Formation of singlet oxygen by urocanic acid by UVA irradiation and some consequences thereof.

Article abstract of DOI:10.1562/0031-8655(2002)075<0565:FOSOBU>2.0.CO;2

Singlet oxygen-initiated decomposition of urocanic acid (UCA) (3-(1H-imidazol-4(5)-yl)-2-propenoic acid) was used to successfully confirm the report that UCA generates singlet oxygen when irradiated with ultraviolet A light (UVA). The UCA-generated singlet oxygen converts UCA to one or more products that then catalyze the further destruction of the UCA with UVA light by singlet oxygen formation. Some nicking of the phiX-174 supercoiled plasmid DNA was observed when UCA was irradiated with UVA to complete destruction of the starting material, and the product mixture was then mixed with the plasmid in the dark. More extensive nicking was seen when the photoproduct mixture and the plasmid were irradiated with UVA light. An "aged" (4 days) solution of UCA photoproduct no longer caused nicking in the dark but retained the capability to nick the plasmid when irradiated. There is evidence for the presence of hydroperoxides in the UCA photolysis product mixture, and the quenching studies with 2-propanol indicate that free radicals are involved in the plasmid-nicking photochemistry. Singlet oxygen does not appear to play a role in the nicking of the plasmid.

Full text of DOI:10.1562/0031-8655(2002)075<0565:FOSOBU>2.0.CO;2

Formation of Singlet Oxygen by Urocanic Acid by UVA Irradiation and Some
Consequences Thereof
Author(s): Elton L. Menon and Harry Morrison
Source: Photochemistry and Photobiology, 75(6):565-569.
Published By: American Society for Photobiology
DOI: http://dx.doi.org/10.1562/0031-8655(2002)075<0565:FOSOBU>2.0.CO;2
URL: http://www.bioone.org/doi/full/10.1562/0031-8655%282002%29075%3C0565%3AFOSOBU
%3E2.0.CO%3B2
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Photochemistry and Photobiology, 2002, 75(6): 565–569
Formation of Singlet Oxygen by Urocanic Acid by UVA Irradiation and
Some Consequences Thereof¶
Elton L. Menon and Harry Morrison*
Department of Chemistry, Purdue University, West Lafayette, IN
Received 26 December 2001; accepted 22 March 2002
ABSTRACT
0.7% of the dry weight of the epidermis (6). Although there
is no significant absorption above 320 nm in the UV ab-
sorption spectrum of UCA at neutral pH, there are reports
on E–Z photoisomerization using broadband light with
wavelengths between 340 and 400 nm (7). In addition, it has
recently been reported that excitation of UCA with mono-
chromatic light at 351 nm leads to singlet oxygen emission
(8). This is the first published report on UCA-generated sin-
glet oxygen formation using UVA light.
Singlet oxygen–initiated decomposition of urocanic acid
(UCA) (3-(1H-imidazol-4(5)-yl)-2-propenoic acid) was
used to successfully confirm the report that UCA gener-
ates singlet oxygen when irradiated with ultraviolet A
light (UVA). The UCA-generated singlet oxygen converts
UCA to one or more products that then catalyze the fur-
ther destruction of the UCA with UVA light by singlet
oxygen formation. Some nicking of the
␾X-174 super-
Photoacoustic calorimetry (PAC) of UCA does, in fact,
indicate the presence of a transition between 320 and 380
nm (8). The maximum for this transition has been estimated
to occur at ca 340 nm (8). In addition, time-dependent den-
sity functional theory calculations have confirmed the exis-
tence of transition at ca 340 nm (9). It was noted that the
PAC spectrum resembles an earlier-published action spec-
trum (2) for the UVA photoaging (e.g. photosagging) of skin
(8). Because there is considerable evidence that UVA aging
of skin is at least partly attributable to ROS, Hanson and
Simon conjecture that the UVA photolysis of UCA may play
a role in the photoaging phenomenon (8). In this article we
(1) verify that the irradiation of UCA with UVA light pro-
duces singlet oxygen; (2) demonstrate that this irradiation
also leads to the creation of radicals; (3) show that excitation
coiled plasmid DNA was observed when UCA was irra-
diated with UVA to complete destruction of the starting
material, and the product mixture was then mixed with
the plasmid in the dark. More extensive nicking was seen
when the photoproduct mixture and the plasmid were
irradiated with UVA light. An ‘‘aged’’ (4 days) solution
of UCA photoproduct no longer caused nicking in the
dark but retained the capability to nick the plasmid when
irradiated. There is evidence for the presence of hydro-
peroxides in the UCA photolysis product mixture, and
the quenching studies with 2-propanol indicate that free
radicals are involved in the plasmid-nicking photochem-
istry. Singlet oxygen does not appear to play a role in the
nicking of the plasmid.
