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Chapter 3
MOLECULAR AND CELLULAR STUDIES
SUMMARY
A decrease in stratospheric ozone has biological
consequences that reflect two processes: (1) the increase
in the intensity of W -B reaching the earth as a function
of decreasing wavelength, and (2) the increase in bio-
logical sensitivity with decreasing wavelength. The
latter function is called an action spectrum. Knowledge
of action spectra is important in evaluating the hazards
of ozone depletion but is not sufficient for making
quantitative predictions.
In the past few years, extensive advances have been
made in understanding the effects of single-wavelength
(monochromatic) light on simple biological systems such
as bacteria and cultured mammalian cells. It is now known
that the biological effects of W -B are quite similar to
those of W -C (with which almost all experiments in the
literature have been done), and that DNA is the target
for many deleterious effects of UV. The action spectrum
for damage to DNA is well characterized. However, despite
the similarity between the DNA action spectrum and the
action spectra for killing, mutating, or transforming
mammalian cells in culture, irradiation of such cellular
systems with broad bands of radiation does not give
results that would be predicted from the sum of the
effects at discrete wavelengths. There are good
indications that the longer wavelengths in W-B, or W-A,
may modify the effects of UV-B. For example, in a
process called photoreactivation, cellular enzymes use
longer wavelength light energy to reverse the effects of
shorter wavelength light. Longer wavelengths may delay
the growth of cells, leaving more time for repair
processes to act on the short-wavelength damage. Thus
47
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48
since sunlight comprises a broad band of wavelengths,
simple action spectra do not permit reliable predictions
of the responses of biological systems to sunlight.
One of the more important recent findings concerns the
fate of photoproducts in skin cells of animals and humans.
Photoproducts result from the absorption of photons
(quanta of light energy) by molecules. Sensitive ways
have been designed to measure the most important UV-C
photoproducts, pyrimidine dimers in DNA, in intact skin
irradiated by sunlamps. The presence of these dimers has
severe biological consequences, including mutation and
cell death. Studies have shown that there are two
processes that remove dimers from cellular DNA in viva;
one of these repair mechanisms acts in the dark (excision
repair), and the other acts in the presence of longer
wavelength UV and visible radiation (photoreactivation).
Photoreactivation is highly specific for pyrimidine
dimers. Preliminary estimates indicate that photo-
reactivation is very rapid in humans and takes place to
an appreciable extent even while human skin is being
irradiated by the W-B in sunlight. Consequently, the
level of pyrimidine dimers in cellular DNA in viva depends
upon the relative intensities of the dimer-forming wave-
lengths in UV-B and the dimer-splitting wavelengths in
UV-A and visible radiation. This argument suggests that
the effects of sunlight exposure on people might depend
upon their exposure habits. For example, exposure in the
afternoon might be much less deleterious than exposure in
the morning. This is because the intensity of W-B in
relation to that of UV-A and visible light increases in
the morning and is greatest at noon. If exposure ended
at noon, dimer formation would be at a peak relative to
dimer splitting. If exposure occurred in the afternoon,
more photoreactivating (dimer splitting) activity would
occur as the relative intensity of W-A and visible light
to UV-B increased. These findings underscore the
importance of obtaining quantitative measures of life
style and effective exposure.
Both excision repair and photoreactivation have now
been found to occur more slowly in mice than in humans;
this fact must be taken into account in extrapolating
data from rodents to humans.
An added complication in attempting to estimate the
effects of UV on, say, skin cancer induction is the effect
of UV-B on the immune systems of animals and humans.
preliminary action spectrum has been determined for
W-induced immunosuppression in mice. If this spectrum
A
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49
were the correct one to use in estimating the carcinogenic
effects of W on humans, the predicted effects of ozone
depletion would be significantly smaller than those
obtained by using an action spectrum determined for DNA
alteration or for production of erythema in human skin.
INTRODUCTI ON
The studies described in this chapter aim at supplying
the basic knowledge required to estimate the shapes of
dose-response curves, to extrapolate from high to low
dose rates, to determine the appropriate action spectra,
and to assess synergistic and antagonistic phenomena in
analyzing biological effects of ozone depletion. Without
such data it is not possible to extrapolate from cellular
studies to predict the effects on ecosystems and humans.
