245

Biochimica et Biophysica Acta, 1(180(1991) 245-25! © lgql El~vier Science Publishers B.V. All rights reserved 0167-483~/91/$03.50

BBAPRO 34038

Synergistic induction of 6-aminolevulinate synthase by glutethimide and iron: relationship to the synergistic induction of heine oxygenase Edward E. Cable *, John F. Healey, Yvonne Greene *, Chheng-Orn Evans and Herbert L. Bonkovsky * Departments of Biochemist~" and Medicine. Erupt, Unicersit~"School of Medicine. Atlanta. GA (U.S.A, (Received 15 January 1991) (Revised manuscript received 15 July 19911

Key words: ~i-Aminolevulinate synthase: lteme oxygenase; Glutethimide: Iron

Relationships between activities of ~-aminolevulinate synthase and heme oxygenase, respectively the rate-limiting enzymes of heme biosynthesis and degradation, have been studied in chick embryo liwr cell cultures following exposure of the cultures to glutethimide and iron, a combination known to produce a synergistic induction of both enzymes. In time-course experiments, synergistic induction of heme oxygenase activity by glutethimide and iron preceded that of ~-aminolevulinate synthase by 4 h. Effects of selective inhibitors of both heme synthesis and degradation have also been studied with respect m effects on ~-aminolcvulinate synthase and heme oxygenase activities. The synergistic induction of heme oxygenase by glutethimide and iron appears to be dependent upon cellular heme synthesis because addition of inhibitovs of heme biosynthesis, 4,6-dioxoheptanoic acid or N-methylmesoporphyrin abolishes this synergistic induction. Exposure of cultures to tin-mesoporphyril~, a potent inhibitor of heme oxygenase, prevented the synergistic induction of ~-aminolevulinate synthase produced by glutethimide and iron, or, when added after induction was already established, promptly halted any further induction. These results suggest that the level of activity of heme oxygenase can reciprocally modulate intracellular heine levels and thus activity ~,f ~-aminolevulinate synthase.

Introduction Regulation of hepatic heine metabolism is thought to occur via a regulatory heine pool. This model was first proposed [1] and further developed [2] by Granick, who suggested that heine is first used for formation of cellular hemoproteins; second, any 'excess' heme is used to regulate 6-aminolevulinate (ALA) synthase (E.C. 2.3.1.37) and effectively turn off heme biosyr,* Present Address: Division of Digestive Diseases and Nutrition. Department of Medicine, University of Massachusetts Medical Center, Worcester, MA (U.S.A,) Abbreviations: ALA, cS-aminolevulinate: ATP. adenosine triphoslahate; CoA, coenzyme A: 4,6-DHA, 4,6-dioxoheptanoic acid: FeNTA, ferric nitrilotriaeetate, Correspondence: E.E. Cable. Division of Digestive Diseases and Nutrition, Department of Medicine, University of Massachusetts Medical Center, 55 Lake Avenue North. "fforcester. MA 01655,

U.S.A.

thesis; and third, if the cell is presented with too much heme, heme oxygenase (E.C. 1.14.88.3) is induced. In contrast, when the cell requires additional heme, (by definition, no 'excess" hem,: v,ouiti exist), heine oxygenase activity would be reduced and ALA synthase would be induced leading to increased heme ~nthesis (for reviews see Refs. 3-6). In this model heme oxygenas~ was assigned the role of primarily degrading excess amounts of heme presented to or produced de novo within the cell [2]. Evidence for this view included (i) the low affinity of heme oxygenase for heme (Km = 1-5 #M, [7,8]). compared to heme affinities of cytochromc P-450 and other major cellular hempproteins; and (it) the low concentrations of heme (0.020-0.05n t-tM, [9]) needed for repression of A L A synthase, compared to those needed for induction of heme ox3'genase ( > 2 p.M, [10-12]). In contrast, others proposed that changes in activity of heine oxygenase, presumably acting by reciprocal alterations in the size of the regulatory heine pool, were capable of exerting

