ARCHIVES

OF BIOCHEMISTRY

Cytochrome

AND

BIOPHYSICS

P-450 Heme and the Regulation Oxygenase Activity’

D. MONTGOMERY Department

of Medicine

176, 91-102 (1976)

and the Liver

BISSELL’ Center,

AND

University

Received

January

of Hepatic

LYDIA

E. HAMMAKER

of California,

San Francisco,

Heme

California

94143

9, 1976

The degradation of cytochrome P-450 heme in the liver has been studied by a new approach. In rats, hepatic heme was labeled by administration of a tracer pulse of 15“C18-aminolevulinic acid (ALA), and its degradation was analyzed in terms of labeled carbon monoxide (%O) excretion, which is a specific degradation product of the labeled heme. Within minutes after administration of 15-‘4C1ALA, ‘“CO was detectable and increased after 2 h to an “early peak,” reflecting the elimination of labeled heme from a rapidly turning over pool in the liver. Beyond the early peak, the rate of ‘CO production decreased in a log-linear manner, consistent with the degradation of heme in stable hepatic hemoproteins. From the rate at which ‘CO production declined during this phase, from the predominant labeling of cytochrome P-450 heme by the administered [5“CIALA and from the known turnover characteristics of this hemoprotein in the liver, it could be inferred that production of ‘%O -between 16 and 30 h after administration of labeled ALA-largely reflected degradation of cytochrome P-450 heme. This approach, which permits serial measurements in a single animal, was used to study the effect on cytochrome P-450 heme of administered heme or endotoxin, both of which are potent stimulators of hepatic heme oxygenase activity. Both of these substances caused marked acceleration of the degradation of cytochrome P-450 heme, the effect occurring over the same dose range as that for stimulation of hepatic heme oxygenase. The findings suggest that stimulation of this enzyme activity in the liver is closely related to the rate of degradation of cytochrome P-450 heme.

Microsomal heme oxygenase is a wellcharacterized enzyme system which in vitro converts heme” to biliverdin and carbon monoxide (CO) (1). The system has been termed “substrate-inducible” because it is stimulated in rats after administration of heme. Intravenous administration of hemoglobin or damaged erythrocytes stimulates heme oxygenase in hepatic pa-

renchymal cells or sinusoidal cells, respectively (2). If the amount of administered hemoglobin exceeds the haptoglobin-binding capacity of plasma, heme oxygenase also increases in the renal parenchyma (3). The enzyme system can be stimulated in peritoneal macrophages (4) or in choroid plexus (51, depending on the route of injection of heme. The major implication of these findings is that heme oxygenase plays a central role in the catabolism of circulating hemoglobin (6). However, a number of questions remain unresolved, particularly with respect to the stimulation of heme oxygenase in the liver. Although the enzyme system in hepatic parenchymal cells readily responds to injected hemoglobin, it is also stimulated by a number of nonheme substances, e.g., ep-

’ This work was aided by USPHS Grants GM21042, AM-11275, and (NIAMDD) P50 AM-18520, and by the Walter C. Pew Fund for Gastrointestinal Research. e Recipient of Research Career Development Award l-K04-GM-00149 from the National Institutes of Health, Bethesda, Md. S “Heme” refers to iron-protoporphyrin IX, without regard to its oxidation state or protein ligand, unless this is stated. 91 Copyright D 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

92

BISSELL

AND

inephrine (71, endotoxin (81, cobalt (91, and others (7, 101. There is no evidence that these agents cause intravascular hemolysis (11). It has been suggested that the effect of these substances may represent direct enzyme induction (8, 9). The theoretical possibility that perturbation of intrinsic hepatic heme metabolism may cause stimulation of heme oxygenase activity in the liver, has received little attention, and is the subject of the present investigation. This investigation was prompted by the observation that in a primary culture of adult rat hepatocytes (121, cytochrome P450 decreases during the early stage of cell incubation. This change in hemoprotein is accompanied by a striking, reciprocal increase in heme oxygenase activity (13). Since the cells were incubated in the absence of serum or other proteins, stimulation of heme oxygenase under these conditions could not be attributed to exogenous heme compounds. These observations, detailed in a separate report,” clearly raised the possibility that hepatic heme oxygenase activity is linked to degradation of intrinsic microsomal heme - in particular, that of cytochrome P-450. In extrapolating these findings to the intact animal, we postulated that administration of endotoxin, for example-and possibly heme itself- might initiate a similar perturbation of hepatic cytochrome P450 and thereby stimulate heme oxygenase. For testing this possibility, it was necessary to devise a convenient and reproducible method for measuring degradation of cytochrome P-450 heme in vim. The existing method involves injection of a group of animals with a tracer pulse of radioactive &aminolevulinic acid (ALA),” which labels hepatic heme (14, 15). At intervals thereafter, animals are sacrificed, and the amount of labeled heme remaining in cytochrome P-450 is determined from microsoma1 “particles” (16, 17). This approach requires a large number of animals and consequently would be expected to entail con4 Bissell, D. M., Guzelian, P. S., and Hammaker, L. E., in preparation. 5 Abbreviation used: ALA, S-aminolevulinic acid.