Damage to DNA
Many of the deleterious effects of W arise from the
damage it does to DNA. Thus a great deal of effort has
gone into understanding the biological effects of
specific damages (photoproducts) in DNA. Early work in
this field established definitively the shape of the
action spectrum for damage to viral or bacterial DNA. It
was supposed that this action spectrum was the appropriate
one to use to calculate the biologically effective solar
W dose for humans and the change in this dose as a
result of ozone depletion (Setlow 1974). This Presumption
was a big extrapolation. There were no reliable action
spectra for W effects on mammalian cells, and there was
a good possibility that the correct action spectrum might
be different from that for bacterial DNA, because in
higher organisms DNA is not a naked polymer but is closely
associated with proteins. This association might change
the action spectrum and might give rise to other
deleterious photoproducts, for example, cross-links
between protein and DNA.
The effects of solar W on DNA are emphasized here
because in much of the current work on carcinogenesis
is assumed with reasonable assurance that DNA is an
important target for initiating carcinogenic events. We
enumerate in Appendix G a number of reasons for this
presumption. It is important to realize, however, that
other external or internal cellular factors, such as
it
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50
tumor promoters, hormones, or immunosuppressors, may be
important in the development of cancer. In addition, UV
could be both an initiator and a promoter, which would
complicate the interpretation of dose-response curves.
DNA Repair
Although dosimetry for UV damage to cells is accurate and
one can enumerate easily the DNA photoproducts existing
immediately after irradiation (Setlow and Setlow 1972),
the biological effects depend not only on the presence of
such photoproducts but also on their lifetimes in cells.
Most cells have repair mechanisms that remove the photo-
products or permit cells to ignore them (Friedberg et al.
1979, Hanawalt et al. 1979, Setlow 1978). A great deal
is known about two of these repair mechanisms, photo-
reactivation and excision repair.
Photoreactivation is a process by which visible light
or W-A may reverse the effects of W-B or UV-C radiation.
In this process, an enzyme binds to a DNA molecule
containing pyrimidine dimers. The complex of enzyme and
damaged DNA can absorb W-A or visible light, which causes
the dimer to split, thereby repairing the damage. When
the photoproducts are removed, the enzyme dissociates
from the DNA. Photoreactivation is highly specific for
pyrimidine dimers (Setlow and Setlow 1972), and when it
is observed for a particular biological effect (such as
survival, chromosome breaks, or mutation), it is taken as
evidence that dimers caused that effect.
Many cells, including normal human cells, contain
repair systems such as excision repair that operate in
the dark. In excision repair, products of W irradiation
are removed from one strand of a DNA double helix by
specific enzymes. The opposite, unaltered strand is then
used as a template on which a new, unaltered strand is
reconstituted. Excision repair is a very active process
in normal human cells. Cells from individuals with a
genetically inherited, sunlight-sensitive, cancer-prone
disease called xeroderma pigmentosum are in almost all
cases defective in excision repair. The high prevalence
of skin cancer in such individuals is ascribed to the
defect (Kraemer 1980). Mouse cells in culture also are
defective in excision repair, and this defect must be
taken into account in attempting to extrapolate from mice
to humans.
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51
ADVANCES IN KNOWLEDGE
Transformation of Cells in Culture
Transformation, an inheritable alteration of cells, can
lead to cancer formation. W -C and W-B are able to
transform mouse, hamster, and human cells in vitro, so
that their growth on surfaces is no longer inhibited by
contact with neighboring cells. The cells grow into
piled-up clumps of cells instead of monolayers and can
grow without being attached to a surface
.
(Chan and Little
1976, DiPaolo and Donovan 1976, Sutherland et al. 1980a).
The fraction of transformed cells per surviving cell
increases with dose.
of W-transformed rodent cells are usually tumorigen~c
when injected into certain mouse strains, but no tumori
genicity has been shown for the W-transformed human
cells described in the experimental results shown in
Figure 3.1. Transformation of mouse and hamster cells is
accomplished by single acute doses of W , but transforma
tion of human cells thus far has only been effected by
several small W doses (Sutherland et al. 1980a) or after
a single dose under rather special conditions (Milo et
al. 1981).
