with unwanted eects of
sunlight are to an increasing extent based on
developing chemicals that absorb ultraviolet radi-
ation. Some factors of importance are discussed here
since they provide an interesting comparison to the
evolution of natural UV-screening compounds, and
an insight into the motives that have driven people
to attempt to develop articial compounds that
allow them to tolerate increased exposure to UV-B
radiation. Furthermore, as the properties and
characteristics of natural compounds are better
understood, a convergence of human needs and
natural responses is likely to become increasingly
important.
The rst compound to be used widely (Natow,
1986; Patel, Highton & Moy, 1992) was para-
aminobenzoic acid (PABA); supercially an ap-
pealing choice because it is a natural product. It
absorbs UV-B quite strongly and so protects
eciently against sunburn, and has been used since
the 1920s. Although it is a crystalline solid which
dissolves poorly in water, it penetrates the stratum
corneum. It does so even if it is suspended in an
emulsion at pH 4n2; when it is largely insoluble, and
can be recovered in the urine. When applied as a
solution in ethanol it can accumulate in the skin
(Algra & Knox, 1978), so that it can be a very
eective photoprotective agent. But drawbacks
gradually became evident. PABA can stain both
natural (cotton) fabrics and synthetic ones such as
nylon and polyesters, perhaps because it is photo-
sensitive, and a number of people became sensitized
to 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 the
reports of potential problems with DNA damage,
notably an ability to sensitize the formation of
thymine dimers in DNA in cultured cells, started to
appear (Sutherland, 1982; Sutherland & Grin,
1984). The search resulted in, among other things,
the introduction of esters of PABA derivatives. One
in particular is worth considering in some detail. It
is 2-ethylhexyl-4 dimethylaminobenzoate, in which
the carboxyl group of PABA has been converted into
a branched chain ester, and the amino-group has
been converted into a dimethylamino group. This
material, 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 particularly
attractive because it is a colourless, oily liquid that is
virtually insoluble in water and did not stain
clothing. It became, for a while, one of the most
widely used UV-screens, and oers very good
protection against sunburn. As assayed by Ames tests
using yeast, it did not appear to have the potential to
damage DNA, at least in the absence of deliberate
illumination (Bonin et al., 1982), and so appeared to
have many advantages.
However, it is important to remember that,
despite their name, UV-screens and sunblocks
cannot simply eliminate the light energy which falls
on them. They have to do something with it, and the
chemicals involved are really energy transducers
which absorb UV energy and convert it to some
other form. It is important to understand exactly
what happens, because compounds such as PABA
and Padimate-O can form excited states. Such states
are not easy to characterize, but can be intensely
reactive, raising the possibility that sunscreens could
have unwanted side-eects, as indeed was shown for
PABA (Sutherland, 1982; Sutherland & Grin,
1984).
PABA sensitizes the formation of thymine dimers
in DNA. It does so because it forms a triplet state
which can react directly with DNA, forming thymine
dimers. However, there are other possibilities. Trip-
let PABA can react with oxygen to form singlet
oxygen (Allen, Gossett & Allen, 1996). It can also
generate solvated electrons, which react with oxygen
to make the superoxide radical anion O
[
#
(Allen et
al., 1996; Martincigh, Allen & Allen, 1997). Proton-
ation of the superoxide radical anion followed by
dismutation generates O
#
and hydrogen peroxide.
Hydrogen peroxide can react with the superoxide
radical anion in the HaberWeiss reaction, or with
Fe#
+
(inevitably present in biological systems) in the
Fenton reaction to produce hydroxyl radicals, which
can damage DNA in various ways. This means (a)
that any mutagenic eects of PABA cannot neces-
sarily be attributed to the formation of thymine
dimers, and (b) that PABA could have undesirable
side-eects, and it is possible that this is why natural
selection has not chosen PABA as a common
UV-screening compound.
What about Padimate-O? On a simple view, one
might expect it to behave in the same way, but in
fact it appears to behave rather dierently. The rst
clue came from a structural comparison with a
functionally identical compound, ethyl-4-dimethyl-
aminobenzoate, which is simply the corresponding
ethyl ester. It is used as an industrial photoinitiator
of polymerization (Wayne, 1988), which it catalyses
because it generates carbon-centred radicals under
UV illumination (Forster & Hester, 1981). As
carbon-centred radicals can, in the presence of water
and oxygen, react in various ways to form highly
reactive hydroxyl and peroxy radicals, one would
expect the sunscreen Padimate-O to have the
potential to inict damage on a variety of bio-
logically important molecules. Experiments using
yeast showed that it is killed by illuminated
Padimate-O, and suggested that DNA damage also
occurs. It could contribute to the toxicity because
mutations were generated and rapidly-dividing cells
that were decient in DNA repair were more
sensitive than non-dividing, repair-procient cells
(Knowland et al., 1993). This work raises the
theoretical possibility that the benecial eects of
sunscreens in reducing UV exposure could be
balanced by potentially harmful eects caused by
sunscreen penetrating the skin, which both PABA
and 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 investigate
this possibility it is essential rst to dene the kind of
damage that any given sunscreen inicts when it is
illuminated. The simplest way to do this is to study,
in preliminary experiments, what happens with
DNA in vitro. It emerges that, unlike PABA,
Padimate-O does not sensitize the formation of
thymine dimers in DNA. Rather, it generates direct
strand breaks and oxidative lesions, mainly but not
exclusively at GC base pairs, that can be detected in
a variety of ways (McHugh & Knowland, 1998).