of UCA in this region can sensitize the nicking of ␾X-174
plasmid DNA; and (4) provide evidence that an intermediate
photoproduct may play a major role in these outcomes.
INTRODUCTION
Chronic exposure to ultraviolet A (UVA) light (320–400
nm) can cause premature aging and cancer of the skin (1–
4). The mechanisms by which these processes occur have
not been completely determined. However, there is strong
evidence that reactive oxygen species (ROS) plays a role,
presumably generated by an endogenous UVA-absorbing
sensitizer (1,5). (E)-urocanic acid (UCA) (3-(1H-imidazol-
4(5)-yl)-2-propenoic acid) is a metabolite of histidine and a
substantial constituent of the stratum corneum, making up
MATERIALS AND METHODS
Chemicals. (E)-UCA was obtained from Sigma (St. Louis, MO) and
was used as received unless otherwise stated. The (E)-UCA was
recrystallized as reported previously (10). A photostationary-state
mixture of (E)- and (Z)-UCA was obtained by irradiation of an Ar-
saturated (E)-UCA solution using 311 nm light. Superoxide dismu-
௡
௡
tase (SOD), Trizma hydrochloride, Trizma base, boric acid, cata-
lase and -histidine monohydrochloride were also from Sigma. (Z)-
L
UCA was prepared as reported previously (11). Silica-bound rose
bengal was prepared as reported previously (12). NaOD solutions
10% in D2O were prepared by dissolving 1 g of NaOH in 10 mL
of D2O, evaporating the solvent and reconstituting the solids with
10 mL of D2O three times. Glycerol and ethidium bromide were
from Aldrich (Milwaukee, WI). Bromophenol blue was from Fisher
(Fairlawn, NJ). Ethylenediamine tetraacetic acid (EDTA) was from
ICN Biomedical Inc. (Aurora, OH). Agarose was from Bethesda
¶Posted on the web site on 27 March 2002.
*To whom correspondence should be addressed at: Department of
Chemistry, Purdue University, West Lafayette, IN 47097-1393,
USA. Fax: 765-496-2592; e-mail: morrison@science.purdue.edu
Abbreviations: EDTA, ethylenediamine tetraacetic acid; Form I, cir-
cular supercoiled plasmid DNA; Form II, circular relaxed plasmid
DNA; HPLC, high-performance liquid chromatography; PAC,
photoacoustic calorimetry; ROS, reactive oxygen species; SOD,
superoxide dismutase; UCA, urocanic acid; UVA, ultraviolet A
radiation at 320–400 nm.
