Photochemistry and Photobiology Vol. 48, No. 2, pp. 153-156, 1988 Printed in Great Britain. All rights reserved

oO31-8655/88 $03 .M)+O.OO Copyright 0 1988 Pergamon Press plc

UROCANIC ACID PHOTOBIOLOGY. PHOTOOXIDATION AND SUPEROXIDE FORMATION"

HARRY MORRISONt and ROSE MARIE DEIBEL Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA

(Received 2 December 1987; accepted 16 February 1988)

Abstract-Photolysis of urocanic acid (UA) and nitro blue tetrazolium (NBT"), under conditions wherein the NBTZ+ absorbs the light, leads to oxidation of the UA by the NBT2+ excited state. When UA is itself photolyzed in the presence of oxygen and NBT2+, NBT2+ reduction again occurs, but in this event via the intermediate formation of superoxide.

INTRODUCTION

Urocanic acid (2-propenoic acid, 3-( 1H-imidazol- 4-yl)(UA$) has been a subject of intense recent photobiological interest because of its substantial presence in the skin, its potential role as a natural photoprotecting agent, its photochemical incorpor- ation into DNA and bovine serum albumin, and its apparent involvement in the phenomenon of photo- induced immunosuppression (for leading references and a review, see Ross et al . , 1986; Deibel and Morrison, 1987; Morrison and Deibel, 1986). To date, the photochemistry of UA which has been best characterized has been the EiZ isomerization and 2+2 cycloaddition chemistry characteristic of the acrylic acid functionality (Morrison and Deibel, 1986). As part of our ongoing interest in the photo- chemical and photophysical properties of this com- pound, we have now studied its ability to act as an electron donor, using as our probe the highly electron-affinic reagent, 3,3'-(3,3'-dimethoxy-[l ,l'- biphenyl]-4,4'-diyl)bis[2-(4-nitrophenyl)-5-phenyl- 2H-tetrazolium] dichloride, (p-nitro blue tetra- zolium chloride, NBT2+ (Tsou et al . , 1956). The facile reduction of such ditetrazolium salts to difor- mazans is well known (Hooper, 1969), in this case leading to 111'-(3,3'-dimethoxy[ 1,l'-biphenyl]-4,4'- diy1)bis [5-(4-nitrophenyl)-3-phenyl]formazan, (p- nitro blue diformazan, DF). The reaction proceeds through a series of one-electron transfers as shown in Fig. 1 (Bielski et al . , 1980). Typically, NBT2+ reduction has been used as an assay for the presence of superoxide (Miller and Kerr, 1966; Beauchamp and Fridovich, 1971), in which case reduction of the NBT2+ occurs by the series of steps shown in Eqs. 1-4 (Bielski et al., 1980). The reaction is followed spectrophotometrically by monitoring the develop- ment of absorption in the visible region

*Organic Photochemistry, 75. Part 74. L. De Cardenas

tTo whom correspondence should be addressed. $Abbreviations: UA, urocanic acid; NBT'+, nitro blue

tetrazolium; SOD, superoxide dismutase; EDTA, ethyl- enediamine tetraacetic acid; 6MP, 6-mercaptopurine.

et al. (1987) J . org. chern. 53, 219-221.

NBTZ' + 0; + NBT+. + 0 2

2NBT+. + H 2 0 + NBT*+ + (MF+---OH-)

(1)

(2)

(NBT+.--OH-) + NBT+. + H20 +

NBT2+ + (MF+--OH-) + OH- (3)

2(NBT+.---OH-) + H20 +

NBT2+ + (MF+--OH-) + 20H- (4)

(usually at 560 nm) generated by the formation of the colored mono and diformazans (Hooper, 1969; Eadie et a l . , 1970; Bielski et al . , 1980). Because of the complexity of the chemistry associated with the reduction, it has been noted that this assay is most reliable when employed as a qualitative or semi- quantitative indicator (Eadie et a l . , 1970; Bielski et al . , 1980).

In this study we demonstrate that both the ground and excited states of urocanic acid can participate in electron transfer reactions, and that in the latter case, the presence of oxygen leads to the formation of superoxide ion.

MATERIALS AND METHODS

Chemicals. E-Urocanic acid.2H20 (recrystallized from water) was from Aldrich. Nitro blue tetrazolium, super- oxide dismutase and riboflavin were from Sigma.

Methods. Ultraviolet analyses were done on a modified Beckmann DU spectrophotometer. Photolyses were carried out with a Canrad-Hanovia 450 watt high pressure mercury lamp (model 679A), filtered either through uran- ium yellow (passing wavelengths >330 nm) or Corex (pass- ing wavelengths >275 nm) glass. Vycor tubes were used in a turntable immersed in a thermostatted cooling bath.