Thus, experiments intended to reveal the kind of
damage inicted on human cells by sunlight in the
presence of Padimate-O must take this into account.
The same is true for other organic sunscreens, but
their photochemistry in relation to potential DNA
damage is, in most cases, very poorly understood.
Other aspects also merit further attention. For
example, it appears that illumination can catalyze
breakdown of organic sunscreens (Roscher et al.,
1994; Schwack & Rudolph, 1995), and it is not clear
what the consequences could be.
As an alternative to organic sunscreens, physical
sunscreens have also been developed. They are based
on titanium dioxide and zinc oxide, which are both
white pigments. The former is widely used in paint.
If the particle sizes are relatively large, creams
containing these pigments simply appear white,
which makes them cosmetically unattractive. In
order to avoid this problem, manufacturers reduce
the particle size to the range 2050 nm, because in
this size range the interaction with light obeys
Rayleighs laws of light scattering, whereby the
intensity of scattered light is inversely proportional
to the fourth power of the wavelength (Judin, 1993).
This means that they scatter the short, UV wave-
lengths far more eciently than visible ones. To the
extent that scattering is backwards rather than
forwards, it is protective. However, the oxide
particles also absorb UV light to a substantial
extent, and, like organic sunscreens, they interact
with that energy. Both titanium dioxide and zinc
oxide are semiconductors. Interaction with UV
promotes electrons from the valence band to the
conduction band, generating single electrons and
electron decient states, known as holes . Any
wavelength shorter than approx. 385 nm will
achieve this, so that these materials are excitable by
both UV-A and UV-B. The electrons and holes
either recombine or migrate rapidly (in less than
approx. 0n01 ns) to the surface. In aqueous environ-
ments, holes react with water or hydroxyl ions, and
electrons react with oxygen, forming hydroxyl
radicals and superoxide (Serpone, 1996):
h
+
jOH
4OH[,
e
jO
#
4O
#
[
,
Both hydroxyl radicals and superoxide are highly
reactive, so that ZnO and TiO
#
are eective
photocatalysts of oxidation. Indeed, TiO
#
has been
extensively investigated in connection with oxidation
of environmental pollutants (Bahnemann et al.,
1994). Although it is not clear whether TiO
#
and
ZnO as used in UV-screens can penetrate human
skin or not, where the evidence is limited and
uncertain (see Dunford et al., 1997 and references
therein), it is clear that small particles of TiO
#
can
enter human cells (Kubota et al., 1994) and that
larger ones can too (Wamer, Yin & Wei, 1997).
Consequently, it is important to investigate the
potential of both TiO
#
and ZnO to attack DNA,
taking into account both the type of damage which
might be inicted and whether it actually occurs.
Here, it is particularly important to study the
preparations actually used in UV-screens, because
they are often coated with other materials which are
intended to reduce or eliminate photoactivity,
although whether such coatings are invariably
successful is hard to judge. It is also important to
consider the type of TiO
#
used, because its activity
depends on crystal structure, the anatase form being
more active than the rutile form. Anatase TiO
#
with
a particle size of 450 nm clearly generates hydroxyl
radicals, as demonstrated using the spin trap 2,2,6,6-
tetramethylpiperidine-N-oxyl (TEMPO), and it
catalyses 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 wide
range of ability to catalyse photooxidation, but that
all were active to some extent (Dunford et al., 1997).
The same survey also demonstrated DNA damage in
cultured human cells illuminated in the presence of
TiO
#
with light intensities very similar to those
found under the stratum corneum. The damage
found was exactly that which would be predicted
direct strand breaks and oxidative lesions attribut-
able to hydroxyl radicals. In that study, damage to
nuclear DNA was found, in contrast to the work of
Wamer et al. (1997), which, using a dierent assay
and much larger particles of TiO
#
, did not report
damage to nuclear DNA. Although it is clear that
much more work needs to be done, it would appear
that the potential of sunscreens to damage DNA is
not conned to organic sunscreens.
It is not the purpose of this review to consider in
any detail the many studies that have been con-
ducted using direct biological approaches, because
that has been done many times before, but a few
comments may be relevant to the more chemical and
molecular work outlined. It has been shown that
certain UV-screen preparations reduce the tumours
found in control hairless mice, although whether this
should be interpreted in terms of prevention of
tumourgenesis (Kligman, Akin & Kligman, 1980) or
simply delay (Wulf et al., 1982) is not entirely clear.