-
In numerous exper~ments, co~on~es
Photoproducts in DNA
Evidence that pyrimidine dimers are one of the major W-C
photoproducts in cells of higher organisms comes from
studies on photoreactivation. About 65 percent of the
lethal damage to frog cells, which have high levels of
photoreactivating activity, is photoreversible (Rosenstein
and Setlow 1980). Since under experimental conditions
only 80 percent of the pyrimidine dimers in the cells are
returned to monomers, the results indicate that
approximately 0.65/0.8 (~80 percent) of the lethal
damage can be ascribed to pyrimidine dimers. In other
systems with high levels of photoreactivating activity
(frog cells and chicken embryonic fibroblasts), there is
extensive photoreactivation of W -C-induced chromosome
aberrations and sister-chromatic exchanges (Griggs and
Bender 1973, Natarajan et al. 1980). Between 75 percent
and 95 percent of the dimers are reduced to monomers, and
the effective reduction in sister-chromatic exchanges was
calculated to be between 65 percent and 80 percent,
indicating again that a major fraction of this particular
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52
0°
_
~ 2 _
O -1
~ 10
LLI
c, 5
He
Is
2
L1J
CL 1o2
I1J
~ 5
> 2
103
LLI
~5
~\~:
2 _
Human Cells
0 Killing (Kantor et al. 1980)
· Transformation (Sutherland et al. 1981 )
Chinese Hamster V79 Cells
Killing (Rothman and Setlow 1979)
~ Dimers (Rothman and Setlow 1979)
Syrian Hamster Cells
O Kil I ing ( Doniger et al. 1981 )
· Dimers (Doniger et al. 1981 )
O Transformation ( Doniger et al. 1981 )
Mouse Cells
X Mutation (Jacobson et al. 1981 )
Frog Cells
V Killing (Rosenstein and Setlow 1980)
Dimers (Rosenstein and Setlow 1980)
DNA Absorption Spectrum (Sutherland and
Griffin 1981)
\~\ ~Action Spectrum for Lethal Effects in
1 \ Bacteria and Viruses (Setlow 1974)
I\\
\
- 4 1 , 1 1 1 1 '
10 250 270 290 310
WAVE LE NGTH ( n m )
FIGURE 3.1 Points on action spectra of a variety of higher organism cells (normal-
ized to 1.00 at 265 nary). Also shown are the absorption spectrum of purified mam-
malian DNA (solid line) and the action spectrum for lethal effects In bacteria and
viruses (dashed line).
type of chromosomal damage arises from pyrimidine dimers
in DNA (Natarajan et al. 1980). The initiation by UV-C
irradiation of the transformation of human cells has now
also been found to be photoreversible (Sutherland et al.
1980a).
That these conclusions may be extended to the UV-B
region is strongly indicated for certain lesions. The
killing of frog cells is photoreversed with equal
effectiveness at each damage-producing wavelength tested
between 252 nm and 313 nm (Rosenstein and Setlow 1980),
and transformation of human cells by W -B is photo-
reactivable (Sutherland et al. 1980a).
W-C makes other photoproducts in DNA in addition to
pyrimidine dimers, but these products have generally not
been analyzed for their biological consequences. Examples
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53
include other photoproducts of thymine, DNA-protein
cross-links, and single-strand breaks (but of a different
character than those made by ionizing radiation) (Erickson
et al. 1980, Setlow and Setlow 1972). The ratio of other
photoproducts to pyrimidine dimers appears to be a
function of the wavelength of irradiation (Hariharan and
Cerutti 1977). If other products were important, they
might distort an action spectrum for affecting DNA from
the shape of the action spectrum for dimer formation.
The action spectrum for dimer formation itself is a
complicated function of wavelength since all types of
pyrimidine dimers may be formed, such as thymine-thymine,
thymine-cytosine, and cytosine-cytosine. The ratio of
cytosine-thymine to thymine-thymine dimers appears to
increase with wavelength from 290 nm to 313 nm (Ellison
and Childs 1981).