246 major effects on activity of ALA synthasc [13] and on levels of cytochromc P-450 [14-16]. Indeed an inverse relationship between activity of heine oxygcnase and levels of cytochrome P-450 is frequently observed [i5,17,18], although no obligatory inverse relationship exists between these enzymes [11,19,20]. Exposure of chick embryo liver cells to a phenobarbital-like drug and iron produces a synergistic induction of ALA synthase [21,22], a finding similar to those observed in rodents [23,24]. In addition, recent work from our laboratory showed that heme oxygenase is induced synergistically in chick embryo liver cells by the combination of the phenobarbital-like drug, glutethimide and iron [22,25]. This induction is associated with parallel increases in the levels of mRNA and immunoreactive protein of heine oxygenase, suggesting a stimulatory effect on the rate of heme oxygenase gene transcription and mRNA translation [25]. The synergistic induction of both ALA synthase and heine oxygenase in chick embryo liver cells upon tn, atment of the cells with glutethimide and iron would not be predicted by the regulatory heme pool model and seems at variance with the view that regulatory heme is solely responsible for the regulation of heme metabolism. Two different hypotheses could be proposed that would explain these inductions: first, glutethimide and iron could cause a rapid uncontrolled induction of ALA synthase, exposing the cell to high levels of heme and producing a secondary induction of heine oxygenase; or second, glutethimide and iron could cause a rapid induction of heme oxygenase, depleting the cell of heme and producing a synergistic induction of ALA synthase. Our results support the latter view. Materials and Methods

Materials'. Fertilized white leghorn eggs were from Hyline Farms (Mansfield, GA). Levulinic acid was from Aldrich (Milwuakee, W1). 2,4-pentanedione (acetylacetone) was from EM Science (Cherry HiU, NJ). ATP, BSA, CoA, dexamcthasone, 4,6-DHA, p-dimethylaminobenzaldehyde, DNase, glutethimide, heme, NADPH, py~idoxal-5'-phosphate, succinic thiokinase (E.C. 6.2.1.4) and tri-iodothyronine were from Sigma (St. Louis, MO). William's E medium, trypsin and penicillin/streptomycin were from Gibco (Grand Island, NY). Deferoxamine (desferal mesylate) was from ClBA-Geigy (Edison, NJ). N-methylmesoporphyrin, and tin-mesoporphyrin were from Porphyrin Products (Logan, UT). All other chemicals were of the highest purity commercially available. Cell cultures and treatments. Chick embryo liver cell cultures were prepared from the livers of 16-18-day-old chick embryos as described previously [11], except that newborn calf serum and serX-Tend (DuPont Chemical, Wilmington, DE) were omitted. Chemicals were pre-

