Oxidative Stress Resulting from Ultraviolet A Irradiation of Human Skin ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 20, Issue of July 15, PP.14678-14661,1993 Printed in U.S.A.

Oxidative Stress Resulting from Ultraviolet A Irradiation of Human Skin Fibroblasts Leads to aHeme Oxygenase-dependent Increase in Ferritin* (Received for publication, September 11, 1992, and in revised form, January 18, 1993)

Glenn F. Vile$ and RexM. Tyrrell From the Swiss Institute for Experimental Cancer Research, Ch. des Boveresses 155, CH-1066 Epalinges, Lausanne, Switzerland

Heme oxygenase-1 mRNA levels increase following iron that can react with hydrogen peroxide and superoxide, exposure of many mammalian cell lines to oxidative giving rise to the very reactive hydroxyl radical (‘OH) via stress such as ultraviolet A (UVA) irradiation. Here weReactions 1 and 2, and it is the hydroxyl radical that is the demonstrate a 4-fold increase in microsomal heme ox- initiator of biological damage (15-17). ygenase activity and a 40% decrease in microsomal 02 + Fe3++ Fez+ + 0 2 heme content 14 h after treatment of human skin fibroblasts (FEKJ with 260 kJ m-2 of UVA radiation. Fez+ + H202 + ’OH + OH- + Fe3+ Paralleling thiswas a 2-fold increase inferritin levels that was sustained for at least46 h after UVA irradiREACTIONS 1 AND 2 ation. Treatment of fibroblasts with the ironchelating Intracellularly most of the iron that is not metabolized is agent desferrioxamine, after the UVA-dependent induction of heme oxygenase, prevented the increase in sequestered in ferritin as a crystalline core of ferric (Fe3+) ferritin levels. Treatment of fibroblasts with Sn-pro- ions (18).To catalyze oxidative reactions, the iron must first toporphyrin IX (an inhibitor of heme oxygenase) also be released from the core. Although superoxide is able to do prevented the effect of UVA radiation on ferritin lev- this, it is very inefficient (19). Thus ferritin is able to restrict els. Thus we conclude that theeffect of UVA radiation the availability of iron to participate in Reaction 1. on ferritin levels is via the heme oxygenase-dependent It has been shown that a hemin-dependentincrease in heme releaseofironfromendogenoushemesources. We oxygenase protein synthesis activates ferritin mRNA transpropose that the increase inferritin that follows UVA lation in rat fibroblasts (20). Whether this led to an increase irradiation would decrease intracellular free iron such in heme oxygenase activity or ferritin levels wasnot examined. thatiron-catalyzed free radicalreactionswould be Elevated levels of newly synthesized ferritin would result in restrictedduringperiodsofsubsequent oxidative an enhancement of cellular iron sequestering capacity that stress. may confer increased resistance to oxidative stress. This study tests the hypothesis that levels of ferritin in human skin fibroblasts are effected byUVA irradiationand that this Many procaryotic and eucaryotic cells synthesize specific occurs via heme oxygenase. proteins in response to oxidative stress. In some cases these