It should be recognized that cell killing by W -A is
produced mostly by mechanisms that are quite different
from those produced by W-C, although the lethal lesion
is still primarily damage to DNA. This is evidenced by
the fact that bacterial mutants lacking DNA repair
systems are very sensitive to W-A. Roughly 90 percent
of the W-A killing requires oxygen (Webb 1977), whereas
W-C killing does not. Further evidence that W-A damage
is different is that the action spectrum for aerobic
killing has a specific structure that suggests absorption
of W by dyelike molecules (Webb 1977). The fraction of
W-A killing that does not require oxygen (10 percent)
may be due to direct production of damage in DNA, as
evidenced by the fact that the action spectrum below 350
nm is without structure (Webb 1977) and similar to the
absorption spectrum of DNA (Cabrera-Juarez et al. 1976,
Peak and Tuveson 1979, Sutherland and Griffin 1981).
Thus a small fraction of the lethal damage produced by
W-A may be similar in mechanism to that produced by W -C.
There are many indications that the mecnan~sms causing
effects on DNA from W-B irradiation represent a mixture
of the W-C and the W-A mechanisms, although the W-C
mechanisms clearly predominate, as shown, for example, by
the data of Figure 3.1. Thus, even though damage to DNA
from W-B (the waveband critical in ozone depletion
effects) is quite similar to that produced by W-C, it is
not identical to it.
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54
Action Spectra for Affecting Simple Cellular Systems
Action spectra for killing a wide variety of higher
organism cells in culture have been obtained for wave-
lengths up to 313 nm. Action spectra have been obtained
for killing human cells, mutating mouse cells, and bans
forming hamster cells up to 313 nm and for transforming
human cells up to 297 nm. The data points for a number
of action spectra, normalized to a value of 1.00 at 265
nm, are shown in Figure 3.1. All the spectra that could
be drawn for the various sets of points are very similar
and coincide with the spectrum for forming pyrimidine
dimers in the DNA of these cells. Figure 3.1 illustrates
that (1) the action spectra for effects on mammalian and
frog cells are very similar to those for effects on
bacteria and viruses, (2) from 297 nm to 313 nm, the
shapes of the spectra that could be drawn for the effects
on cells of higher organisms are all approximately the
same, and (3) there is a substantial deviation at long
wavelengths between the values for all the action spectra
and the absorption spectrum of purified mammalian DNA.
At long wavelengths, the biological effects indicated in
Figure 3.1 are smaller than would be predicted by the DNA
absorption spectrum, probably because much of the W is
absorbed in the purine (guanine) residues of DNA whereas
the lethal photoproducts are primarily in the pyrimidine
(cytosine and thymine) residues that absorb little at
long wavelengths.
Possible effects at wavelengths longer than 313 nm
have not yet been determined in higher biological systems
because the energies needed are higher than those achiev-
able with the monochromatic UV sources used in past
studies. The lethal responses of a large number of
normal human cell strains have been examined at 254 nm
and 313 nm (Smith and Paterson 1981), and the ratios of
their sensitivities are close to those shown in Figure
3.1. A similar ratio is obtained for xeroderma
pigmentosum cells (Smith and Paterson 1981), indicating
that these repair-deficient human cells show equally
enhanced sensitivity to 254 nm and to 313 nm. Thus for
monochromatic radiation sources and the effects shown in
Figure 3.1, almost all cells follow the DNA action--not
absorption--spectrum. The primary conclusion drawn from
the current understanding of action spectra is that all
have similar shapes and hence the DNA action spectrum for
mammalian cells may be taken to represent an average
spectrum (not drawn in the figure).
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55
Effects of UV on Photoproducts
in Animal and Human Skin
Sensitive enzymatic techniques have recently been
developed to investigate the amounts of pyrimidine dimers
in irradiated animal and human skin. These experiments
indicate that excision repair (toy et al. 1977) and
photoreactivation (toy et al. 1978) are negligible in
mice, except for neonatal mice, in which there is a low
level of photoreactivation (Ananthaswamy and Fisher
1980). For human skin irradiated with one minimal
erythemal dose by a sunlamp, it is possible to measure
the number of pyrimidine dimers immediately after
irradiation. If incubation is continued in viva in the
dark, there is an appreciable loss of dimers within 20
minutes, presumably by excision repair (D'Ambrosio et al.
1981a, Sutherland et al. 1980b). If instead the skin is
illuminated with light from an incandescent lamp
(predominantly visible wavelengths) for 20 minutes
immediately after the sunlamp, there is more loss of
dimers than from incubation in the dark. With even
higher photoreactivating illumination, 80 percent of the
dimers are lost in 4 minutes (D'Ambrosio et al. 1981b).