pared and added as previously described [11]. Appropriate solvent controls showed no effects on heme oxygenase or ALA synthase activity. Heme, tin-mesoporphyrin and N-methylmesoporphyrin were added as albumin complexes with a porphyrin/albumin molar ratio of 5/3. FeNTA was an aqueous solution added to the cultures containing 5 mM FeCI 3 and 10 mM nitrilotriacetate. Enzyme assays. Cultures were harvested and heme o×ygenase was assayed as described [25]. ALA synthase was assayed using the following modifications of published methods [26,27]: cells cultured on 60 mm tissue culture plates were washed twice with room temperature phosphate buffered saline (0.15 M NaCI, 0.1 M Na phosphate, pH 7.4~ and harvested in 750 #1 of assay buffer (35 mM Tris-HCI, 30 mM Na phosphate, 8 mM MgCI 2, 5 mM EDTA, 15 mM citric acid. 15 mM sodium succinate, 10 mM sodium levulinate, 0.5 mM pyridoxal-5'-phosphate, 10 mM ATP, 0.1 mM CoA, 5 mM 13-mercaptoethanol, 100 mM potassium fluoride, 5 U/1 suecinic thiokinase, final pH 7.4). The cells were scraped off the plate with a rubber spatula and the cellular suspension was sonieated for 6 s at a power output that was 35% of the microtip limit (Branson Heat Systems W225 sonicator, Danbury, CT.). The sonicate was split into three 150 ~1 aliquots (one reference and assay duplicates). Incubations were started with the addition of 10 #1 of 1 M glycine (pH 7.0) and were allowed to proceed for 30 rain at 37 ° C. The reactions were terminated by the addition of 100 /.tl of 10% TCA. TCA was added to reference samples immediately and kept on ice through the incubation period. All samples were centrifuged at 801) x g for 10 rain and 150 ~1 of the supernatant was removed and placed in fresh tubes. 101) M of 10% 2,4-pentanedione !n IM sodium acetate was added to the TCA supernatant and incubated at 85°C for 15 rain to form an ALA-pyrrole (2-r.~ethyl-3-acetyl-4-propionic acid pyrrole) from the newly synthesized ALA. The tubes were chilled on ice and 250 gl of modified Ehrlich's reagent 10.134 M p-dimethylaminobenzaldehyde, 13.8 mM mercuric chloride, 16% (v/v) perchloric acid in glacial acetic acid) was added. The resultant mixture was vortexed and allowed to sit at room temperature for 15 min during which t~me a colored Ehrlich's salt of the ALA-pyrrole formed. The colored Ehrlich's salt was quantitated by scanning each of the assay duplicates versus the appropriate reference sample from 500 to 650 nm on an SLM • Aminco DW-2C :~ spectrophotometer and the amount of ALA tormed was calculated from the ,-tAs55_65o using ,Ae = 65 mM -I cm -I. The Ehrlich's salt was assayed within 30 rain to avoid formation of a colorless dipyrrolphenyl methane [28]. RNA preparation and Northern blot analysis. Total cellular RNA was prepared [29] and northern blots done as described [30] except that synthetic oligenu-

247 eleotide probes identical to either a portion of chick heme oxygenase cDNA cloned in our laboratory [31] or a previously synthesized [26] portion of ALA synthase cDNA [32]. Blots were washed in 0.1 × SSC, without SDS, at room temperature for 1 h with 4 changes of wash buffer. Blots were ~:xposed and developed as -reviously described [31]. Results In agreement with results of previous experiments in chick embryo fiver cell eulture~ (Fig. 1 [11], [25]), treatment of the cultures with 50 # M glutethimide and 50 ~M FeNTA led to synergistic induction of heine oxygenase. Increases in hemc oxygenase were associated with increased levels of mRNA and protein (results not shown), probably due to an increase in transcription of heme oxygenase message [25]. Lower concentrations of FeNTA (5 #M) were previously shown to have a modest synergistic effect on heme oxygenase and ALA synthase [22]. The data show that inductions of both ALA synthase and i~cme oxygenase activity by glutethimide and iron * are synergistic, that is, the inductions of both enzymatic activities are greater when cultures are treated with the combination of glutethimide and iron than ',he additive inductions of each treatment individually. To modulate heme oxygenase induction, 50 nM tin-mesoporphyrin, a potent

* Note: iron is used throughout the r e m a i n d e r of the text Io m e a n FeNTA.

inhibitor of heme oxygenase [8,33], proposed as a possible therapy for acute po~phyric attacks [34,35], was used to inhibit heme o~genase activity. Tin-mesoporphyrin, when added with glutethimide and iron 18 h before harvest, inhibited hemc oxygenase activity and suppressed the synergistic induction of ALA synthase (Fig. 1). Tin-mesoporphyrin inhibited heine oxygenase activity under all conditions, but only suppressed ALA synthase induction in the group treated with glutethimide and iron. ALA synthase induction by glutethimide alone was not affected by tin-mesoporphyrin. Further understanding of the synergistic inductions of heme oxygenase and ALA synthase by glutethimide and iron was gained from detailed time-course experiments (Fig. 2). The results show that both ALA synthase and heme oxygenase are synergistically induced between 6 and 18 h. A more detailed time-course of the critical period between 6 and 12 h indicated that the synergistic induction of hemc exygenase activity preceded the synergistic induction of ALA synthase activity (Fig. 2, inset). Specifically, heine oxygenase reached maximum activity by 9 h, while the inceease in ALA synthase activity above that observed at 6 h first became significant at 11-12 h and continued to increase through 24 h. These results suggested that synergistic induction of heme ox3,genase activity may play a role in producing the synergistic induction of ALA synthase by glutethimide and iron. We next tested whether tin-mesoporphyrin could terminate or blunt the synergistic induction of ALA synthasc that had already commenced upon prior 700