HAMMAKER

siderable experimental variation. Moreover, the study of multiple experimental variables by this technique is a formidable undertaking. An alternative approach for measuring degradation of hepatic cytochrome P-450 heme in vivo is suggested by the way in which administered radioactive ALA is metabolized and by the characteristics of cytochrome P-450 heme in the liver. Labeled ALA apparently fails to penetrate erythroid cells and does not label hemoglobin heme (14). By contrast, it enters hepatocytes, and labeled hepatic heme accounts for most of the radioactivity retained by the animal after injection of labeled ALA (14). Within the liver, the amount and rapid turnover of cytochrome P-450, relative to other hepatic hemoproteins (181, suggest that more than 50% of available heme is utilized in its synthesis. If this is true, it should be possible to measure the degradation of cytochrome P-450 in terms of the rate of appearance of labeled degradation products of heme (bilirubin or CO), in animals given a tracer dose of radioactive ALA. Production of labeled bilirubin after administration of [‘“CIALA has been studied previously (15). The initial event is the rapid appearance of an “early labeled peak” of bilirubin, probably representing a heme fraction with a very rapid turnover. Beyond the early peak, production of labeled bilirubin stabilizes, declining slowly over a period of several days (15). The latter phase of bilirubin production presumably represents degradation of heme in stable hepatic hemoproteins such as cytochrome P-450. In this paper, we explore the possibility that the rate of degradation of hepatic cytochrome P-450 heme can be studied as the rate of production of ‘“CO from single animals, given a tracer pulse of [5-14ClALA. The data suggest that 14C0 excretion (when studied beyond the early peak) closely approximates the rate of degradation of cytochrome P-450 heme. With this approach, the effect of stimulators of heme oxygenase has been studied. Both endotoxin and administered heme share the property of accelerating the degradation of cytochrome P-450 heme, and these effects

CYTOCHROME

P-450 AND

occur over the same dose range as does stimulation of hepatic heme oxygenase. On the basis of these observations, we suggest that stimulation of heme oxygenase in the liver results from perturbation of cytochrome P-450 heme. MATERIALS

AND

METHODS

Male Sprague-Dawley albino rats, weighing 180210 g, were purchased from Simonsen (Gilroy, California) and maintained on chow diet ad lib. up to the time of study. Labeled ALA, in the form of the hydrochloride, was obtained from New England Nuclear Corp., Boston, Massachusetts ([5-14C1ALA, 25 Ci/mol); the solution was neutralized with 0.1 N NaOH prior to injection. Endotoxin from Salmonella typhimurium was purchased from Difco (Detroit, Michigan) and, at a concentration of 1 mg/ml, was suspended in isotonic saline and finely dispersed with brief sonication. Methemalbumin was prepared by dissolving 25 mg of hemin (Sigma Chemical Co., St. Louis, Missouri) in 5 ml of NaOH (0.1 N), which contained 12 mg of Tris base and 222 mg of NaCl. To this solution was added 10 ml of 1% human serum albumin (Pentex, Kankakee, Illinois) in water, and the pH of the mixture was reduced to 7.4 by addition of a few drops of 1 N HCl. This preparation, containing 2.5 pmol of heme/ml, was used both for injection and for the heme oxygenase assay. Analytical procedures. Cytochrome P-450 and b, were measured by difference spectrophotometry in a DW-2 UV-VIS spectrophotometer (American Instrument Co., Silver Springs, Maryland). Cytochrome P-450 in whole liver homogenate was determined from the CO reduced minus CO difference spectrum (19); in microsomes and enzyme-digested particles, the method of Omura and Sato was used (20). Cytochrome b, was measured according to Omura and Sato (20) and protein was measured by the method of Lowry et al. (21). Microsomal heme oxygenase was assayed in 18,OOOg supernatant from rat liver homogenate, as described previously (21, at a protein concentration of approximately 2 mg/ml. Preparation of ‘Glabeled hemoglobin. Reticulocytosis was induced in rats (300-400 g) by subcutaneous injection of phenylhydrazine, 40 mglkg, every other day for a total of four injections. Three days after the last injection, blood was harvested with heparin as anticoagulant. The erythrocytes were washed twice with isotonic saline and resuspended in an equal volume of 0.87% NaCl. Disruption of the cells was carried out by nitrogen compression-rapid decompression (900 psi for 20 min) in a Parr Chamber (Kontes, Vineland, New Jersey). Hemoglobin crystallized spontaneously in the lysate at 4°C and the crystals were removed by brief centrifugation so as to reduce the concentration of unlabeled heme present. The supernatant fraction was incubated at