stress proteins have been shown to have an antioxidant role, MATERIALS ANDMETHODS e.g. treatment of Escherichia coli with hydrogen peroxide All biochemicals were from Sigma except where indicated. results inexpression of at least 30 proteins including catalase Cell Culture-Monolayers of the normal human skinfibroblast line (1). The protein most consistentlyactivated by oxidative (FEKJ were grown to 100% confluence in 15-cm dishes over 7 days stress in a wide variety of eucaryotic cells is theheme degrad- in minimum essential media supplemented with penicillin, streptoing enzyme, heme oxygenase-1 (2-5). UVA radiation (320- mycin, glutamine, sodium carbonate, and 15% fetal calf serum. At 380 nm), hydrogen peroxide, and glutathione depleting com- day 7 each dish contained approximately 8 X lo6 fibroblasts. For pounds all enhance heme oxygenase-1 mRNA synthesis (6) some experiments fibroblasts were grown to 40% confluence over 3 days. Fibroblasts were passaged twice a week and used between and result in accumulation of heme oxygenase-1 mRNA in passages 9 and 16. Cellculture materialswere from Life Technologies cultured human skin fibroblasts (FEK,) and other mamma- (Paisley, Scotland), except fetal calf serum, which wasfrom Biological lian cell lines (7). Industries (Haemek, Israel). UVA irradiation of biological molecules givesrise to superUVA Irradiation-Fibroblasts were irradiated with 250 kJ rn-’ of broad spectrum UVA light using a Uvasun 3000 lamp (Mutzhas, oxide and hydrogen peroxide (8-lo), species that maybe involved not only in cell death but also in the carcinogenic Munich, Germany). The UVA dose was measured using an IL 1700 radiometer (International Light, Newbury, USA). Irradiation was effects of UVA irradiation (11).There is good evidence that done at 25 “C. Priorto irradiation, media were removed and retained, the biological damage attributed to superoxide and hydrogen and the fibroblasts were covered with Ca2+/Mg2+(0.01% each) enperoxide is dependent on the presence of iron (12-14). It has riched phosphate-buffered saline (PBS)’as described previously (3). been proposed that there is a small intracellular pool of free After irradiation the original media were added back to the fibroblasts. Control fibroblasts were treated in the same manner except that they were not irradiated. Hemin Treatment-A stock solution of hemin (1mM) was prepared Cancer and The Swiss National Science Foundation (31-30880-91). The costs of publication of this article were defrayed in part by the in KOH (8mM) and phosphate buffer (100 mM, pH 7.4).After removal payment of page charges. This article must therefore be hereby of the media fibroblasts were treated with hemin (4P M ) in PBS for marked “aduertkement” in accordance with 18 U.S.C. Section 1734 1 h. After treatment the fibroblasts were washed thoroughly and the solely to indicate this fact. The abbreviations used are: PBS, phosphate-buffered saline; 3 Recipient of a Swiss Institute for Experimental Cancer Research MOPS, 4-morpholinepropanesulfonicacid. Postdoctoral Fellowship. ~~