Research Lab (Gaithersburg, MD). ␾X-174 plasmid DNA was from
New England BioLabs (Beverly, MA). High-performance liquid
chromatography (HPLC) water was distilled in glass using a Corning
MP-1 water still, whereas other HPLC grade solvents, phosphate
salts, NaOH, acetic acid and ammonium acetate were from Mal-
᭧
2002 American Society for Photobiology 0031-8655/02
$5.00ϩ0.00
565

566 Elton L. Menon and Harry Morrison
Table 1. Effects of wavelength, temperature, pH, singlet oxygen quencher and presence of oxygen and solvent on (E)-UCA photolysis
with UVA light*
Time of
irradiation
(h)
% destruction
Temperature of UCA
%
Sample description
Light source
Solvent
pH or pD
(Њ
C)
(Z ϩ E) isomerization
(E)-UCA
(E)-UCA
(E)-UCA
(E)-UCA
(E)-UCA
(E)-UCA
(E)-UCA
(E)-UCA
Ͼ330
Ͼ330
Ͼ330
Ͼ330
Ͼ330
Ͼ330
Ͼ311
Ͼ330
Ͼ330
20
20
20
20
20
20
0.5
20
20
D2O
D2O
D2O
D2O
D2O
H2O
D2O
D2O
D2O
5.5
7.0
7.8
7.8
7.8
8.0
7.8
7.8
7.8
8
8
8
0
30
30
30
30
30
2.0
3.5
1.8
1.0
1.5
1.0
6.0
50.0
5.0
8.0
15.4
60.2
11.5
80.0
14.0
0
ϩ
15 equivalents of histidine
3.5
0
(E)-UCA under Ar
*In all cases the solution occupies ca 25% of the reaction vessel volume, i.e. there is a large excess of oxygen present.
linckrodt (Paris, KY). Deuterium oxide was from Cambridge Isotope
Laboratories (Andover, MA). EM Quant peroxide test strips were
lutions. After irradiation, 10 ␮L of 10ϫ stop buffer–loading solution
௡
(50% glycerol, 0.2% bromophenol blue, 0.05 M EDTA in 100 mM
Tris buffer, pH 8) was added to each sample, and the samples were
loaded onto a 0.8% agarose gel containing ethidium bromide (0.5
from EM Science (Gibbstown, NJ).
Instruments. An Orion 290A pH meter with a Triode pH elec-
௢
trode was used to measure the pH of solutions. The pD values of
buffered D2O solutions were uncorrected pH meter readings. UV
absorption spectroscopy was done using a Varian Cary 100 scanning
UV–visible spectrophotometer. Samples were analyzed in 1 cm2
matched quartz cells from Wilmad (Buena, NJ). HPLC analyses
were performed using a Varian 5000 series liquid chromatograph
␮
g/mL). The gel was run in 0.5
ϫ Tris-borate EDTA buffer contain-
ing ethidium bromide (0.5 g/mL). After electrophoresis at 132 V
␮
for approximately 1.5 h, the DNA was visualized by UV light using
a NucleoVision gel analyzer. Gel bands were quantified using Gel
Expert software, with the intensities corrected for the decreased
binding of ethidium bromide to supercoiled DNA (i.e. circular su-
percoiled plasmid DNA [Form I] is 1.4 times less responsive than
circular relaxed plasmid [Form II] or linear plasmid DNA) (13,14).
fitted with a 7125 Rheodyne injection valve and a 200 ␮L injection
loop. A Varian 2050 variable wavelength detector set at 254 nm
monitored the column effluent, and the data were recorded using
Packard Radiomatic Flo-One analysis software. An Alltech Econosil
RESULTS AND DISCUSSION
C-8 (4.6 mm
ϫ 25 cm) reversed-phase stainless steel column was
used with the following isocratic condition: 1 mL/min 100% 50 mM
ammonium acetate buffer (pH 5). The retention times were 5.0 and
9.6 min for the E- and Z-isomers, respectively. For quantitative work
the (Z)-isomer area counts were multiplied by a factor of 1.4 to
correct for its lower extinction coefficient compared with that of (E)-
UCA (6).
UCA-sensitized formation of singlet oxygen by UVA
light
In our initial studies we confirmed that the sensitized for-
mation of singlet oxygen by UVA light could be monitored
using the singlet oxygen–initiated decomposition of UCA as
our probe (15). A 2 mL solution of 3 mM (E)-UCA in 50
mM phosphate buffer, pH 8, was saturated with O2 and ir-
Irradiations. Photolyses of UCA with ␭ Ͼ 330 nm light used
cylindrical Pyrex tubes (12
ϫ 150 mm) in a photolysis box in which
samples in a turntable revolved around a water-cooled 450 W Han-
ovia medium-pressure mercury lamp filtered with a uranium yellow
glass filter. The incident light at the tubes was measured as 4.6 mW/
cm2 using a UVX Radiometer with a UVX-36 sensor, from
UltraViolet Products, Inc. (San Gabriel, CA). The samples were im-
mersed in a temperature-controlled water bath. Alternatively, mono-
chromatic 311 nm radiation was provided by Philips Ultraviolet B
low-pressure 311 nm lamps in a fan-cooled photolysis box with a
turntable occupying a central position. Singlet oxygen was generated
by the irradiation of silica-bound rose bengal using a slide projec-
tor’s 500 W tungsten lamp filtered with an LL-500 Corion corp.