8 Herne Oxy~enose

ALA S y n t h o s e 7

.,,

3 ~, "6

t°

.:/ / ~

:~5.

.

300

~; .--""

."= ~,

_ ~ k

! 40C

;,oo/

i//

6oo

o~ .xo

8

6

° •

6

~

~

~

~.~

~

-

o

-

......

4

5

10

~

. . . .

0

) . . . . . . . .

15

20

"

25

Time of Treatment (hours)

Fig. 2. Time-course of induction of ALA synthase and heine oxygenase by glutethimide and ferric nitrik)triacetate (FeNTA). ('hick embryo liver cell cultures were treated with 50 ~ M glutethimide and 50 p M FeNTA and harvested at the times indicated. Assays were peril)treed as described in Materials and Methods. The inset shows a more detailed time-course during the critical time, from 6 to 12 h after the start of treatment. Heme oxygenase reached maximum activity by c) h while ALA synthase continued to rise through 24 h. Results arc mean + S.E.. n = 3. When no error bar is present the standard error of the mean falls within the size of the symbol.

treatment with glutcthimide and iron. This was done to test the hypothesis that in this system early inductions of heine oxygenase would deplete a regulatory hemc pool causing a derepression of ALA syn~hase. When tin-mesoporphyrin was added 14 h after the treatment of the cultures with glutethimide and iron, heme oxygenase activity was decreased within 2 h and any further

is F

increases in ALA synthase activi~ were halted (Fig. 3). Since tin-mesoporphyrin probably affects induction of ALA synthase by virtue of its ability to increase a regulatory heme pool, hern.,e (10 ~M) was added to the cultures to determine if it would have an effect similar to that of tin-mesoporphyrin. This concentration of heme was used since it gave maximal induction of

700

÷

I ALA Synthose

I

Heine Oxygenase

&

>--.

t:

17"

\

]

""h-,

%r!"

,

"O

\

O

5~ 9, il

_.__.~.

\",,

..6

. F-I- ~SnU ~ @ 14hours

2O0

I [~'

100 "C)" ' SnMesaa~x~Hecne@ O!

10

....

i

'

I '

'

15 20 Time ot T r e a t m e n t (hours)

/

'

25

10

15 20 Time of T r e a t m e n t ( h o u r s )

~0 9~

Fig. 3. Effects of l0 # M heme and 100 nM tin-mesoporphyrin added to chick embryo liver cell cultures treated with 50 ~.M glutethimide and 50 ~M ferric nitrilotriacetate (FeNTA). Heine, tin-mesoporphyrin, or a combination of both, were added after culture,', had been treated for 14 h with g|utethimide and FeNTA, The ce!!s were harvested zt the indicated times and assays were performed as de~ribed in Materials and Methods. Results are mean + S.E., n = 3. When no error bar is present the standard error of the mean fa;Is within the size of the symbol.

249 TABLE I

Effects o f inhibitors o f heine biosyntheA;.~ on m tit itws o]" A L A ~ynthas,, attd heine oaygenase Chick e m b r y o dyer cell cultures were treated fi)r 18 h as indicatcd with 2 m M 4,6-dioxoheptanoic acid (4,h-DttA). Ill() qM N-mcthyb mesoporphyrin or 10 # M heine. Cells were harvested and assays p e r f o r m e d as described in Materials and Methods. Results are m e a n + S.E.. n = 3. Treatment

Activity of A L A synlhase (nmol A L A mg i h r - I )

Activity of h e m e oxygenase (pmol bilirubin mg t m i n ~ l )