* This study was supported in part by the Swiss League Against

14678

14679

Induction of Ferritin by UVA Occurs via HemeOxygenase original media was added back to thefibroblasts. Inactivation of Heme Oxygenase-To inactivate heme oxygenase, fibroblasts were treated with Sn-protoporphyrin IX (Porphyrin Products, Logan, UT). Immediately after UVA irradiation the PBS was removed from the fibroblasts and and Sn-protoporphyrin IX (100 p ~ in) PBS was added. Fibroblasts were kept in the dark during treatment with Sn-protoporphyrin IX to minimize photoreactions of the porphyrin. After 2 h of treatment a t 37 "C the Sn-protoporphyrin IX was removed, fibroblasts were washed and theoriginal media was replaced. A stock solution of Sn-protoporphyrin IX (1mM) was made up in KOH (8 mM) and phosphate buffer (100 mM, pH 7.4). Desferrioxamine Treatment of Fibroblasts-To bind low molecular weight intracellular iron fibroblasts were treated with desferrioxam) Geigy, Basel, Switzerland) in PBS at ine (Desferal) (500 p ~ (Ciba 37 "C after removal and storage of the media. After incubation for 1.5 h the desferrioxamine was removed and fibroblasts were washed thoroughly with PBS before adding back the original media. Heme Oxygenase-1 mRNA-Total RNA was isolated by the guanidinium thiocyanate-phenol-chloroform method 3 h after irradiation (21). RNA (15 pg/well) was electrophoresed in a MOPS/HCHO 1.3% agarose gel (22), transferred onto a Genescreen nylon membrane (NEN Research Products, Regensdorf, Switzerland), and hybridized with the 1000base pair EcoRI fragment of the human heme oxygenase cDNA clone 2/10 (3). After autoradiography blots were rehybridized with the PstI fragment (1300 base pairs) of rat glyceraldehyde-3phosphate dehydrogenase cDNA. The glyceraldehyde-3-phosphate dehydrogenase RNA signal was used as an internal control for the loading error between samples. Fibroblast Extracts-Fibroblasts (8 X 10') were washed thoroughly with ice-cold PBS, harvested with a rubber policeman and homogenized with a Potter Elvehjem homogenizer (Bellco, Fetham, United Kingdom) at 4 "C. Cell debris was removed by centrifugation at 5000 X g, and an aliquot of supernatant was retained for ferritin analysis. The remaining supernatant was spun at 15,000 X g to remove mitochondria, and the new supernatant was centrifuged at 105,000 X g for 60 min. The resulting microsomal pellet was resuspended in phosphate buffer (100 mM, pH 7.4). All centrifugation steps were performed at 4 "C. The protein content of extracts was determined using the method of Bradford (23)standardized with bovine serum albumin. Heme Oxygenase Determination-The method of Shibihara et al. (24) was used to determine heme oxygenase activity. Microsomes , (100-200 pg of protein) were incubated with hemin (20 p ~ )bovine serum albumin (0.064%), NADPH (100 p ~ ) and , crude biliverdin reductase extract (0.29 mg ml-'), prepared according to the method of Tenhunen et al. (25). Reactions took place in phosphate buffer (100 mM, pH 7.4), and samples were gently mixed in the dark at 37 "Cfor 30 min. The reaction was stopped by placing on ice, tubes were centrifuged at 5000 X g for 1 min at 4 "C, and theabsorbance of bilirubin at 465 nm was measured against a base-line absorbance at 520 nm (e4% = 40,000 M" cm-'; Ref. 26). Ferritin Content-The ferritin assays were performed with a polyclonal enyme-linked immunosorbent assay kit (Boehringer, Mannheim, Germany). Supernatants (10-20 pg of protein) from the 5000 X g centrifugation step after homogenization of the fibroblasts were analyzed for ferritin according to theprocedure supplied with the kit. Heme Content-Mitochondria1 and microsomal pellets, prepared from fibroblasts as described above, were resuspended in 1 ml of concentrated formic acid (Fluka, Buchs, Switzerland), and the heme content of the solution was measured at 398 nm (27). The concentration of heme was calculated from a standard curve constructed from treatment of cytochrome c with concentrated formic acid.

10 20 30 40 50 Time after treatment ( h ) FIG. 1. Heme oxygenase activity in FEK, fibroblasts following UVA irradiation (260 kJ m-'). Fibroblasts were harvested at

0

or control treatment the times shown after UVA irradiation (e-) ( - -0--). Eachpoint is the mean -+ S.D. for 3-5 determinations.

TABLE I Effect ofUVA irradiation (250 kJ m-') on heme oxygenase and ferritin content of FEK, fibroblasts in the presence and absence of desferrioxamine and Sn-protoporphyrin IX Data shown are the means f S.D. for 3-4 determinations.

? ~ ~ ~ ~ Heme

Treatment

oxygenase Ferritin content activitv

h

nmol min" mgprotein-l

22 0.11 46

0.11 f 0.04 0.12 f 0.02 92 f 0.04

98 f 10 f 15 1 O O f 18

0.14 -t 0.06 0.34 f 0.2 fO.04

95 f 3 180 f 40 186 f 2 6 f 21

Non-irradiated 4

Irradiated

4 22 0.12 46

+ desferrioxam-

Irradiated ine (500 p Irradiated phyrin IX

0.28 22

&

0.02 109

ng mgprotein"

~ )