filter that transmits ␭ Ͼ 500 nm. The presence of peroxide in solu-
radiated at 30ЊC with ␭ Ͼ 330 nm for 20 h. This is a region
where UCA shows negligible light absorption (8,16). HPLC
analysis indicated that 6% of the initial (E)-UCA had un-
dergone isomerization, whereas 14% of the total UCA (i.e.
E- plus Z-isomers) had been destroyed. The reaction de-
pended on the presence of oxygen and was greatly enhanced
when D2O was used as solvent. In the latter case there was
as much as 80% destruction of the total UCA after 20 h of
irradiation. Also, reaction in D2O was quenched by histidine,
a known singlet oxygen scavenger (17). In addition, this re-
action showed temperature and pH dependence, being more
reactive at higher temperatures and at alkaline pH. At pH 8,
UCA exists predominantly as the carboxylate anion (6). Nei-
ther spectroscopy nor theoretical calculations give evidence
for any significant electronic perturbations at wavelengths
greater than 320 nm across the pH 6–8 region (9). It is in-
teresting that histidine also shows a strong increase in its
reactivity with independently generated singlet oxygen on
going from pH 6 to 8 in a manner that matches well with
the histidine pK curve (18), i.e. the protonated imidazole
ring is relatively unreactive with singlet oxygen. The salient
data are summarized in Table 1.
௡
tion was detected using EM Quant peroxide test strips.
Photocleavage of
phosphate buffer, pD 7.8, was added to 30
174 phage DNA to obtain a DNA solution, which was 205
base pairs. Two different solutions were irradiated with DNA: (1) a
3 mM solution of 12 recrystallized (E)-UCA in 50 mM phosphate
␾
X-174 phage DNA. Sixty microliters of 50 mM
L of commercial X-
M in
␮
␾
␮
ϫ
buffer, pD 7.8; and (2) a 15 mL 3 mM solution of recrystallized (E)-
UCA in 50 mM phosphate buffer, pD 7.8, which was saturated with
O2, capped, sealed with parafilm and irradiated with ␭ Ͼ 330 nm
for 34 h. These solutions were saturated with O2 or Ar, and 7
was placed into end-tapered tubes containing 3 L of X-174 phage
DNA solution. These were inserted into Corning 10 75 mm Pyrex
␮L
␮
␾
ϫ
test tubes, the tubes were capped with septa and parafilm sealed,
and the atmosphere was saturated with O2 or Ar. Some experiments
included histidine hydrochloride (45 mM) to scavenge singlet oxy-
gen, 2-propanol (0.5 M) as a free radical scavenger, catalase (25
mL) and SOD (25 g/mL). Controls also included unirradiated
DNA, irradiated DNA free of drug and unirradiated DNA–UCA so-
␮g/
␮
These results are in contrast with what one observes when

Photochemistry and Photobiology, 2002, 75(6) 567
Figure 1. Change in absorption spectra as a function of irradiation
time when (E)-UCA is photolyzed with light 330 nm in D2O, pD
Ͼ
Figure 2. Time study for the photodestruction of various samples
7.8. From left to right in the figure, the time points are 0, 5, 10, 15
and 20 h of irradiation.
of UCA in D2O, pD 7.8, with light
Ͼ 300 nm.
photooxidative degradation of the histidine is accompanied
by the formation of a pale yellow polymer. The reaction is
faster in basic media, quenched by 2-propanol and facilitated
by a histidine photoproduct, with a discernible induction pe-
riod. This photoproduct is attributed to a histidine hydroper-
UCA is irradiated at 311 nm (Table 1) and are actually more
similar to the consequences of irradiation at 254 nm. It has
been demonstrated that photolysis of UCA at these wave-
lengths excites the molecule into two different singlet states
that are embodied within one absorption envelope having a
•
oxide that is formed from His reacting with ground-state
␭
at 280 nm at pH 7.2 (19–21). In particular, we have
oxygen. The authors suggest that the histidine triplet state
initiates the chemistry by generating singlet oxygen (22).