Control G l u t e t h i m i d e + iron Glutethimide +iron +4,6-DHA G l u t e t h i m i d e + iron + N methylmesoporphyrin Glutetbimide + iron + 4 , 6 - D H A + heine Glutethimide + iron + N-methylmesoporphyrin + heme

1).8 + 0.1 7.2 + 11.I 10.9+ 1.4

43 _+ 5.2 413 4- 33 98:~ 12

8.9 + I).4

84 -+ 18

7.1 4-11,2

426 _+42

4.0 + 0,1

1494- 2.9

heme ox'ygenase (data not shown). Heine dot only prevented further increases in A L A synthase activity, but actually caused a reduction in ALA synthase activity within 4 h after hcme addition (Fig. 3). The combination of tin-mesoporphyrin and heme, when added at 14 h after treatment with glutethimide and iron, reduced A L A synthase activity by 70% within 2 h after treatment, more rapidly than addition of heme alone. These data further support the concept that heme oxygenase activ;ty can influznce activity of A L A synthase by modulating levels of a regulatory heine pool. In an effort to deplete tl-.e regulatory heine pool by a different means, an inhibitor ot heme synthesis, either 4,6-dioxoheptanoic acid(4,6-DHA), an inhibitor of A L A dehydrase (EC 4.2.1.24) [36], or N-methylmesoporphyrin, an inhibitor of ferrochelatase (E.C. 4.99.1.1 ) [37], was added to the ct:,'tures. Addition of either inhibitor simultaneously with glutethimide and iron suppressed the synergistic induction of heine oxygenase (Table i), indicating that this induction requires intracellular heme synthesis, iv accordance with previous findings [37]. When 10/xM exogenous heine was added along with 4,6 DHA, glutethimide and iron, heme oxygenase activity was induced to a level coinciding with heine oxygenase activities following treatment with glutethimide and iron alone. When 10 p.M heme was added with N-methylmesoporphyrin, glutethimide and iron, heine oxygenase activity increased but did not return to fully induced levels. A L A synthase acttvity was further increased when either 4,6-DHA or Nmethylmesoporphyrin was added to the culture along •with glutethimide and iron (Table 1), indicating that if a regulatory heme pool is depleted, very marked induction of ALA synthase can occur. As expected. 10 ~ M

cxogcm,us hcmc diminished the inducti,m of ALA synthasc by glutcthintide, iron and 4.h-DHA or Nmethylmesoporphyrin (Table !). Discussion

The ability of iron in combination with phenobarbital-like drugs to produce synergistic induction of hepatic ALA synthase has becn known for many years, although the prccise mechanisms underlying this synergism have remained obscure [6,21-241. Perhaps the best established general mechanism for regulation of hepatic ALA svnthase is an inverse relationship between activity of the enzyme and the relative size of a small hypothetical pool of intracellular heine that has been called the 'regulatory" or 'unassigned' heine pool (for reviews see Refs. 3, 4 and 6). lntracellular heme can control the activity of ALA synthase via several different mechanisms: (1) possible transcriptional or translational repression [38]; (2) effects of home on the halt-life of ALA synthase mRI';A [39-43]; and (3) inhibi:ion ef the mitochonurial uptake of immature ALA .'~ynthase [44,45]. Although not measured directly, there seems little doubt that the relative size of a regulatory heine pool does, in fact, play a role in modulating activity of both ALA synthase, the rate controlling enzyme of heme synthesis, and heme oxygenase, the rate-controlling enzyme of heine catabolism. The recent observation that the combination of iron and glutethimide produced synergistic induction of heme oxygenase (Fig. !) [li,22,25], to a level that is produced by the most potent inducers, prompted us to investigate the hypothesis that induction of heine oxygenase per se could deplete the regulatory heme pool sufficiently to contribute to synergistic induction of ALA synthase. This hypothesis would be considered unlikely using the model proposed by Grankk [1,2] for control of ALA syr~thase, because the concentrations of heine needed to saturate or induce heine oxygenase (>_ 2 ,uM) arz orders of magnitude greater than those thought t~ be required to repress ALA synthase [2]. However, data to support the hypothesis that induction of heme oxygenase can lead to induction of ALA synthase were later presented by others [13]. Our detailed time-course experiment (Fig. 2, inset) shows that marked synergistic induction of heine oxygenase activity by glutcthimide and iron did, in fact, precede by 4-8 h the synergistic induction of ALA synthase. Addition of tin-me~porphyrin, a potent inhibitor of heine oxygenase, prevented (Fig. 1) or promptly blunted (Fig. 3) the synergistic induction of ALA synthase by glutethimide and iron. When heme was added during the A L A synthase induction, not only was the induction arrested, but repression of A L A synthase activity occurred within 4 h. When tin-mesoporphyrin and heine were added concurrently, ALA synthase was repressed