These data indicate that normal human skin has both an
active excision repair process and an active photoreacti-
vation process, as was inferred from experiments on cells
in culture (see the section below, "Mitigation and
Enhancement of W -B Effects by Light at Other Wave-
lengths"). The experiments imply that the illumination
of human skin by sunlight results in a rather complex set
of reactions for exposures that cover an appreciable
period of time. W -B exposure makes pyrimidine dimers,
but during the exposure dimers are being excised, and the
W -A and visible components of sunlight are reversing the
dimers by enzymatic photoreactivation. Thus in some
. ~
· · ~ . ~ _ _
situations low, chronic UV exposures might have little
effect, especially if the exposure continues into the
later parts of the day when the W-B component of sunlight
is relatively low and the photoreactivating (W -A)
component is relatively high.
Action Spectra for Immune Responses
Irradiation of certain strains of mice with sunlamps
( W-A and UV-B) suppresses two immune responses,
rejection of W-induced tumors (DeFabo and Kripke 1979,
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56
Fisher and Kripke 1977) and contact hypersensitivity to
the chemical trinitrochlorobenzene (TNCB) (see Chapter
5). Such systemic effects in mice irradiated at low
doses (less than 1 minimal erythemal dose) could well be
a contributing factor to the efficacy of W as a
carcinogenic agent.
The abolition of contact hypersensitivity to TNCB
requires much lower doses than those needed for the
lowered rejection of UV-induced tumors. This makes it
experimentally possible to obtain an action spectrum for
inhibition of contact hypersensitivity. Preliminary
descriptions of this spectrum (De Fabo and Noonan 1980)
indicate that it is in the UV-B region and falls off
rapidly as wavelength increases above 290 nm. The action
spectrum matches closely the absorption spectrum of
several compounds known to be in mammalian skin, such as
urocanic acid, and its values are appreciably greater
than the absorption spectrum of DNA (Figure 3.1) at
wavelengths greater than 290 nm. However, the target(s)
(as yet unknown) for this effect may lie below the
surface of the skin, and light absorption of skin is
greater at shorter wavelengths. Hence, if this action
spectrum were expressed in terms of quanta incident on
the target as they are in Figure 3.1, rather than on the
surface of animals, the values for the shorter wavelengths
would be increased in relation to the values for the
longer ones. As a result, the shape of the action
spectrum for the depression of contact hypersensitivity
would be closer to, but probably not identical to, the
DNA absorption spectrum.
If the suppression of these immune responses is
important in UV carcinogenesis, and if their biological
sensitivities at longer wavelengths are greater than that
for damage to DNA, the effects of ozone depletion would
be less than those computed for a DNA action spectrum.
This is because it is the steepness of the action spectrum
in the UV-B that makes ozone depletion important (see
Figure 2.2).
Experiments using broad-band sunlamps have shown
suppression of contact hypersensitivity in mice. The
shapes of the dose-response curves are similar to those
of the narrow-band UV used to determine the action
spectrum (Noonan et al. 1981a). It is important to
determine how the effects of heterochromatic radiation on
these immune responses compare quantitatively with the
sum of the effects of monochromatic radiation.
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Mitigation and Enhancement of W-B Effects
by Light at Other Wavelengths
Sunlight and UV-B in Bacterial Systems
Much of what is known concerning photobiological effects
in animal cells originates from studies of bacteria.
Bacteria are very small, have large populations in a
small volume, and have a very short generation time,
permitting efficient study of rare events, such as
mutation. Although bacteria are different in many ways
from human cells, the fundamental biochemistry and
genetics are analogous. It is generally true therefore
that advances in bacterial photobiology have preceded and
have pointed to advances in the photobiology of cells of
higher organisms.
It is now evident that UV-B acts on bacteria in much
the same way as do shorter wavelengths (W-C), namely,
through absorption of energy in DNA. Sunlight, however,
contains much more of the longer wavelength W-A than
UV-B. Although UV-A by itself can kill and mutate
bacteria, it does this with only very low efficiency; the
primary effect of the UV-A in full spectrum sunlight is a
modification of the action of W-B. This modification
may be either antagonistic or synergistic. Because of
these modifications, it is clear that most actions of
sunlight on biological systems cannot be understood from
experimental work using monochromatic light alone.