HEPATIC

HEME

OXYGENASE

93

pH 8.0 with [14C]ALA and fresh rat serum, as described elsewhere (22). At the end of the incubation, the solution was brought to pH 6.8, concentrated with Lyphogel (Gelman Instrument Co., Ann Arbor, Michigan) and placed at 4°C for 18 h. The newly synthesized labeled hemoglobin crystallized during this period and was washed free of labeled ALA by several centrifugations through 0.01 M KIHPOI, pH 7.4. The specific activity of hemoglobin heme, extracted and crystallized from these preparations, varied between 2000 and 6000 dpm/Fg. Immediately prior to use, the hemoglobin crystals were dissolved in a small amount of 0.05 N NaOH, and the solution was diluted with 0.01 M phosphate buffered isotonic saline, pH 7.8. Measurement of f’T!Iheme. Whole liver homogenate or subcellular fractions containing labeled heme were mixed with measured amounts of carrier heme. The carrier consisted of washed rat erythrocytes suspended in isotonic saline at a hematocrit of approximately 50%. The hemoglobin concentration of the suspension was estimated as cyanomethemoglobin with the Hycel reagent (Hycel, Houston, Texas). Carrier erythrocytes, 1 ml, were added to 1 ml of tissue sample. Heme was extracted and crystallized by the method of Labbe and Nishida (23), with a recovery of approximately 50%. A weighed amount of the dried crystals (ca. 0.5 mg) was dissolved in 0.2 ml of 0.1 M NaOH in a count vial and bleached with 0.2 ml of 30% hydrogen peroxide. Scintillation fluid (Dimilume-30, Packard Instrument Co., Inc., Santa Clara, California) was added, and radioactivity was determined in a Beckman LS200 spectrometer with an efficiency of 80-90%. From the specific activity of the isolated heme and the quantity of carrier, the radioactivity in heme in the original sample was determined. The contribution of heme from the sample itself was assumed to be negligible in relation to the amount of carrier added. Measurement of labeled heme in duplicate samples varied less than 10%. Analysis of expired ‘CO. Rats were injected either intraperitoneally or intravenously with [5IIClALA, which serves as label for the a-methene bridge carbon of the heme ring (18); on cleavage of the heme ring, this carbon is converted to 14C0 (6). The 14C0 exhaled by the animals was collected in a system similar to that described previously (24), except that two trains were arranged in parallel from the same vacuum source, so that pairs of animals could be accommodated. In this method, air from the animal chamber is drawn first through a series of traps that remove 14C0,. The ‘CO then passes over heated hopcalite for conversion of CO- to CO,, which is trapped in 4 ml of ethanolamine:methoxyethanol (1:l). One milliliter of this solution was added to 4 ml of methanol, 1 ml of methoxyethanol, and 12 ml of Liquifluor (New England Nuclear Corp., Boston, Massachusetts), and

94

BISSELL

AND

radioactivity was quantitated by liquid scintillation spectrometry with a counting efficiency of 92%. The efficiency of the CO train is 90-lOO%, as determined by the recovery of a known amount of ‘CO released in the animal chamber and collected in the usual manner. Preparation of cytochrome P-450 particles and quantitation of [‘Tlheme in cytochromes p-450 and 6,. Cytochrome P-450 particles were prepared by two approaches, using either lipase (25, 26) or bacterial subtilisin (27) for digestion of rat liver microsomes. Use of two different methods in parallel seemed desirable to check for systematic errors that might be associated with a single approach. Since the two techniques provided comparable results (see below), the choice of a particular method in a given experiment was dictated largely by logistical constraints. The lipase technique permits direct assay of specific activity of [“C-hemelcytochrome b,, while the subtilisin method provides a direct value for labeled cytochrome P-450 heme. With either method, the specific activity of the “other” cytochrome was calculated from the total [‘“Clheme content of the microsomes and their measured cytochrome P-450 and cytochrome b, concentrations. This calculation is based on the assumption that microsomal heme represents the sum of hemes in cytochrome P-450 and cytochrome b,-an assumption that has been verified experimentally (20, 28). In the lipase method, the liver was excised without perfusion and homogenized in 3 vol of 0.1 M potassium phosphate buffer, pH 7.4. Microsomes were prepared from this homogenate by sequential centrifugations at 10,OOOgfor 20 min and the supernatant fraction at 105,OOOgfor 60 min. The microsoma1 pellet was resuspended in the same buffer and centrifuged at 105,OOOgfor 30 min. The washed microsomes then were suspended in 4 ml of the phosphate buffer, of which a l-ml aliquot was used for extraction of [Ylheme and measurement of cytochromes and protein. The remaining 3 ml of the microsomal suspension were diluted to 15 ml with phosphate buffer (final concentration, ca. 4 mg protein/ml) and mixed with 30 mg of lipase, type II (Sigma, lot No. 105B-1960). Incubation was carried out under air, without shaking and in the dark at 37°C. After 30 min, the solution was centrifuged at 4°C and 105,OOOgfor 120 min. The supernatant contained 40-50% of the total microsomal cytochrome bj, with negligible amounts of cytochrome P-450 or P-420 (26). After spectrophotometric quantitation of the cytochrome b,, the solution was concentrated by lyophilization, mixed with carrier heme and extracted, as described above, for determination of the amount of [‘Clheme in the original lipase supernatant. From this, the specific activity of [‘“Chemelcytochrome bj was calculated. The specific activity of cytochrome P-450 then was determined from microsomes by subtraction.