250 within two h, even more rapidly than the repression that occurred with hcmc alone (Fig. 3). This suppressive effect of tin-mesoporphyrin on tnc synergistic induction of A L A synthase activity, by glutethimidc and iron, is unlikely to bc due to a 'direct' or home-like effect on A L A synth~sc by tin-mesoporphyrin because: (1) a low concentration of tin-mcsoporphyrin (50 or I(10 qM) was used; (2) tin-mesoporphyrin had no effect on the induction of A L A synthase by glutethimide alone; and (3) 5/.tM tin-mesoporphyrin had no effect on A L A synthase activity in vitro (data not shown). T h e s e data thus provide further evidence that A L A synthase activity is regulated via a regulatory heine pool and that a sufficient a m o u n t of heine in the regulatory pool can repress A L A synthase activity. They strongly mJggest t h a t u n d e r at least some conditions, increases in h e m e oxygenase activity can lead to synergistic increases in activity of A L A synthase. However these data do not resolve the issue of w h e t h e r depletion of the regulatory heme pool per se is sufficient to induce A L A synthase activity. A t t e m p t s to deplete the regulatory h e m e pool by impeding heme synthesis within the cell using 4,6-DHA or N-methylmesoporphyrin show that h e m e oxygenase induction by glutethimide and iron is d e p e n d e n t upon heme formation (Table I). "lhus, inhibitors of h e m c synthesis suppressed induction of heme oxygenase. They also led to even greater inductions of A L A synthase, demonstrating that, w h e n h e m e synthesis is blocked in the presence of glutethimide, m a r k e d induction of A L A synthase occurs. T h e s e data are in agreement with previous reports [46,47]. W h e n the deftciency of regulatory heine, p r o d u c e d by the inhibition of heme synthesis, was corrected by the addition of exogenous heine, the effects on induction of A L A synthase were blunted or abolished (Table I). H e m e alone, in this system, is as effective at inducing h e m e oxygenase as glutethimide a n d iron [11]. As shown in Fig. 3, effects of h e m e to repress A L A synthase are rapid and impressive. T h e lesser effects observed in Table I are due to the long incubation time 118 h). N-methylmesoporphyrin is not a direct inhibitor of heine oxygenase in vitro [8]; ihus, the reason that h e m e in the presence of N-methylmesoporphyrin failed to restore heme oxygenase activity to levels comparable to those obtained with the glutethimide and iron treatment is not clearly understood. The mechanisms of effects of glutethimide alone (or of similar phenobarbital-like drugs) on A L A synthase were not the focus of this paper. However, others recently reported that such drugs alone (in the absence of iron) induce m R N A of A L A synthase [26,40]. Although not yet resolved, such m R N A induction is probably not due solely to depletion of regulatory heme, e.g., as a result of induction of cytochrome /'-450 [26], or of heine oxygenase, (our data following t r e a t m e n t

with glutcthimidc and tin-mesoporphyrin Fig. 1, discussed above). In summary, our rcsults support the hypothesis that alterations in activity of h e m e oxygenase can m o d u l a t e the size of a regulatory h c m c pool and thereby affect thc activity of A L A synthase. Additionzl studies are n e e d e d to learn w h e t h e r this interrelationship between the r a t c - c ntrolling enzymes of h e m e synthesis a n d h e m c breakdown hold true w h e n h e m e oxygenase is induced by metal ions, which act by a h e m e - i n d e p e n dent mechanism [7,11,48], and ,~hat effect, if any, t h e r e is on the half-life of A L A synthase m R N A a n d the translocation of i m m a t u r e ,~LA synthase into the mitoc h o n d r i a u n d e r these conditions.