Among the known antagonistic processes are photoreacti-
vation and photoprotection. Photoreactivation was
defined earlier as a process in which UV-A or visible
light eliminates the pyrimidine dimers produced by UV-B.
The great excess of UV-A in sunlight suggests that much
of the W-B damage will be repaired in the same exposure
to sunlight that produced the damage (Webb 1977). Such
effects have been observed in bacterial systems, which
are normally killed (to a 10 percent survival level) by
about 30 minutes of exposure to bright sunlight, but
which have been shown to be more sensitive to killing by
sunlight under conditions where photoreactivation is
prevented (by low temperature or by using a system
possessing defective photoreactivating enzyme). It is
not easy to demonstrate such effects in animal cells
irradiated with sunlight, but one may confidently expect
that any animal tissue (such as human skin) that contains
photoreactivating enzyme will in fact have some of its
lethal damage repaired in this way (see the section
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58
above, "Effects of W on Photoproducts in Animal and
Human Skin").
Another way in which UV-A may decrease the damage
caused by W -B is through the phenomenon of photoprotec-
tion, that is, protection resulting from a preceding
illumination with W-A (see the review by Webb (1977)).
Photoprotection in bacteria may be induced by as little
as 10 minutes of exposure to bright sunlight. It
involves the induction by UV-A of a delay in growth,
allowing more time after W -B irradiation is completed
for error-free dark-repair systems to repair the damaged
DNA (Tsai and Jagger 1981). In addition, the UV-A
effective in Photoprotection may actively inhibit
error-prone repair (Turner and Webb 1981). In wild-type
Escherichia cold bacteria, for example, it has been shown
that sunlight does not induce mutations. The UV-A
wavelengths effective in reducing mutations are the same
as those that delay growth, so this process may be a
Photoprotection effect (Tyrrell 1980). Photoprotection
and growth delay in E. cold are produced by the
absorption of light by an unusual base in transfer RNA,
4-thiouracil (Tsai and Jagger 1981), but this base has
not yet been shown to exist in the cells of higher
animals. However, growth delay in Bacillus subtillis has
been shown to be due to the absorption of light by
quinones in the bacterial membrane (Taber et al. 1978).
The process could also occur in the cells of higher
organisms, although it is not yet tested experimentally.
Synergistic effects have been observed in E. cold
between W-A and W-B wavelengths, at high doses of W-A
(Turner and Webb 1981, Tyrrell 1978, Webb et al. 1978).
Some of these synergisms appear to be due to the destruc-
tion of error-free DNA repair systems by the UV-A radia-
tion. Error-prone recombination repair is responsible
for mutation induced by all UV wavelengths in bacteria.
At high W-A doses, the destruction of error-free repair
systems results in an enhancement of mutation; at low
doses, enhanced mutation is seen only in cells that are
defective in error-free repair systems and thus are not
capable of photoprotection. Such synergisms may operate
in the sunlight induction of skin cancer in those humans
whose skin is defective in error-free repair systems,
such as those with excision-defective xeroderma
pigmentosum (Maher and McCormick 1976).
Finally, consideration must be given to UV-induced
repair systems. In the W-C region, for example, W-C
itself induces the error-prone repair system that is
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59
responsible for most of the mutation produced by UV-C in
bacteria (Witkin 1976). It has recently been discovered
that UV-A will induce a repair system in bacteria that is
capable of repairing damage caused by UV-A (Peters and
Jagger 1981). It is not yet known if the W -A system
will repair damage caused by UV-B, or if it is
error-prone and would therefore produce mutation.
Sunlight or Broad-Band Radiation and Mammalian Cells
In mammalian cells, comparisons have been made between
254-nm radiation, sunlamp radiation in the range from 290
nm to 365 nm, sunlamps filtered to remove wavelengths
below 300 nm, and sunlamps filtered to remove radiation
below 310 nm. The results depend on the response being
studied. For example, in Chinese hamster ovary cells,
sister-chromatic exchanges are proportional to the
amounts of pyrimidine dimers made by 254-nm radiation or
by sunlamp radiation at wavelengths greater than 290 nm
(Reynolds et al. 1979). However, the ratio of killing
efficiency to dimer production, or mutation efficiency to
dimer production, increases as the shorter wavelengths
are removed from the radiation bands with mutation per
dimer increasing more rapidly (Zelle et al. 1980).