To examine the nature of (E)-UCA photodegradation by
its photoproducts, we irradiated a 3 mM stock solution of
max
noted in the earlier work (21), and see again here, that 311
nm produces efficient E–Z isomerization and little decom-
position. Photolysis at 254 nm gives a higher ratio of de-
composition to isomerization, at least in part because of the
10-fold less efficient isomerization chemistry at this wave-
length (21). These results indicate that excitation beyond 330
nm leads to different chemistry from that seen at 311 nm.
During this work we noted that the self-sensitized oxida-
tion of UCA leads to the development of a yellow color in
the photolysis solutions after ca 10 h of irradiation. This is
evident in the UV–visible absorption spectrum of the reac-
tion mixtures (Fig. 1).
12
ϫ
recrystallized (E)-UCA in 50 mM phosphate buffer, pD
330 nm to complete the destruction of the
7.8, with light
Ͼ
UCA. Additional (E)-UCA was then added to the photolyzed
mixture to form a 1 mM (E)-UCA solution. Irradiation of
this solution under O2 with light
Ͼ 330 nm for 4 h gave a
92% destruction of the UCA. Irradiation under Ar resulted
only in isomerization and not in destruction of the UCA.
Photolysis in oxygen in the presence of 15 molar equivalents
of histidine or 0.5 M 2-propanol led to 9 or 84% destruction
of UCA, respectively. These results clearly indicate that the
photodegradation of UCA by its photoproducts is oxygen
dependent and that the formation of singlet oxygen plays a
major role in the sensitized degradation. We believe that it
is also more than coincidental that the amount of degradation
nonquenchable by histidine (9%) correlates well with the
diminution in UCA destruction (8%) in the presence of the
hydroxyl radical trap, 2-propanol (see also subsequently).
UCA has been shown to be a better hydroxyl radical scav-
enger than other 4-(5-) substituted imidazole derivatives
such as histidine (23). It is noteworthy that a freshly irra-
diated solution of UCA, taken for the total destruction of
UCA, tested strongly positive for the presence of hydrogen
In a more quantitative analysis of the reaction as a func-
tion of reaction time, we irradiated four sets of 2 mL solu-
tions of 3 mM UCA in oxygen-saturated D2O containing 50
mM phosphate buffer, pD 7.8, at 30ЊC with ␭ Ͼ 330 nm.
The UCA samples consisted of commercial (E)-UCA, a sam-
ple of (E)-UCA that had been recrystallized five times, syn-
thetic (Z)-UCA (12) and a mixture of (E)- and (Z)-UCA
prepared by 311 nm photolysis of the commercial E isomer.
The reactions were monitored at 5, 10, 15 or 20 h. The total
loss of the UCA in each case is presented in Fig. 2.
It is clear from this figure that the photodestruction of the
UCA involves an induction period, which we visually ob-
serve to be accompanied by the generation of the yellow
color. This induction period is present in all the samples.
௡
peroxide or hydroperoxides (or both) using an EM Quant
However, the (Z)-UCA and the 5
ϫ
recrystallized sample of
peroxide test strip.
(E)-UCA show the slowest rate of destruction in the postin-
duction process. The commercial sample of E is faster than
either of these, and the (E)–(Z) mixture is faster yet. The
latter observation is consistent with our observation that the
reaction of purified (E)-UCA, (Z)-UCA or an (E)–(Z) mix-
ture with independently generated singlet oxygen using pho-
tosensitization by silica-bound rose bengal indicates that the
Z isomer is ca 1.4 times more reactive toward singlet oxygen
than the E isomer is (data not shown). There are some strik-
ing similarities between our observations and those made
with histidine, albeit upon its photolysis at 254 nm (22).