Acknowledgements We t h a n k Richard W. L a m b r e c h t for helpful discussions on this p a p e r and J o s h u a W. Hamilton et al. for a p r e p r i n t of their p a p e r [43]. S u p p o r t e d by N I H grant ( D K 38825) a n d a grant from T h e A m e r i c a n Porphyria Foundation.

References I Granick. S. (Igb~) J. Biol. Chem, 241, 1350-1375. 2 Granick, S., Sinclair, P,R.. Sassa. S. and Grieninger, G. 11975) J. Biol. Chem. 250. 9215-0225. 3 Kappas. A., Sassa. S., Gali~raith, R.A. and Nordmann, Y. 11989) in The metabolic basis of inherited disease (Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle. D.. eds.), pp. 1305-1365. McGraw-Hill. New York. 4 May, B.K., Borthwick, I.A., Srivaslava, G., Pirola, B.A. and Ellion. W.H. (1986) Curr. Topics Ceil Reg. 28. 233-262. 5 May. B.K. and Bawden. M.J. 119891Sere. Hematol. 26. 1511-156. 6 Bonk~wsky. tt.L. (199tl) in Hepatology a textb~x~kof liver disease (Zakim. D. and Boyer. T.D., eds.), pp. 378-424. W.B. Saunders Company. Philadeplphia. 7 Kikuchi. G. and Yoshida. T. (198"~)Mol. Cell. Biochem. 53/54. 163-183. 8 Bonkovsky.H.L.. Healey, J.F. and Pohl. J. (19911)Eur. J. Biochem. 189, 155-166. 9 Srivastava, G., Brooker, J.D., May, B.K and Elliou. W.H. 1198111 Biochem. J. 188. 781-788. 10 Sassa. S., Kappas. A.. Bernstein. S.E. and Alvares, A.P. 119791J. Biol. Chem. 254, 729-735. 11 Lincoln, B.C., Healey. J.F. and Bonkovsky, tt.k. 119881Biochem. J. 250. 189-196. 12 Lincoln, B.C., Aw, T.Y. and Bonko~,sky, !I.k. 119891 Biochim. Biophys. Acta 992. 49-58. 13 Srivastava, G., Brooker, J.D.. May. B.K and Elliott, W.H 119801 Biochem. Int. 1.64-70. 14 Maines, M. and Kappas, A. 119771Science 198, 1215-1221. 15 KuUy. R.K., Daniel, R.F,, Ryan, D.E.. Levin, W. and Maines, M.D. 119881Arch. Biochem. Biophys. 260. 638-644. 16 Maines, M.D. (1988) FASEB J. 2. 2557-25t~8. 17 Sardana. M.K., Sassa, S. and Kappas, A. 119801 J. Biol. Chem. ~95, 11320-11323. 18 Maines. M.D, 119841CRC Crit. Rev. Toxicol. 12. 241-314. 1O P,~ine. A.J. and Legg. R.F. (1978) Bio~cm. Biophys. Res. Commun. 81. 672-679.