+ Sn-protopor-

22

0.00 f 0.01

95 f 8

(100 p M )

after UVA irradiationand were still maximal 46 hafter irradiation (Table I). Heme oxygenase activity had returned to control levels 46 h after irradiation(Fig. 1).The UVA dose used (250 kJ m-') was equivalent to less than 30 min of exposure to a typical tanning lamp. Thus theUVA-dependent increase in heme oxygenase activity and ferritin levels we show here represent a response to a physiologicallevel of oxidative stress. Heme oxygenase-1 mRNA levels were increased 13 f 5 RESULTSANDDISCUSSION fold (mean f S.D. of 10 determinations) following irradiation This study shows that treatment of human skin fibroblasts of fibroblasts with 250 kJ m-2 of UVA 3 days after seeding (FEK4) with 250 kJ m-'ofUVA irradiation elevated heme (Fig. 2). Fibroblasts irradiated 7 days after seeding (100% oxygenase activity 4-fold, 14 h after irradiation (Fig. 1).The confluent) showed an even greater increase in heme oxygenincrease in heme oxygenase activity after UVA irradiation we ase-1 mRNA (Fig. 2). Previous work from this laboratory has show here extends previous results from this laboratory that shown accumulation of heme oxygenase-1 mRNA in fibrohave shown induction of heme oxygenase protein levels and blasts irradiated 2-4 days after seeding (30-50% confluent) increased rate of heme oxygenase-1 RNA accumulation after (7). For all investigations in the present study we used fibroUVA irradiation of FEK, fibroblasts (3,6). We now show that blasts 7 days after seeding because they were not undergoing the UVA-dependent increase in heme oxygenase activity of so rapid a rate of growth as that seen in 3-day fibroblasts FEK4 fibroblasts is paralleled by an increase in ferritinlevels. (data not shown) and therefore more closely represent the Levels of ferritin were increased approximately 2-fold, 22 h growth characteristics of cells in uiuo. The apparent discrep-

Induction of Ferritin by UVA Occurs via Heme Oxygenase

14680

-e L

c o

a

E o

>

3

0

3

>

1-

-e

a

HO

.&- I

G A P D H ~ ~

"

3 days

7 days

FIG. 2. Effect of UVA irradiation (250 kJ m-2) on heme oxygenase-1 mRNA content of FEK, fibroblasts. Fibroblasts were irradiated either 3 or 7 days after seeding and probed with heme oxygenase-1 mRNA then glyceraldehyde-3-phosphatedehydrogenase mRNA. The autoradiograph shown is of a typical experiment.

TABLEI1 Effect of UVA irradiation (250 kJ m-*) on heme content of miCmwrnes and mitOchondria Prepared from FEK4 fibrobhts Heme content was determined 14 and 22 h after irradiation. Data shown are the means S.D. of 3-4 determinations. ND, not determined.

*

Treatment

Non-irradiated Irradiated After 14 h After 22 h

Microsomal heme content

Mitochondrial heme content

nmol mg protein"

nmol mg protein"