Hasselmann and Laustriat (22) noted that a self-sensitized
In summary, we observe that (1) the UCA forms singlet
oxygen with UVA light, with the reaction most efficient in
D2O, on the basic side of a pD range of 5.5–7.8; (2) the
UCA reacts with singlet oxygen to form products, one or
more of which are colored; (3) the product mixture is re-
sponsible for a much more rapid generation of singlet oxy-
gen with UVA light, possibly because of greater light ab-
sorption at these wavelengths; (4) the photoproducts include
peroxides, and a portion of the UCA degradation involves
free radicals; and (5) the commercial (E)-UCA contains an
impurity that accelerates these processes. We emphasize,
however, that the extensively crystallized (E)-UCA and the

568 Elton L. Menon and Harry Morrison
synthetic, purified (Z)-UCA show the same ability to gen-
erate the yellow intermediate and photosensitize their oxi-
dative self-destruction.
Effects of UVA irradiation of UCA on
supercoiled DNA plasmid
␾X-174
In order to study the effect of UVA irradiation of UCA on
biomolecules, we irradiated 12 recrystallized (E)-UCA
X-174 supercoiled plasmid DNA at a phosphate–UCA
0.1. As with the previous experiments, the samples
ϫ
with
ratio
␾
ϭ
were in 50 mM phosphate buffer, pD 7.8. Agarose gel elec-
trophoresis was used to separate Form I from its nicked
Form II. We found that irradiation with 12ϫ recrystallized
(E)-UCA for 4 h produced only a small (ca 6%) level of
nicking of the plasmid DNA (data not shown). In contrast,
the irradiation of prephotolyzed UCA solutions with the
plasmid generated extensive nicking. After a number of such
experiments, it became clear that aging of the irradiated
UCA solutions affected the plasmid results. We, therefore,
irradiated a 3 mM stock solution of 12
ϫ recrystallized (E)-
UCA in 50 mM phosphate buffer, pD 7.8, in D2O to the
complete loss of the UCA (and the concomitant appearance
of a yellow color) and divided the solution into two parts.
One portion was immediately mixed with the plasmid DNA,
and the solution was irradiated for an additional 4 h. The
second portion was allowed to stand in the dark for 4 days
and then irradiated with the plasmid. The results are shown
in Fig. 3.
There are several noteworthy observations. (1) UVA light
has no effect on the plasmid; the results in Lane 1 are typical
of what we see for the plasmid when left in solution in the
dark. (2) There is a modest, but apparently real, nicking of
the plasmid by the fresh solution in the dark that is not
evident for the ‘‘aged’’ solution (Lane 2 vs Lane 1). (3)
Irradiation of either solution produces extensive nicking (cf
Lane 4). To determine the effect of potential quenchers, we
use the data from Lanes 3 to 8 vs Lane 2, relative to the
results for Lane 4 vs Lane 2. (4) The histidine provides no
protection against nicking (Lane 6), i.e. there is no evidence
for singlet oxygen as a source of the nicking. Likewise, Ar
degassing provides no protection (Lane 8). The contrasting
effects of histidine and Ar here, relative to those observed
for the sensitized self-destruction (see previously), may be a
consequence of the very high sensitivity of UCA toward
singlet oxygen (24). Note that Ar degassing is by necessity
Figure 3. Irradiation of UCA photodecomposition products with
plasmid DNA using light 330 nm for 4 h (see Materials and
Methods for details). A fresh photoproduct solution was used for
the experiment shown in the top gel; a 4 day aged solution was used
Ͼ
for the bottom gel. Lane 1, DNA
products of UCA in the dark; Lane 3, DNA
UCA catalase (25 g/mL) light; Lane 4, DNA
of UCA light; Lane 5, same as lane 4 2-propanol (0.5 M); Lane
6, same as lane 4 histidine (45 mM); Lane 7, same as lane 4
SOD (25 g/mL); Lane 8, same as lane 4 under Ar.
ϩ
light; Lane 2, DNA
photoproducts of
photoproducts
ϩ photo-
ϩ
ϩ
␮
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
␮
presence of hydroperoxides in the UCA photoproduct mix-
ture (see previously). Organic peroxides and hydroperoxides
absorb in the UVA region and can photolytically generate
free radicals in an oxygen-independent mechanism (26).