251 211 Drummond. G.S. Rt)senberg. D.W, and Kappas, A. (19821 Biochem J. 2112, 59-66. 21 Shedhffsky, S.i.. Bonkossky, |t.L., Sinclair, P,R.. Sinc}:lir, J.F. and Bement, W.J. (19821 ttepatology 2, 732 (abstract), 22 I~mkovsky, !t L. (IqSql Hepalology IlL 354-364. 23 Stein, J.A., Tschudy. D.P.. Corcoran. P L and Collins, A. (U)7{)) J. Biol. Chem. 245. 2213-2218. 24 Bonkowsky, lt.L., Sinclair, P.R. and Sinclair, J.F, {1~701 Yale J, Biol. Med. 52, 13-37. 25 Cable, E., Greene, Y., Healey. J,, Evans, C.-O. and ~mkovsky. I-i. (Igq0) Biochem. Biophys. Res. Commun. 168, 176-181. 26 ilamilton, J.W., Bement, W J . , Sinclair, P R . . Sinclair. J.F. and Wetterhahn, K.E, (19881 Biochem. J. 255. 267-275. 27 Sinclair, P.R. and Granick. S. (Ig77) Anal. Biochem. 79. 381}-303. 28 Lien, L-F. and Beattie, D.S. (Ig82) Enzyme 28, 12(I-132. 29 Chomczynski, P. and Sacchi, N. (19871 Anal. Biochem. 162. 156-159. 30 Li, K., Warner, C.K., tlodgc. J.A., Minoshima, S., Kudoh, J., Fukuyama, R., Maekawa, M., Shimizu, Y., Shimizu, N. and Wallace, D.C. (19891 J. Biol. (?hem. 264, 13998-14(~14. 31 Evans. C.-O., ttealey, J.F.. Greene, Y. and Bonkovsky. It.L. (1991) 13iochem. J. 273, 6 5 g - ~ . 32 Borthwick, I.A., Srwastava, G., Day. A R . , Pirola, B.A., Snowell, M.A., May, B.K. and Elliot. W J I . (19851 Eur J. Biochem. 1511. 481-484. 33 Drummond. G.S.. Galbraith. R.A., Sardana, M K . and Kappas, A. (19871 Arch. Biochem. Biophys. 255. 64-74,

Galhrailh, R.A.. D[ummond, (}£. and K:~ppas. A. (1085t J. ( l i n Invest. 76. 243~. 2439. 35 (;albraith. R.A. and Kapp~l~. A. I IC~},9) ttcpatoh~i,~ '), 882 S ~ . 36 Tschudy, D.P.. Itcss. R,A :rod F~kholm, H('. ~l~}Sl} J Bier! ('hem. 25B. qq 15 - ¢~q23. 37 l)c Matlci~, F. and Marks, G.S.(t'~¢~,) FEHS l e l l i ~(J, 127-131 3g Yamamoto. M., Kure. S., EngeL J.D. and Ihr:Jga. K. f l g X S ) J. Biol. Chem. 263, 15973-1507q. 3cJ Ades, i.Z., Stevens, T.M. and t~=~~., ~'.D. ~,i ~;/~ Arch. |$1OChCnL Biophys 253. 297-3~14. 4(1 Dre'*, P.D., A d e s I 7 119891 Bit~chem. Bioph'¢~, R c s ('ommun. 162, 11/2-1(17. 41 Lorher. B.J. and Adcs, I.Z. (19891 Int. J. Biochcm 21, 43q-443. 42 Iwasa. F,. Sassa, S. and Kappas, A. (It189) Biochem. I, 262, 81t7-813. 43 Itamillon. J.W.. Bemenl. W.J,. Sinclair, P R . , Sinclair, J.F., AIcedo. J.A. and Wetterhahn, K,E, { l'U,q ) Arch. Biochem. Biophys, in press. 44 Yamauchi, K., Itayashi. N, and Kikuchi. (3 (lt)RIH J. Biol. ('hem. 255, 1746-1751, 45 ttayashi, N.. Watanabe. N. and Kikuchi, (}. (19~3t Biochcm. Biophys. Res. Commun. i I;,. 7~!--'{~. 46 Scht~nfeld, N., Greenblal, Y., Epslein, 0 and Alsman, A. ( Iq,~21 Bk~him. Biophys. Acta 721.40g-417. ,17 Giger. U. and Meyer. U . A 110831 FEBS l_eu. 153. 335-33~. ,.18 Maines, M.D. and Kappas. A. (It17`1) Proc. Nail Acad. SCL USA 71, 42(13- 4297, 34