It is also clear that some can reduce the formation of
thymine dimers in mouse skin under illumination
which under-represents UV-A (Ananthaswamy et
al., 1997). It is hard to know whether suggestions
from work on a melanoma-susceptible hybrid sh
(Setlow et al., 1993) showing that UV-A is par-
ticularly important in that species apply to mice and
men, but if UV-A is indeed a relevant risk factor,
then much of the earlier work, conducted before
good solar simulators which adequately represent
natural sunlight became available, may have to be
re-assessed. One problem with these whole-animal
experiments is that they cannot easily distinguish
between the eects of screens applied to skin and the
eects of that which diuses into the skin, but on the
other hand they do address an important biological
end-point : the formation of a tumour. The overall
conclusion has to be that as many approaches as
possible need to be applied to the question of the role
that sunscreens play in protecting us from the
unwanted eects of the sun.
Natural sunscreens may provide some solutions.
An interesting parallel has occurred in research.
As we have begun to understand the potential
photoxidative problems associated with some of the
articial sunscreens, so we have also begun to
elucidate the role that many natural UV-screening
compounds have in anti-oxidant activity. This
functional link may not be a coincidence. UV-
screening avonoids are important anti-oxidants in
plants (Larson, 1988). In corals, the UV-screening
MAA, mycosporine-glycine, is a strong anti-oxidant
(Dunlap & Yamamoto, 1995). Thus, it is possible
that within the complement of natural UV-screening
compounds, some are eective not just in passive
screening, but also at quenching reactive oxygen
species. They may provide an additional line of
defence against photooxidative damage as well as
other compounds that are presumed to have a
dedicated anti-oxidant role, such as carotenoids.
Compounds such as avonoids and mycosporine-
glycine are likely to be of great interest in the
development of human sunscreens. Thus, a conver-
gence of the study of natural UV-screens and the
development of sunscreens for human requirements
seems inevitable.
Secondly, of course, research on human sunscreens
may provide directions for research on natural
UV-screening compounds. Having investigated the
fate of energy produced by UV screening in human
suncreams the question arises as to how signicant
this is in natural populations and what happens to
energy produced by natural UV-screening com-
pounds. How necessary are the antioxidant proper-
ties of some MAAs, avonoids and some carotenoids
in mitigating UV-induced damage generated by the
UV absorbance of these UV-screening compounds
in the rst place? What is the nature of this
interaction between antioxidant activity and UV
screening in natural compounds ? Answers to these
questions are likely to have many natural science
and human commercial implications.
XII. CONCLUSIONS
(1) UV-screening compounds are represented in
well-dened groups (for example cyanobacterial
scytonemin, MAAs, avonoids and eumelanins)
across a great diversity of organisms. This suggests
that the specic screening of UV radiation has been
a strategy for coping with its damaging eects for a
considerable time. The screening compounds dis-
covered to date use the to * transitions associated
with conjugated bond structures, aromatic or
indolic derivatives to screen UV radiation, providing
a common chemical theme.
(2) Since a UV-screening compound is by
denition a passive process, it is important to ensure
that any experimentally observed screening is indeed
physiologically relevant. A series of basic rules have
developed that allows the experimenter to check the
role of a compound in UV screening. By these
criteria both scytonemin, and in some cases MAAs,
have been found to have a UV-screening role as well
as many plant avonoids and animal eumelanins.
Some compounds such as sporopollenin and melanin
are examples of compounds that may provide
constitutive, but physiologically relevant UV
screening.
(3) Although compounds provide an eective
UV-specic shield, particularly for photosynthetic
organisms, it is also clear that other UV mechanisms
such as phototaxis and particularly protein and
nucleic acid repair processes may provide an eective
UV response and in many cases may actually reduce
or even negate the need for UV-screening com-
pounds. Simple measures of concentrations and
screening characteristics of compounds do not
provide insights into an organisms UV tolerance.
More detailed studies of UV-screening compounds
and their screening abilities in relation to other
modes of UV response will be an important avenue
of future research, particularly in attempting to
understand 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 to
continue work to understand the representation of
dierent types of UV-screening compounds in a
greater diversity of organisms. This work will have
great evolutionary interest. It can provide more
detailed insights into how ubiquitous and necessary
UV-screening compounds are in many related
species and during the course of their evolutionary
development. Furthermore, through such work we
can gain better insights into the role of UV-screening
compounds on early earth. Studies on the deepest
branches of the phylogenetic tree will elucidate the
origins of UV screening.
UV radiation has been a ubiquitous feature of the
exposed surface of earth since the origin of life itself.
It is the one of the most damaging forms of radiation
that penetrates to the surface of the earth and is
therefore a highly important regulator of organism
survival and ecosystem balance. We can expect the
work that has been completed to date to provide a
foundation for increasing our understanding of the
role of UV-screening compounds in survival under
the various UV regimes of the earth, past and
present and future.
XIII. ACKNOWLEDGEMENTS
Acknowledgements are due to two anonymous reviewers
for thoughtful and helpful ideas and suggestions on the
manuscript.
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