UCA solutions irradiated to complete destruction with light
Ͼ
330 nm and tested immediately after irradiation gave a
much more (ca 10-fold) positive hydroperoxide test than
aged solutions did. Such hydroperoxides could also be re-
sponsible for the dark nicking seen for freshly irradiated
UCA solutions.
incomplete because the 3 ␮L solution of DNA was not de-
CONCLUSIONS
gassed to avoid mechanical nicking of the plasmid. How-
ever, we would have expected at least partial protection by
degassing the UCA solution. (5) The addition of 2-propanol
provides significant protection (Lane 5), evidence for radical
intermediates as part of the nicking process. (6) There is
evidence for protection by SOD (Lane 7), especially in the
fresh solutions. Though UCA has been shown to generate
superoxide (25), it is hard to reconcile this result with the
Ar studies at this point. Similarly, though the observation is
reproducible, we have no explanation as yet for the catalase
result in the aged solution (Lane 3).
We have successfully confirmed the report that UCA gen-
erates singlet oxygen when irradiated with UVA light. The
singlet oxygen converts UCA to one or more products that
are more effective in causing the destruction of UCA with
UVA light than UCA is. The photodestruction of UCA by
its photoproduct(s) is caused by the generation of singlet
oxygen. A freshly generated UCA photoproduct solution
causes some nicking of Form I plasmid DNA. This dark
nicking is not evident for an aged (4 days) photolysis solu-
tion. UVA irradiation of the UCA product mixture with the
plasmid causes extensive nicking that is inhibited by 2-pro-
panol but not by histidine or by Ar degassing. Thus, the
formation of singlet oxygen does not appear to play a role
The combination of a lack of protection by Ar degassing,
quenching of nicking by a free radical scavenger and sus-
ceptibility to further UVA irradiation is consistent with the

Photochemistry and Photobiology, 2002, 75(6) 569
10. Meher, A. H. and H. Tabor (1953) Deamination of histidine to
form urocanic acid in liver. J. Biol. Chem. 201, 775–784.
11. Mohammad, T. and H. Morrison (2000) A convenient synthesis
of cis-urocanic acid and endogenous immunosuppressant. OPPI
Briefs 23, 581–583.
12. Tamagaki, S., C. E. Liesner and D. C. Necker (1980) Polymer-
based sensitizers for photochemical reactions. Silica gel as a
support. J. Org. Chem. 45, 1573–1576.
in the nicking of the plasmid. The UCA photoproduct so-
lutions test positive for the presence of hydroperoxides, and
these are likely to be major contributors to the plasmid-nick-
ing chemistry. The studies designed to elucidate the structure
of the UCA photoproduct(s) and the mechanisms of the dark
and photoinitiated nicking of plasmid DNA are in progress.
We are also further elaborating the potential photobiological
consequences of UCA–UVA photochemistry.
13. Lloyd, R. S., C. W. Haidle and D. L. Robberson (1978) Bleo-
mycin-specific fragmentation of double-stranded DNA. Bio-
chemistry 17, 1890–1897.
14. Hongtao Y., Y. Wok, L. H. Hurley and S. M. Kerwin (2000)
Efficient, Mg2ϩ-dependent photochemical DNA cleavage by the
antitumor quinobenzoxazine (s)-A-62176. Biochemistry 39,
10236–10246.
15. Mohammad, T., A. Kasper and H. Morrison (1994) Urocanic
acid photobiology. Purine assisted photooxidation to 1H-imid-
azole-4(5)-carboxaldehyde. Tetrahedron Lett. 35, 4903–4906.
16. Hanson, K. M., B. Li and J. D. Simon (1997) A spectroscopic
study of the epidermal ultraviolet chromophore trans-urocanic
acid. J. Am. Chem. Soc. 119, 2715–2721.
Acknowledgements We thank the Purdue Gerontology Program for
a grant in support of this research. E.L.M. also acknowledges a
training grant 5T32O9634 from the National Cancer Institute of the
NIH administered by the Purdue Cancer Center. We thank Dr. Taj
Mohammad for observations made at the outset of this project and
Professor Tad Sarna for helpful discussions.