HAMMAKER In the subtilisin method, the initial handling of the liver was similar to that in the lipase procedure, except that prior to excision the liver was perfused in situ with phosphate buffer containing 10 mM nicotinamide and 2 mM glutathione (27). The initial microsomal pellet was suspended in the same buffer supplemented with glycerol to 20% (w/v) and incubated with subtilisin (Sigma “protease,” type VII) for 20 h at 4°C. By this procedure, the microsomes are stripped of virtually all cytochrome b,, with removal of only minor amounts of cytochrome P-450. There is no measurable conversion of cytochrome P450 to cytochrome P-420. Hence, the treated particles permit direct estimation of the specific activity of [‘lC-heme]cytochrome P-450. The specific activity of cytochrome b, is then calculated by subtraction, using microsomes as described above. To test the validity of this approach, a single batch of microsomes containing [‘4Clheme-labeled hemoproteins was divided into equal parts, one of which was incubated with lipase and the other with subtilisin. The specific activity of the 1’4C-hemelcytochrome P-450 obtained by the two methods differed by less than 10%. RESULTS

Experimental Approach: Evaluation of 14C0 Production as a Measure of the Rate of Degradation of Cytochrome P450 Heme in the Liver The time course of ‘%O production in a rat given a tracer dose of [5J4ClALA is shown in Fig. 1. Labeled CO appeared 10.000 -

FIG. 1. Production of 14C0 after administration of [5-‘C]ALA. Rats were injected with 5 &i of labeled ALA and placed immediately in the CO train for quantitation of 14C0 production, as described in Methods. The data shown are those of a typical experiment.

CYTOCHROME

P-450 AND

HEPATIC

within 30 min of the injection, increased rapidly to a peak at about 2.5 h, and then fell during the subsequent 7 to 9 h. Beginning about 12 h after the administration of label, 14C0 production assumed an apparently log-linear decline. The overall shape of this curve closely resembles that reported previously for production of labeled bilirubin after a dose of radioactive ALA (15). The apparent first-order kinetics of the log-linear portion of the curve suggest that, during this time period, the CO may be derived from a single heme pool in the liver. The slope of the curve suggests a half-life for the heme source of 28 + 5 h (n = lo), which is similar to that reported for the heme of cytochrome P-450 (16, 17) and suggests that this heme fraction gives rise to the labeled CO. This inference was further supported by studies of the distribution of [‘*C]heme in the liver. At a time 22 h after administration of the pulse of labeled ALA, on determination of the [14C]heme content of whole liver homogenate and in cytochromes P-450 and b, (from microsomal “particles”), it was found that 60-70% of the total hepatic [14Clheme was present in cytochrome P450 (Fig. 2). Since the estimated turnover rate of cytochrome P-450 heme is twice that of cytochrome b, and several-fold that of mitochondrial cytochromes (18), these data imply that, between 18 and 30 h after administration of labeled ALA, hemoproteins other than cytochrome P-450 will contribute little to total 14C0 production by the liver. Also shown in Fig. 2 is the fact that 14C0 production is proportional to the amount of [14C]heme in the liver or in cytochrome P-450, i.e., it can be assumed that production of labeled CO at comparable rates by individual animals reflects comparable amounts of 14C-heme in the liver. These findings indicate that the rate of 14C0 excretion- between 18 and 30 h after administration of labeled ALA -closely approximates the rate of degradation of cytochrome P-450 [14C]heme in the liver. Moreover, since animals excreting 14C0 at similar rates are labeled to a similar extent with respect to hepatic 14C-heme, it is possible to study them in pairs, one animal receiving the experimental manipulation,

HEME

I0

f

TOTAL “C-HEME/LIVER “C-HEM

2

800

b :

400

: 6 2 4 %

95

OXYGENASE

IN ClTOCHlOME

~

l-l

P-450

14C-HEME IN CYTOCHROME

b,

400

200

I 100

260

360

4bo

RATE OF 14C0 EXCRETION

560

600

700

(DPM/HOUR)

FIG. 2. Relationship between ‘WO production and hepatic [‘%]heme content after administration of [5-‘*C]ALA. Animals were injected with 1.25, 2.50, or 5.00 &i of labeled ALA. At a point 18 h after the injection, rates of 14C0 production were determined hourly for a period of 3 h. The animals then were sacrificed, and the [‘%!]heme in whole liver homogenate and in cytochromes P-450 and b, was measured, as described in Methods. Recovery of microsomes from the whole homogenate was assessed from the recovery of cytochrome P-450. The average rate of ‘“CO production, shown on the abscissa, for each animal was proportional to the dose of administered ALA.