RESULTS

Selective excitation of N B P + using a uranium yellow filter

Photolysis of NBP' in the presence of UA. Four vycor tubes were each charged with 5 me of a 0.1 M phosphate buffered solution (pH 7.0) of E-UA (2.0 >: M ) . The solutions in two tubes also

153

154 HARRY MORRISON and ROSE MARIE DEIBEL

ditetrazolium ( N BT2*)

tetrazolinyl radicol (NBT+.) tetrazolium

-(e-,H+) e;H+ I 11 1

Rp-N-N

/N--N-R2 I 'C-R, RI-C /

N=N-R,-R,-N=N \\ formazan (MF+) tetrazolium

A l

formazan tetrazolinyl radical

H -(e;H+) e;Ht H

R2-N-N \\

I (T 1 "I-% //N-N-R2

N=N-R,-Rj-N=N diformazan (DF)

R,=C,H, ;R2=p-NO2C6H,;R3=rn-CH3OC6H4

Figure 1. Mechanism of the stepwise reduction of NBTZ+ to diformazan (from Bielski et al., 1980).

contained 64 x M NBT2+ while the solutions in the other two tubes contained 128 x M NBF'. One set of tubes with each of the NBT2+ concentrations was bubbled with argon for 40 min; the second set was bubbled with oxygen for 40 min. All four tubes were irradiated at 10°C for 2 h in the turntable using the uranium yellow filter and the absorbances read at 560 nm. The data are shown in Table 1.

Dependence of N B F + reduction by UA on pH. Solutions of E-UA (2.0 x lo-' M) and NBT2+ (64 x lop6 M) were made to pH values ranging from 4.0 to 8.5 using phosphate buffer, degassed with argon for 0.5 h and irradiated through uranium yellow at 10°C for 2 h. Absorbances were read at 560 nm and are shown in Table 2.

Comparison of E-UA and ethyl urocanate as N B F + reductants. The ethyl ester of E-UA was compared to UA by photolyzing 5 me argon degassed and air saturated pH 7.0 solutions (2.0 x

M ) at 10°C through uranium yellow for 2 h with the turntable. For UA, the A5M) values were 0.12 and 0.02 (argon

M ) of each with NBT2+ (64 x

ogous values were 0.07 and 0.01. The presence of SOD was found to have no affect on the extent of reduction in the air saturated solutions.

Co-excitation of UA and N B F + using a Corex filter

Photolysis of N B F + and CIA (or ethyl urocanate) with and without SOD. Four vycor tubes were each charged with 5 me of a 0.1 M phosphate buffered solution (pH 7.0) of E-UA (2.0 x M) and NBT2+ (64 x M). Two of the tubes also con- tained 0.5 mg of SOD. One set of tubes with and without SOD was bubbled with argon for 45 min and a second set was bubbled with oxygen. All four tubes were irradiated at 10°C for 100 min in the turntable using the Corex filter and the absorbances read at 560 nm. The data are shown in Table 3.

An analogous experiment was run using both UA and ethyl urocanate, with air rather than oxygen. The phosphate buffered solutions were 2.0 x M in the UA or ester and 64 X M NBT2+, and one set contained 0.5 mg of SOD. The solutions were bubbled with air or argon for 30 min and the solutions irradiated through Corex for 2 h at 10°C. The results are presented in Table 4.

Photolysis of NBP' and UA as a function of pH. Five vycor tubes were each charged with 5 mP of phosphate buffered solutions of E-UA (2.0 X

M); the solutions

Table 1. Photolysis of NBTZ+ in the presence of E-UA

NBTZ+ Concentration

M) and NBT2+ (64 x

(CLW OJAr As,,,

64

128

Ar 0.23 0 2 0.03

Ar 0.35 0, 0.05

Table 2. pH dependence of the photo- reduction of NBT2+ by E-UA

4.0 0.01 5.5 0.06 6.9 0.21 7.8 0.43 8.5 0.58

Table 3. Photolysis of NBT2+ and UA with and without SOD through Corex

Ar 0.63 + Ar 0.56 - 0 2 0.16

-

+ 0 2 0.01 and air respectively); for ethyl urocanate, the anal-

Urocanic acid photooxidation 155

ranged in p H from 4.6 to 8.4. The solutions were bubbled with argon for 40 min and irradiated for 100 min at 10°C in the turntable using the Corex filter. Absorbances were read at 560 nm and are shown in Table 5. Included in Table 5 are data for a comparable 120 min photolysis run in air rather than argon.