REFERENCES
17. Wilkinson, F. and J. G. Brummer (1981) Rate constants for the
decay and reactions of the lower electronically excited singlet
state of molecular oxygen in solution. J. Phys. Chem. Ref. Data
10, 809–1000.
18. Matheson, I. B. C. and J. Lee (1979) Chemical reaction rates
of amino acids with singlet oxygen. Photochem. Photobiol. 29,
879–881.
19. Hanson, K. M. and J. D. Simon (1998) The origin of the wave-
length-dependent photoreactivity of trans-urocanic acid. Pho-
tochem. Photobiol. 67, 538–540.
20. Ryan, W. L. and D. H. Levy (2001) Electronic spectroscopy
and photoisomerization of trans-urocanic acid in supersonic jet.
J. Am. Chem. Soc. 123, 961–966.
1. Urbach, F. (1992) Biological Responses to Ultraviolet A Radi-
ation. Valdenmar, Overland Park, KS.
2. Bissett, D. L., D. P. Hannon and T. V. Orr (1989) Wavelength
dependence of histological, physical, and visible changes in UV-
irradiated hairless mouse skin. Photochem. Photobiol. 57, 763–
769.
3. Bissett, D. L., D. P. Hannon and T. V. Orr (1987) An animal
model for solar-aged skin: histological, physical and visible
changes in UV-irradiated hairless mouse skin. Photochem. Pho-
tobiol. 46, 367–378.
4. Simon, J. D. (2000) Spectroscopic and dynamic studies of the
epidermal chromophores trans-UCA and eumelanin. Acc. Chem.
Res. 33, 307–313.
5. Pourzand, C. and R. M. Tyrell (1999) Apoptosis, the role of
oxidative stress and the example of solar UV radiation. Photo-
chem. Photobiol. 70, 380–390.
6. Mohammad, T., H. Morrison and H. HogenEsch (1999) Uro-
canic acid photochemistry and photobiology. Photochem. Pho-
tobiol. 69, 115–135.
21. Morrison, H., C. Bernasconi and G. Pandey (1984) A wave-
length effect on urocanic acid photoisomerizaton. Photochem.
Photobiol. 40, 549–550.
22. Hasselmann, C. and G. Laustriat (1976) Photochemical study of
L-histidine in aqueous solution. J. Chim. Phys. 73, 767–774.
23. Kammeyer, A., T. A. Eggelte, J. D. Bos and M. B. M. Teunissen
(1999) Urocanic acid isomers are good hydroxyl radical scav-
engers: a comparative study with structural analogues and with
uric acid. Biochim. Biophys. Acta 1428, 117–120.
24. Haralampus-Grynaviski, N., C. Ransom, T. Ye, M. Rozanows-
ka, M. Wrona, T. Sarna and J. D. Simon (2002) Photogeneration
and quenching of reactive oxygen species by urocanic acid. J.
Am. Chem. Soc 124, 3461–3468.
25. Morrison, H. and R. M. Deibel (1988) Urocanic acid photobi-
ology. Photooxidation and superoxide formation. Photochem.
Photobiol. 48, 153–156.
26. Calvert, J. G. and J. N. Pitts (1966) Photochemistry, pp. 447–
450. John Wiley, New York.
7. Weber, L. J., E. Wang and E. C. DeFabo (1997) The effects of
UVA-I (320–400 nm), UVA-II (320–340 nm) and UVA-I
ϩ II
on the photoisomerization of urocanic acid in vivo. Photochem.
Photobiol. 66, 484–492.
8. Hanson, K. M. and J. D. Simon (1998) Epidermal trans-uro-
canic acid and the UV-A-induced photoaging of the skin. Proc.
Natl. Acad. Sci. USA 95, 10576–10578.
9. Danielsson, J., J. Ulicny and A. Laaksonen (2001) A TD-DFT
study of the photochemistry of urocanic acid in biologically
relevant ionic, rotameric and protomeric forms. J. Am. Chem
Soc. 123, 9817–9821.