the other serving as control. This permits analysis not only of differential rates of 14C0 production but also of the disappearance of labeled heme from cytochrome P450. With these considerations in mind, a standard protocol was devised, which has been used in the studies to be described below (Fig. 3). Pairs of animals, matched for sex, weight, and age, were injected with 5 PCi of [5J4ClALA. After an interval of 16-18 h, the rats were placed in individual metabolic glass cages, and basal rates of 14C0 production were determined hourly, for the indicated period. If rates of production of labeled CO were comparable, one animal was subjected to experimental manipulation, according to protocol, while the other served as control. The rate of 14C0 production was measured serially or until the animals were sacrificed for determination of total labeled heme and specific activity of heme in cytochrome P-450. In both control and endotoxin-treated animals (see below), the amount of 14C0 produced over a given period of time accounted for approximately

96

BISSELL

AND

FIG. 3. Protocol for study of ‘CO production in animals injected with a tracer pulse of [5-“CIALA.

70% of the [‘4Clheme that disappeared concomitantly from the liver. Effect of Endotoxin on Hepatic Heme Oxygenase Activity and 14C0 Production In examining the relationship between degradation of cytochrome P-450 heme and stimulation of hepatic heme oxygenase, the first series of studies involved administration of bacterial endotoxin. In confirmation of previous findings (8), endotoxin was found to be a potent stimulator of hepatic heme oxygenase. The time-course of this response is shown in Fig. 4. Though endotoxin stimulates heme oxygenase in both the parenchymal and the sinusoidal cells of the liver (8), the sinusoidal cells comprise less than 5% of the total liver volume (29) and, therefore, represent an insignificant fraction of the total cellular protein in the homogenate. The response to endotoxin is dose-dependent over a range of 0.5 to 1.5 mg/kg (Fig. 5). Though all animals survived these doses, larger amounts of endotoxin resulted in substantial mortality and therefore were not used. When animals were pulse-labeled with 15-‘*ClALA and treated with endotoxin or control injection, according to the protocol in Fig. 3, endotoxin was found to cause a marked stimulation of ‘“CO production (Fig. 6). Accelerated production of labeled CO was apparent about 3 h after administration of endotoxin, reaching a peak after about 10 h (Fig. 6). This response was maximal at 1.5 mg/kg and was negligible at 0.5 mglkg. Thus, the dose-response range for stimulation of 14C0 production was similar to that for stimulation of hepatic heme oxygenase by endotoxin. After administration of [14Clheme to a rat, pro-

HAMMAKER

I,

IO-

{ E

II 20

IO

c

30

HOURS

FIG. 4. Stimulation of hepatic heme oxygenase by endotoxin. Endotoxin was administered intraperitoneally in isotonic saline. At various times thereafter, heme oxygenase activity was determined in 18,OOOg supernatant of liver homogenate. Each point represents the average value of a pair of animals. The shaded area represents the mean value 5 SD for six pairs of animals injected with saline and sacrificed in parallel with the endotoxin-treated rats.

c

1

I

I

I

I

0

5

10

1.5

DOSE ENDOTOXIN

J

(mg kg)

FIG. 5. Relationship of dose of administered endotoxin to stimulation of heme oxygenase activity. Rats were injected with varying doses of endotoxin or with comparable volumes of isotonic saline; 12 h later, hepatic heme oxygenase activity was determined as described in Fig. 4. Each point represents mean 2 SE for six to eight animals.

duction of 14C0 lags slightly (about 30 min) behind [14Clbilirubin output in bile, possibly because of binding of CO to circulating hemoglobin (24). Despite this fact, peak stimulation of 14C0 production in the

CYTOCHROME

P-450 AND

HEPATIC

A

8

12

16

20

24

97

OXYGENASE

tion of endotoxin. Although basal 14C0 production, under the standard protocol (Fig. 3) seems clearly to be derived from cytochrome P-450 heme, it cannot be assumed a priori that 14C0 resulting from experimental perturbation also is derived from this hemoprotein, since the liver contains labeled heme in addition to that of cytochrome P-450. Accordingly, at various time-points after injection of endotoxin, pairs of labeled animals were sacrificed and the amount of [‘4C]heme in microsomes and in enzymatically isolated cytochrome P-450 was determined. As is shown in Table I, at time points corresponding to the peak effect of endotoxin on 14C0 production and beyond, the endotoxin-treated animal of each pair exhibited reduced [‘“CJheme in microsomes and in cytochrome P-450. These data verify that endotoxin acts to accelerate the degradation of cytochrome P-450 heme. The amount of labeled heme in cytochrome b5 or in mitochondria, in addition to being relatively small (cf. Fig. 21, was not consistently altered by administration of endotoxin. Table I also shows that, although the amount of [14Clheme in cytochrome P-450 was reduced after endotoxin, the specific activity of [‘4C-hemelcytochrome P-450, on the av-

r

2000

lOOi 0

HEME

28

HOURS

FIG. 6. Effect of endotoxin on ‘%O production in rats labeled with [5-‘%]ALA. Rats were injected with a tracer pulse of labeled ALA and studied according to the protocol shown in Fig. 3. After a period of basal 14C0 production, one animal received endotoxin (1.5 mg/kg) while the other received a comparable volume of isotonic saline.