0.63 & 0.07

0.76 f 0.22

0.38 f 0.15 0.57 0.03

0.73 0.24 ND

*

*

ancy between the -fold increaseinmRNAandthe -fold increase in enzyme activity after UVA irradiation is likely to be because the totalcellular heme oxygenase activity consists of the inducible heme oxygenase-1 isozyme and the constitutive heme oxygenase-2 isozyme (28). In the rat liver there is a 2:l ratio of heme oxygenase-2 to heme oxygenase-1 activity (29), and in rat testes almost all the heme oxygenase activity has been attributed to hemeoxygenase-2 (30). Therefore the -fold increase in heme oxygenase-1 activity of FEK4 fibroblasts following UVA irradiation is almost certainly greater than we show here due to the contribution (currently unknown) of heme oxygenase-2 activity to the basallevels. T o examine theeffect of increased heme oxygenase activity on cellular heme, we measured total heme content of microsomes(endoplasmic reticulum) and mitochondria prepared from UVA-irradiated fibroblasts. The heme content of microsomes of irradiatedfibroblasts wasdecreased to approximately 60% of the level in non-irradiated fibroblasts (Table 11), at the time when the UVA-dependent increase in heme oxygenase activity was maximal, i.e. 14 h after UVA irradiation (Fig. 1).The heme content of mitochondria from irradiated fibroblasts was unchanged 14 h after irradiation (Table 11).The decrease in microsomal heme contentwas transitory, since theheme content of microsomes increased from 60%of the level in non-irradiated fibroblasts 14 h after irradiation to 90% of the level in non-irradiated fibroblasts 22 h after irradiation (Table 11). We did not examine the type of microsomalheme that was decreased after the UVA-dependent induction of heme oxygenase activity. In addition to cytochrome P450and bs (31), there may also be a small amount of free heme associated with microsomes (32). Cytochrome P450 has been shownto be degradedto biliverdin by areconstituted heme oxygenase-1system (33) and liver microsomesprepared from ratstreatedwithcobalt show an increase in heme oxygenase activity and a decrease in cytochrome Pd50(26). Cytochrome b5 levels were unchanged (26). The unsuitability of cytochrome bs as a substrate for heme oxygenase-2 has been demonstrated in a reconstituted system (30). The decrease inheme content we show in microsomes with enhanced heme oxygenase activity is much greater than could be ac-

counted for by consumption of theheme alone free (32). Therefore we conclude that isit likely tocytochrome be P450 that isdegraded upon UVA induction of heme oxygenase. Intracellular free iron hasbeen shown to be involved in the UVA-dependent induction of heme oxygenase-1 mRNA accumulation (34). Therefore, in orderto test theeffect of iron released from endogenous heme ferritin on levels, we added desferrioxamine a t a time when the rate of heme oxygenaseRNA synthesis had reached a maximum, i.e. 1 h after UVA irradiation (6). Chelation of iron at this time blocked the increase in ferritin levels attributed to the release O f iron by the induced heme oxygenase (Table I). Thisimplies a role for free iron in theregulation of ferritin levels. For the purposes of this discussion, free iron refers small the to pool of intracellular non-heme, non-protein-bound iron that is chelatable by desferrioxamine and mostlikely exists complexed to citrate or phosphate (35). Treatment of fibroblasts with PBSfor the Same length of time as fibroblasts were treated with desferrioxamine (1.5 h) did not effect the UVA-dependent increase in heme oxygenase activity and ferritinlevels. Non-irradiated fibroblasts showed no change in either ferritinlevels or heme oxygenase activity when treated with desferrioxamine. Other studies have shown that iron added asa low molecular weight salt can enhance the rate of ferritin mRNA translation ain variety of cells (20,36, 37). Iron salts can also enhance act to the rate of ferritin RNA svnthesis. However the effect of this enhancement on ferritin protein levels is modest compared to the effect of free iron on translation (36). It has also been proposed that theiron needs to be associated with porphyrin in order to activate ferritin mRNA translation (38).However our results support the alternative proposal that theprincipal effect of heme on ferritinlevels is via free iron (20, 36). The possibility that UVA irradiation was stimulating ferritin levels by a heme oxygenase-independent mechanismwas tested by treating UVA-irradiated fibroblasts with Sn-protoporphyrin IX. This porphyrin irreversibly inhibits heme oxygenase. Sn-protoporphyrin IX treatment of fibroblasts inhibited heme oxygenase activity almostcompletely (Table I). Under these conditions UVA irradiation had no effect on ferritin levels. This rules out thepossibility that iron, released directly from hemes or ferritin by UVA irradiation (39), was stimulating ferritin synthesis. Thus it canbe concluded that the effect of UVA on ferritin levels is via heme oxygenase. Treatment of fibroblasts with PBS for the same length of time as fibroblasts were treated with Sn-protoporphyrin IX (2 h) did not effect the UVA-dependentincrease in heme oxygenase activityandferritin levels. Treatment of nonirradiated fibroblasts with Sn-protoporphyrin IX for 2 h inhibitedheme oxygenase activity but did not affectbasal ferritin levels. T o demonstrate that the relationshipbetween heme oxygenase andferritin was not a specific effect of oxidative stress, we used hemin insteadof UVA irradiation as a n inducer of heme oxygenase. Fibroblasts treatedwith hemin showed an increase in heme oxygenase activity of 6-fold 14 h after treatment (from 0.11 & 0.03 to 0.68 2 0.07 nmol min" mg protein", mean & S.D. of two determinations) andshowed an almost 2-fold increase inferritin 22 h after treatment (from 102 & 10 to 191 f 14 ng mg protein", mean f S.D. range of two determinations). This study has demonstrated that the inducibility of heme oxygenase by oxidative stress plays an important role in the regulation of the major intracellular iron-binding protein, ferritin. It has been hypothesized that an increase in heme oxygenase activity may lead to anincrease in the antioxidant potential of cells, thereby enhancing cell survival under oxidative stress (34, 40). We now show that a heme oxygenase-