UA degradation and N B F + reduction by ribo- flavin generated superoxide. Superoxide was gener- ated independently using the photolysis of riboflavin (Gibson et al., 1984) and the degradation of U A was monitored. Solutions (5 me) of 2.0 x lo-' M U A , 30 x M riboflavin and 1 X M E D T A in p H 7.0 buffer were bubbled with A r or oxygen for 0.5 h. A third tube containing only a U A solution in buffer was bubbled with oxygen for 0.5 h as a reference. The three samples were photolyzed through uranium yellow at 10°C for 3.6 h and analyzed for loss of U A by hplc. The data (Yo loss of UA): U A alone, 2%; UA/riboflavin/Ar, 4%; UAlriboflavin/Oz, 65%. A separate control con- firmed both the generation of superoxide and its reduction of NBT2+. A solution of 30 x M riboflavin, 64 X M NBT2+ and 1 X lo-' M EDTA in p H 7.0 buffer was divided into two por- tions, and to one was added 0.5 mg of SOD. Each portion was then further divided into two 4 mY solutions, one of which was bubbled with oxygen and the other with Ar for 0.5 h. The four solutions were irradiated at 10°C through uranium yellow for 1.5 h, and absorbances then read at 560 nm (Table 6).

DISCUSSION

Excitation of NBTZ+/UA solutions under two dif- ferent sets of conditions has been described herein, i.e., using either a Corex or a uranium yellow filter. Only light of wavelength >330 nm is transmitted through the uranium yellow filter, and since U A is transparent in this region, absorption of the incident radiation under these circumstances is limited to the NBT2+. In this event we observe:

1) The NBT2+ excited state is reduced by U A and, to a lesser extent, by ethyl urocanate. U A and its ester will be oxidized in this process though attempts to directly observe the U A radical or rad- ical-cation by flash photolysis have so far been unsuccessful (G. Truscott , private communication).

Table 4. Photolysis of NBTZ+ and UA or ethyl urocanate with and without SOD through Corex

Substrate (UA/Ester) SOD AirIAr ASMI

Ar 0.32 UA Ester - Ar 0.21 UA - Air 0.21 Ester - Air 0.12 UA + Air 0.10 Ester + Air 0.07

-

Table 5 , Photolysis of NBT- + and UA through Corex as a function of pH

AriAir PH A,,,,

Ar

Air

4.6 5.8 (3.9 7.7 8.4

4.6 5.9 6.9 7.9 8.7

0.05 0.29 0.65 0.97 0.96

0.02 0.18 0.31 0.71 0.88

Table 6. Riboflavin generation of super- oxide and reduction of NBT"

Arl0,lSOD AS,,,

Ar 0.09 Ar + SOD 0.00 0, 0.79 0, + SOD 0.12

2) Reduction of the NBT2+ excited state by either UA or its ethyl ester is significantly quenched by the presence of oxygen. SOD has no effect on the reductions in the presence of oxygen.

3) Reduction of the NBT2+ excited state by U A is maximized in basic media (Table 2). Though it is tempting (and intuitively logical) to ascribe this increase to greater reduction by the U A anion rela- tive to its zwitterion, the ca. 10 fold increase observed is more than sufficiently accounted for by the factor of 2 increase in the Ash,, value of M F + and the factor of 10 increase in the rate of conversion of NBT.' to MF' upon changing from pH 6 to p H 10 (Bielski et al., 1980). (The absorbance of NBT2+ itself is p H independent between p H 6 and 10 as is the rate of superoxide reaction with NBT2+, Bielski et al., 1980.)

These conclusions are summarized in equations 5-7.

( NBT2+ ) *

(6) (NBT2+)* + O2 + NBT2+ + O2

(NBT2+)* + UA+ NBT.' + UA.' (7) These reactions find ample analogy in the photolytic reduction of NBT2+ by other chemicals, such as riboflavin (Beauchamp and Fridovich, 1971) and 6-mercapto-purine (6MP) (Hemmens and Moore, 1986). For example, irradiation of 6MP and NBT2+ with l JVA in argon leads to a rapid reduction of the NBT2+ and, although it is unclear from the data given as to what proportion of the incident light is absorbed by each of the reactants, there is no doubt

156 HARRY MORRISON and ROSE MARIE DEIBEL

that reactions equivalent to equations 5 and 7 are operating.