present study preceded peak heme oxygenase activity by roughly 3 h. These data indicate that heme degradation precedes stimulation of heme oxygenase in the liver. Additional studies were carried out for determining the [14Clheme source of the excess 14C0 associated with administraTABLE

I

EFFECT OF ADMINISTERED END~TOXIN ON THE [%]HEME CONTENT OF HEPATIC MICROSOMES HW+W CYTWHROME P-450 PARTICLES~ Treatment

Tim@ (h)

Microsomes nmol cytochrome P450 per mg protein

[W]Heme

in cytochrome

P-450

dpm [‘4CJheme per mg protein

Total dpm per sample

dpm/mg protein

dpm/nmol cytochrome P450

Saline Endotoxin

5

0.69 0.63 (91)c

967 795

43,630 28,040 (64)’

1,360 1,078

1,017 1,159

Saline Endotoxin

9

0.87 0.60 (69)

678 649

13,470 8,430 (63)

435 383

410 663

Saline Endotoxin

21

0.97 0.51 (53)

893 445

9,600 3,360 (35)

649 343

538 472

Saline Endotoxin

22

0.83 0.37 (451

587 463

12,896 7,491 (58)

430 340

488 740

o Pairs of animals were pulse-labeled with 15-‘VIALA and studied according to the protocol shown in Fig. 3. The individual animals of a pair were treated either with endotoxin (1.5 mg/kg) or with a comparable volume of isotonic saline, by intraperitoneal injection. b “Time” denotes elapsed time between injection and analysis. c Figures in parentheses represent percentages of the paired control value.

98

BISSELL

.4ND HAMMAKER

erage, was unchanged. This indicates that there has been no dilution of [‘C]heme by newly synthesized unlabeled heme in cytochrome P-450 during the period of accelerated degradation. Effect of Administered duction

Heme on ‘“CO Pro-

The studies with endotoxin strongly suggested a causal link between degradation of cytochrome P-450 heme and stimulation of heme oxygenase. We next examined whether administered heme stimulated hepatic heme oxygenase indirectly, like endotoxin perturbing the metabolism of cytochrome P-450 heme. Animals were labeled with [5-14ClALA, according to protocol (Fig. 3) and were injected with heme in the form of methemalbumin. As with endotoxin, administered heme caused a striking stimulation of 14C0 production. The dose-response range for stimulation of labeled CO by heme is shown in Fig. 7. This corresponds closely to the dose-de-

pendent range for stimulation of hepatic heme oxygenase by heme (Fig. 8). The source of excess 14C0 after administration of heme was investigated in experiments similar to those with endotoxin, and the results were similar. Administered heme reduced the [‘4Clheme content of microsomes and of cytochrome P-450. However, in contrast to the effect of endotoxin, the specific activity of [14C-heme]cytochrome P-450 also was reduced, and the concentration of the hemoprotein in microsomes was only slightly affected (Table II). Heme administered as hemoglobin had an effect on 14C0 production similar to that of methemalbumin (Fig. 9). Because both of these manipulations increase heme flux in the hepatocyte (2), we considered the possibility that the peak of 14C0 represented not accelerated degradation of cytochrome P-450 heme but merely exchange of unlabeled administered heme for labeled microsomal heme. If this were true, the labeled exchanged heme should be degraded with the bulk of the administered heme. To test this possibility, we studied the time-course of degradation of 14C-labeled hemoglobin, which had been prepared biosynthetically from [5-14ClALA. Degradation of hemoglobin, labeled in this

00 HOURS

FIG. 7. Relationship of dose of administered heme to stimulation of ‘CO production, The data shown are a composite of three studies, each with a pair of animals, studied according to the protocol given in Fig. 3. After a period of basal ‘VO production, one animal of the pair received a dose of heme (2.50, 1.25, or 0.62 pmol) while the other received a comparable volume of heme-free buffered albumin (see Methods). The subsequent rates of ‘“CO production for the three control animals have been averaged and are represented as a single line on the chart (“control”). For purposes of comparing the effect of administered heme at varying doses, the values from the heme-treated animals have been normalized with respect to the “control” curve.

AHEME OXYGENASE ACTIVITY nmoles/min per 1Omg protein

FIG. 8. Proportionality of change in WO production and stimulation of heme oxygenase activity in animals injected with heme. Five hours after administration of heme, as shown in Fig. 7, animals were sacrificed and hepatic heme oxygenase activity was determined. The doses of heme administered were as follows: 0, 0.62 pmol; A, 1.25pmol; 0, 2.50 pmol.