Induction of Ferritin by UVA OccursHeme via dependent increase in antioxidant potential may actually be mediated by ferritin. We propose that the increased levels of ferritin that result from the UVA-dependent induction of heme oxygenase will further decrease intracellular free iron levels and therefore may limit iron-catalyzed oxidative reactions that would occur during subsequentperiods of oxidative stress. Acknowledgment-We thank Sharmila Basu-Modak for preparing the biliverdin reductase extract and for measuring heme oxygenase-1 mRNA. Addendum-The antioxidant roleof ferritin is supported by a study that appeared after submission of this work, showing that the cytotoxicity ofhydrogen peroxide and hemin was inhibited by an increase in cellular ferritin levels (41).Balla et al. (41) induced ferritin levels by pre-exposing porcine aortic endothelial cellsto hemin. Although they observed an increase in heme oxygenase activity the induction of ferritin was not effected by an inhibitor of heme oxygenase, Sn-mesoporphyrin IX. This result suggests that the induction of ferritin in the endothelial cell system occurs hy a mechanism different from the heme oxygenase-dependent pathway we now report to occur in cultured human skin fibroblasts.

Oxygenase

14681

(1985) Photochem. Photobiol. 42,125-128 11. Black, H. S. (1987) Photochem. Photobiol. 4 6 , 213-221 12. Aust, S. D..Morehouse. L. A.. and Thomas. C. E. (1985) . , J. Free Radicals Biol. Med. 1 , 3-25 13. Halliwell, B., and Gutteridge, J. M. C. (1984) Biochem. J. 2 1 9 , l - 1 4 14. Sterrenhurg, L., Julichen, R. H. M., Bast, A,, and Noordhoek, J. (1984) Toxicol. Lett. ( A m s t . )2 2 , 153-156 15. Walling, C. (1975) Acc. Chem. Res. 8, 125-131 16. Halliwell, B., and Gutteridge,J. M. C. (1986) Arch. Biochem. Biophys.2 4 6 , 501-514 17. Borg, D. C., and Schaich, K. M. (1984) Isr. J. Chem. 24,38-53 18. Harrison, P. M., and Hoy, T. G. (1973) in Inorganic Chemistry (Eichhorn, G. L., ed) pp. 253-279, Elsevier Science Publishing Co., Inc., New York 19. Bolann, B. J., and Ulvik, R. J. (1990) Eur. J. Biochem. 1 9 3 , 899-904 20. Eisenstein,R. S., Garcia-Mayol, D., Pettingell, W., andMunro, H. M. (1991) Proc. Natl. Acad. Sci. U. S . A . 8 8 , 688-692 21. Chomczynski, P., and Saachi, N. (1987) Anal. Biochem. 1 6 2 , 156-159 22. Davis, L. G., Dibner, M. D., and Battey, J. F. (1986) Basic Methods in Molecular Biology, Elsevier Science Publishing Co., Inc., New York 23. Bradford, M. M. (1976) Anal. Biochem. 72,248-254 24. Shibahara, S., Yoshida, T., and Kikuchi, G.(1978) Arch. Biochem. Biophys. 1 8 8 , 243-250 25. Tenhunen, R., Ross, M. E., Marver, H. S., and Schmid, R. (1970) Biochemistry 9, 298-303 26. Maines, M. D., and Kappas, A. (1975) J. Biol. Chem. 250,4171-4177 27. Kuross, S. A,, Rank, B. H., and Hebbel R. P. (1988) Blood 71,876-882 28. Maines, M. D. (1988) FASEB J. 2 , 25537-2568 29. Maines, M. D., Trakshel, G. M., and Kutty, R. K. (1986) J . Biol. Chem. 2 6 1 , 411-419 30. Trakshel, G. M., Kutty, R. K., and Maines, M. D. (1986) J. Biol. Chem. 261,11131-11137 31. Estabrook, R. W., and Werringloer, J. (1967) Methods Enzymol. 1 0 , 212'