When NBT2+ and U A are irradiated through a Corex filter (A> 275 nm), both substrates are excited and chemistry resulting from the formation of UA* in the presence of NBT2+ ground state is superimposed on reactions 5-7. Thus we now observe:

1) Reduction of the NBT2+ is again quenched by oxygen, but by a factor of 4 (Table 3) rather than 8, the value observed when NBT” is selectively excited (Table 1). We attribute the less efficient quenching by oxygen to the involvement of a non- quenchable U A excited state andlor the introduc- tion of a new, superoxide-mediated, pathway for reduction of the NBT2+.

2) In contrast to the uranium yellow results noted above, there is a significant effect of SOD on the NBT2+ reduction in the oxygenated or air-saturated solutions (cf. Tables 3 and 4). Since oxygen is quite effective in eliminating reduction due to (NBT2+)* (Table l), the large and virtually ‘completely quenchable by SOD’ residual component under Corex conditions (Table 3) requires that, with this filter, all of the NBT2+ reduction in the presence of oxygen is due to superoxide generated by UA*.

3 ) Ethyl urocanate exhibits chemistry identical to that of U A but in each case (Ar, air, SOD) at a rate somewhat lower than that of the acid. (A qualitative observation only, given the complexity of light absorption by the multicomponent mixture with the Corex filter.)

4) Reduction of NBT2+ using Corex is again max- imized in basic media, both in argon and in air. In the latter case, the UA* initiated superoxide mechanism should be dominant. However, as was noted above, there are several potential sources of such a rate acceleration in base.

Thus, equations 8-10 are indicated by the Corex filter data.

hv A> 275 nm UA- UA*

UA* + 0 2 + U A * + + 0 2 . - (9)

0 2 . - + NBT2++ 0 2 + NBT.+ (10)

In summary, we have presented evidence that (NBT2+)* and O2 are capable of oxidizing the U A ground and excited states respectively, in the latter

case, with the concomitant production of super- oxide. There is good evidence to indicate that U A is degraded by reaction with superoxide, as it is by singlet oxygen (Morrison and Deibel, 1986). The nature of the products formed in these reactions, and the significance of these observations for the in vivo environment remain to be determined.

Acknowledgements-This investigation was supported by PHS Grant Number CA18267, awarded by the National Cancer Institute, DHHS, and by a grant from the Amer- ican Cancer Society to R.M.D.

REFERENCES Beauchamp, C. and I. Fridovich (1971) Superoxide

dismutase: Improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276-287.

Bielski, B. H. J. , G . G . Shine and S. Bajuk (1980) Reduction of nitro blue tetrazolium by C0,- and 0,- radicals. J . Phys. Chem. 84, 830-833 and references therein.

Deibel, R. M., H. Morrison and W. M. Baird (1987) Urocanic acid photobiology. Photochemical binding of urocanic acid to bovine serum albumin. Photochem. Photobiol. 45, 421-423 and references therein.

Eadie, M. J., J. H. Tyrer, J. R. Kukums and W. D. Hooper (1970) Aspects of tetrazolium salt reduction relevant to quantitative histochemistry. Histochemie 21, 170-1 80.

Gibson, S. L., H. J. Cohen and R. Hilf (1984) Evidence against the production of superoxide by photoirradiation of hematoporphyrin derivative. Photochem. Photobiol. 40, 441448.

Hemmens, V. J. and D. E. Moore (1986) Photochemical sensitization by azathioprine and its metabolites-I. 6- Mercaptopurine. Photochem. Photobiol. 43, 247-255.

Hooper, W. D. (1969) Recent chemistry and uses of formazans and tetrazolium salts. Rev. Pure Appl. Chem.

Miller, R. W. and C. T. Kerr (1966) Dihydroorotate dehydrogenase. 111. Interactions with substrates, inhibi- tors, artificial electron acceptors and cytochrome C . J . Biol. Chem. 241, 5597-5604.

Morrison, H. and R. M. Deibel (1986) Photochemistry and photobiology of urocanic acid. Photochem. Photo- biol. 43, 663-665.

Rasanen, L., C. T. Janzen, T. Reunala and H. Morrison (1987) Stereospecific inhibition by cis-urocanic acid of epidermal cell interleukin-1 secretion and HLA-DR expression. Photodermatol. 4, 182-186.

Ross, J., S. E. M. Howie, M. Norval, J . Maingay and T. Simpson (1986) Ultraviolet irradiated urocanic acid suppresses delayed-type hyper-sensitivity to Herpes sim- plex virus in mice. J . Invest. Dermatol. 87, 630-633.

Tsou, K-C., C-S. Cheng, M. M. Nachlas and A. M. Seligman (1956) Syntheses of some p-nitrophenyl sub- stituted tetrazolium salts as electron acceptors for the demonstration of dehydrogenases. 1. Am. Chem. SOC. 78, 6139-6144.

19, 221-241.