CYTOCHROME

P-450 AND HEPATIC

99

HEME OXYGENASE

TABLE II EFFECT OF ADMINISTERED METHEMALBUMIN ON THE [WIHEME CONTENT OF HEPATIC MICROSOMES OR CYIWHROME P-450 PARTICLE@

Treatment

Tim@

(h)

[“‘C]Heme in cytochrome P-450

Microsomes nmol cyto- dpm[W]chrome P- heme per 450 per mg mg protein protein

Total dpm per sample

dpm/mg protein

dpm/nmol cytochrome P-450

Saline Heme

4

0.75 0.76

980 720

41,800 25,700

1,300 870

1,188 754

Saline Heme

20

0.60 0.49

650 380

15,220 7,290

525 270

544 320

u Animals were labeled with [5-WIALA and matched for WO production, as described in Table I. At the start of the study, animals were injected intravenously with methemalbumin equivalent to 2.5 pmol of heme or with an equal volume of heme-free albumin solution (see Materials and Methods). b “Time” denotes elapsed time between injection and analysis.

lation of heme oxygenase by hemoglobin which, as with endotoxin, clearly occurs subsequent to accelerated catabolism of cytochrome P-450 heme. DISCUSSION

These findings indicate that diverse stimulators of hepatic heme oxygenase activity - both nonheme substances and heme itself - are capable of perturbing the metabolism of cytochrome P-450. Accelerated degradation of this heme fraction in FIG. 9. Effect of administered hemoglobin on the liver appears to be closely linked to production of ‘%O by rats labeled with a pulse of 15- stimulation of heme oxygenase. These ‘%lALA. The experimental protocol is shown in Fig. studies were greatly facilitated by meas3. The dose of hemoglobin was 20 mg/lOO g rat urement of a degradation product, CO, of weight in a total volume of 1.0 ml. The control cytochrome P-450 heme, in addition to animal received a similar volume of isotonic saline. measurement of the loss, over time, of labeled heme from cytochrome P-450 “partifashion, yields 14C0 and, therefore, can be cles” (16, 17). We chose to measure CO measured under the conditions used for rather than bilirubin, because collection of analyzing degradation of cytochrome P-450 CO from rats requires neither the operaheme. The time-course of degradation of tive procedure nor physical restraining of labeled hemoglobin is shown in Fig. 10 animals that is needed for studies of biliru(left curve). For purposes of comparison, bin excretion. The system has proved to be the time-course of 14C0 production from virtually free of systematic error, to yield cytochrome P-450 heme, after a comparareproducible results, and to be highly senble dose of unlabeled hemoglobin, is shown sitive to the experimental variable introin the same figure (center curve). The tem- duced. The assumptions involved in measporal separation of these two processes es- uring heme degradation in this fashion sentially rules out the possibility that appear to be well founded, i.e., the fact quantitative exchange occurs between un- that heme is the only physiological source labeled heme of hemoglobin and [14Clheme of CO (30-32) and that, in normal animals, in cytochrome P-450. Also shown in Fig. 10 bilirubin and CO are produced in equimo(right curve) is the time-course for stimular amounts (6). A shortcoming of this ap-

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may arise as altered apocytochrome P-450 and not be strictly related to heme degradation in the cell. This problem, discussed elsewhere (13, 341, is under study at the present time. In relating stimulation of heme oxygenase to degradation of cytochrome P-450 heme, the conclusions of these studies may be applicable only to the liver. While cytochrome P-450 is abundant in liver, it is detectable only with difficulty in spleen or in peritoneal macrophages, despite the FIG. 10. Time course of degradation of adminisfact that spleen normally exhibits high tered heme compared to that of cytochrome P-450 heme oxygenase activity (1, 10) and peritoheme and to stimulation of heme oxygenase activity neal macrophages can be stimulated to in the liver. The figure is a composite of three sepahigh activity (4). Thus, the heme moiety of rate experiments. In all studies, rats were injected this cytochrome may not be a factor in intravenously with 20 mg/kg of hemoglobin. For the the regulation of heme oxygenase in cells data of the left-hand curve, ‘%-labeled hemoglobin was injected, and its degradation to ‘%O was meaother than the hepatocyte. Indeed, endosured. A second pair of animals was studied accordtoxin fails to stimulate the enzyme system ing to the protocol in Fig. 3, and the injected hemoin macrophages incubated in suspension globin was unlabeled. These data are shown in the (8). center curve. A final group of animals was injected The data appear to clarify a number of with hemoglobin or saline and sacrificed at intervals puzzling features of heme oxygenase in the thereafter for determination of hepatic heme oxyliver, particularly with regard to the nongenase activity, as shown in Fig. 4. The shaded area heme stimulators of the enzyme system (right) represents the range of heme oxygenase ac(7-10). Previous explanations for the effect tivity in the saline-treated control rats. of these substances included the possibility that they directly induced heme oxygenase preach is that the precise source of CO can activity (9) or that they caused an influx of reticuloendothelial cells into the liver (10). be deduced only indirectly, unless supplementary studies of the disappearance of It now appears likely that they act by inlabeled heme are carried out, as has been creasing the degradation of heme from done in the present work. cytochrome P-450. Though we present data and administered The results indicate a close link between only for endotoxin degradation of cytochrome P-450 heme and heme, it is reasonable to assume that stimulation of heme oxygenase activity. other stimulators of the enzyme system act These changes are linked both temporally in a similar fashion, as suggested by preand quantitatively, after administration of liminary experiments with cobalt and epieither endotoxin or heme. Since the rate of nephrine. Starvation of rats for 48 to 72 h also causes an increase in hepatic heme heme degradation changes prior to stimulation of heme oxygenase, the data appear oxygenase (71, accompanied by a decrease to be consistent with a substrate-mediated in the concentration of cytochrome P-450 induction of the enzyme system, as has in the liver (35). The latter phenomenon been postulated previously (2-4, 6, 10). In may reflect accelerated degradation of the albeit decreased synthesis the case of hepatic parenchymal cells, the hemoprotein, substrate may be cytochrome P-450 heme. may also play a role. The mechanism (or mechanisms) However, a stringent proof of heme-meof heme or endodiated induction has not been offered in whereby administration toxin accelerates degradation of cytothis paper, and other possibilities exist. chrome P-450 heme are conjectural. The For example, the reciprocal relationship suggests between cytochrome P-450 and heme oxy- diversity of these perturbations genase in hepatocyte cell culture (13, 33) that their effect is indirect, with a final common pathway yet to be delineated. In suggests that heme oxygenase activity I