Similar results are obtained with Chinese hamster V79
cells, where the longest wavelength band used (greater
than 310 nm) produces, in the time of a typical
irradiation, no cell killing but appreciable mutation,
and with mouse cells, where it produces very little
killing but considerable transformation (Elkind et al.
1978, Suzuki et al. 1981).
Thus it seems as if heterochromatic light in the
longer wavelength regions of UV-B does not act as the sum
of a series of monochromatic wavelengths. On the other
hand, it is not clear which wavelengths are interacting
to give the apparent synergistic effects for mutation and
transformation. The interaction may arise between wave-
lengths in the 310-nm to 315-nm region and longer wave-
lengths such as WV-A. At present, the quantitative
response to an enhancement in UV intensity in the region
of 305 nm to 310 nm as a result of ozone depletion, with
the other, longer UV wavelengths remaining constant, is
not known.
There is some evidence for the existence of UV-induced
repair systems in mammalian cells (Bockstahler and Lytle
1977, Rommelaere et al. 1981). Experiments investigating
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60
these mechanisms have used UV-C irradiation, and, in view
of the shape of the average mammalian action spectrum in
Figure 3.1, one would expect similar findings for UV-B
radiation. The evidence for the existence of W-induced
repair is the ability of irradiated cells to reactivate
W-irradiated viruses that are used to infect the cells.
Irradiation of cells before virus infection results in an
enhanced survival of the infecting W-irradiated viruses,
and in some instances a higher frequency of mutations is
observed in irradiated viruses infecting irradiated cells
(Des Gupta and Summers 1978). The magnitudes of the
observed effects are small, and the extrapolation of such
data from effects on viruses to effects on the cells
themselves has not been made.
RESEARCH RECOMMENDATIONS
The following list of unclarified issues is intended as a
guide for future research. The list is not exhaustive.
It has been limited to those issues that should receive
attention first, but it is not organized according to
priority.
1. It would be useful to know the shape of the action
spectrum for affecting so-called DNA functions of
mammalian cells at wavelengths greater than 313 nm.
2. An understanding is needed of why broad bands of
UV (heterochromatic radiation) do not seem to act like a
simple sum of monochromatic wavelengths in terms of their
effects on DNA. Studies of synergistic effects between
W-A and W-B (for example, in bacteria) are fundamental
to understanding the mechanisms of cancer induction by
sunlight.
3. The quantitative aspects of the immune response of
mice to monochromatic wavelengths versus the response to
broad bands of W -B should be explored. The molecular
and cellular mechanisms for immune system effects and
wavelength dependence should be investigated.
4. An understanding of the mechanism of neoplastic
transformation by UV in vitro is needed. In some rodent
systems, the level of transformation is so high--close to
100 percent--that this transformation process looks
suspiciously like a triggering mechanism that controls
the regulation of cell growth rather than like an effect
on a specific gene or genes (Kennedy et al. 1980).
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5. The characteristics of W-A-induced repair systems
in bacteria need to be determined. The possibility of
the existence of such systems in mammalian cells should
be examined.
6. Data are needed on the rates of repair, in the
dark and in the light, of UV-irradiated human skin cells
as a function of UV dose. The differences, if any,
between acute and chronic irradiations should be deter-
mined. One might be able to study the responses of
individuals who are exposed to high levels of W-B as
part of therapy for psoriasis. The aim of such studies
would be to determine whether the kinetics of dark repair
of damage from dimers in human skin show two components,
a slow and a fast one, as is true for human cells irradi-
ated in vitro. (The fast component represents repair of
DNA in the so-called linker regions of chromatic, and the
slow reaction is the repair in the core regions of
chromatin (Cleaver 1977, Smerdon et al. 1978). The
latter is not as readily accessible to enzymes as is the
former.) Equally important questions are, what other
types of biologically important damages occur in skin,
what are their lifetimes, and are any of them persistent?
Representative terms from entire chapter:
pyrimidine dimers