220

1. 2. 3. 4. 5. 6. 7.

8. 9. 10.

REFERENCES Christman, M. F., Morgan, R. W., Jacobson, F. S., and Ames, B. N. (1985) Cell 41,753-762 Keyse, S. M., and Tyrrell, R. M. (1987) J. Biol. Chem. 2 6 2 , 14821-14825 Keyse, S. M., and Tyrrell, R. M. (1989) Proc. Natl. Acad. Sa. U. S. A. 8 6 , 99-103 Hiwasa, T., and Sakiyama, S. (1986) Cancer Res. 4 6 , 2474-2481 Taketani, S., Kohno, H., Yoshinaga, T., and Tokunaga, R. (1989) FEBS Lett. 2 4 5 , 173-176 Keyse, S. M., Applegate, L. A., Tromvoukis, Y., and Tyrrell, R. M. (1990) Mol. Cell. Biol. 1 0 , 4967-4969 Applegate, L, A,, Luscher, P., and Tyrrell, R. M. (1991) Cancer Res. 5 1 , 974-978 McCormick, J. P., Fisher, J. R., Pachlatko, J. P., and Eisenstark, A. (1976) Science 191,468-469 Czochralska, B., Kawczynski, W., Bartosz, G., andShugar, D. (1984) Biochim. Biophys. Acta801,403-409 Cunningham, M. L., Johnson, J. S., Giovanazzi, S. M., and Peak, M. J.

32. Gr&jck, S., Sinclair, P., Sassa,S., and Grieninger, G. (1975) J. Biol. Chem. 250,9215-9225 33. Kutty, R. K., Daniel, R. F., Ryan, D. E., Levin, W., and Maines, M. D. (1988) Arch. Biochem. Biophys. 260,638-644 34. Keyse, S.M., and Tyrrell, R. M. (1989) Carcinogenesis 11, 787-791 35. Baker, M. S., and Gehicki, J. M. (1986) Arch. Btochem. Biophys. 246,581# F I N 3

36. Coccia, E. M., Profita V., Fiorucci G., Romeo, R., Affabris E. Testa U., Hentze, M. W., andBattistini, A. (1992) Mol. Cell. Biol. i2.5015-3622 37. Rogers, J., and Munro, H. (1987) Proc. Natl. Acad. Sei. U. S. A. 8 4 , 22777781

38. LinrjT-J., Daniels-McQueen, S., Patino, M. M., Gaffield, L., Walden, W. E.,and Thach, R. E. (1990) Science 2 4 7 , 74-77 39. Auhallly, M., Santus, R., and Salmon, S. (1991) Photochem. Photobiol. 6 4 , 7GQ-772

40. Stocker, R. (1990) Free Radical Res. Cornrnun. 9,101-112 41. Balla, G., Jacob, H. S., Balla, J., Rosenberg, M. E., Nath, K. A,, Apple, F., Eaton, J. W., and Vercellotti, G. M. (1992) J. Biol. Chem. 2 6 7 , 1814818153