c

CYTOCHROME

P-450 AND

HEPATIC

vitro studies have suggested that the heme of cytochrome P-450 may be degraded by peroxidative processes (36). However, this mechanism may not be operative in uiuo, since pretreatment of animals with antioxidant, in doses sufficient to prevent lipid peroxidation (37), fails to prevent the effect of endotoxin on heme oxygenase activity.6 Moreover, the decrease in cytochrome P-450 and increase in heme oxygenase that occur in hepatocyte culture are unaffected by addition of a variety of antioxidants to the incubation medium (13). A noteworthy feature of any of these stimulators of heme oxygenase is the fact that effective doses, in general, fall in the pharmacologic range and may be associated with stress or frank toxicity. The amounts of epinephrine, endotoxin, cobalt, or zymosan (7-10) used for maximal stimulation approach the LD,, of these substances for rats. The dose of glucagon (71, in addition to stimulating heme oxygenase, produces autophagocytosis in rat liver (38). Also, the amounts of methemalbumin or hemoglobin approximate, in an intravenous bolus, the fractional daily catabolism of circulating hemoglobin in a rat, much of which normally may be degraded not in hepatocytes but in the reticuloendothelial system. In other words, it is not clear whether the response to any of these substances represents a specific physiologic effect or rather a secondary reaction to a pharmacologic challenge. In this regard, it is relevant that numerous kinds of physical stress provoke increased activity of heme oxygenase in the liver. The effect of starvation has been described (7). We have observed also that rigid restraining of rats, as is required for collection of bile from cannulated animals, leads to increased activity of heme oxygenase. The latter phenomenon may be responsible for experimental artifacts, as suggested by studies several years ago of the conversion of administered 14C-labeled hemoglobin to 14C!-labeled bilirubin (39). In the course of this work, it was noted that the specific activity of bilirubin in bile from cannulated animals was lower than expected; this implied a dilution of the la6 Bissell, D. M., and Hammaker, lished observations.

L. E., unpub-

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101

beled pigment by unlabeled bilirubin. The source of this unlabeled fraction was unknown at the time but, in retrospect, may have been intrinsic hepatic heme, the degradation of which was stimulated by the operative procedure and restraining of the animals. The mediation of these stressproducing manipulations - if stress is their common feature-at a subcellular level is under investigation. Hepatic heme oxygenase appears to be insensitive to large doses of adrenal corticosteroids, administered to normal animals (71, while studies in adrenalectomized animals have not yet been reported. In addition to the regulation of hepatic heme oxygenase, the fate of cytochrome P450 heme at the microsomal level is of interest. If heme is not degraded directly on apocytochrome P-450, then it presumably dissociates from its apoprotein prior to degradation. If this is so, the main effect of administered endotoxin or heme may be on the affinity of heme for apocytochrome P-450, such that dissociation of the holocytochrome is facilitated. In the context of this hypothesis, heme dissociated from apocytochrome P-450 may constitute a labile “free” pool, destined ultimately for degradation to bile pigment but possibly with metabolic effects prior to its degradation. Numerous previous studies have suggested that ALA synthetase, the mitochondrial enzyme that mediates the ratedetermining step in hepatic heme synthesis, is subject to regulation by a pool of endogenous heme (18, 40). The possibility that “free” heme from cytochrome P-450 augments this postulated regulatory pool and therefore affects ALA synthetase is explored in the companion paper. The findings emphasize the fact that the heme of cytochrome P-450 is a rapidly turning over cell component, the metabolism of which is readily perturbed. Such perturbation appears to have consequences both for heme oxygenase activity and, as will be shown, for the regulation of ALA synthetase activity in the liver. Numerous previous studies have suggested that the activity of ALA synthetase in the liver reflects synthesis of cytochrome P-450 heme (401. The present data suggest that heme oxygenase activity may be a compar-

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