the induction in vitro of the synthesis of s ... · the induction in vitro of the synthesis of...

TIIE JOURNAL OF I%IOLOGICAL CHEMISTRY Vol. 241, No 6, Iesue of March 25.1966

hinted zn U.S.A.

The Induction in Vitro of the Synthesis of s-Aminolevulinic

Acid Synthetase in Chemical Porphyria: A Response

to Certain Drugs, Sex Hormones,

and Foreign Chemicals*

(Received for publication, July 21, 1965)

S. GHANICK

WITH THE TECHNICAL ASSISTANCE OF WILLIAM CUMMING AND RITA LAU

From The Rockefeller- Cniversity, New York, New York 10021

SUMMARY

1. A method is described for the primary growth of chick embryo liver cells on cover slips in culture, and the factors of the culture medium are considered that affect the induction of porphyrin formation with certain chemicals.

2. A method is described for following &aminolevulinic acid synthetase (ALA synthetase) activity by the determination of porphyrin fluorescence developed in the cells on cover slips after induction with chemicals or drugs.

3. A nonchromatographic method is described for the determination of b-aminolevulinic acid and aminoacetone in the range of 10-s mole.

4. With allylisopropylacetamide as inducing chemical, an &fold increase in the mitochondrial ALA synthetase of chick embryo liver has been found. No other tissue tested was inducible.

5. After induction, porphyrin increased at almost a logarithmic rate for the first 12 hours.

6. The inducing action of the chemical is reversible. 7. The half-life of ALA synthetase in chick embryo liver

cells in culture is 4 to 6 hours. 8. The chemicals and drugs which induce a porphyria in

the chick embryo liver cells in culture may be separated into four classes: the barbiturates which contain three chemical groups that can individually induce; the collidines which contain two chemical groups; the sex steroids; and a miscellaneous class.

9. Evidence is presented that the control of ALA synthetase in the liver is by feedback repression in which heme may be the corepressor. No feedback inhibition was found.

10. The site for induction is proposed to be a site on a repressor which is competed for by heme and an inducing chemical.

11. Hepatic porphyria is postulated to be caused by a mutation in an operator gene that is poorly responsive to the repressor. The delay in appearance of symptoms of this

* This investigation was supported in part by Grant GM-02922 from the Division of Research Grants and Fellowships of the National Institutes of Health, Unit,ed St,ates Public Health Serv- ice.

disease until puberty suggests that sex steroids are the inducing chemicals.

A number of chemicals of diverse structure are known that

cause a porphyria in various animals. This porphyria mimics the inheritable human disease, hepatic porphyria, especially in the excretion of porphyrins and their precursors (I, 2). The more immediate action of one of these chemicals, 3,5-dicarbethoxy- 1,4-dihydrocollidine, was shown by Granick and Urata (3) to be a marked increase in the Caminolevulinic acid synthetase activity of the mitochondria of the hepat.ic parenchyma cells of the guinea pig. This enzyme forms Caminolevulinic acid from succinyl coenzyme A and glycine as first shown by Shemin (4).

In this paper, we describe a method by which chemical induction of ALA synthetasel activity can be studied in chick embryo liver cells in primary culture. With this method, the properties of the inducing mechanism have been investigated. Some as- pects of this study have been reported in preliminary communi- cations (5-7).

Studies on a number of cell types, including aerobic bacteria (8), the differentiating erythrocyte (9, lo), and the liver cell (5), suggest that there is a basically similar mechanism for the control of the rate of porphyrin and heme biosynthesis. The control is on the activity of the first enzyme of this biosynthetic chain, ALA synthetase. All of the other enzymes of this chain are present in nonlimiting amounts.

Two general mechanisms for the control of the first enzyme of a biosynthetic chain based on bacterial studies have been proposed by Jacob, Monod, and Wollman and summarized by

1 The abbreviations used are : ALA, &aminolevulinic acid; aminoacetone-pyrrole, the pyrrole made by the condensation of aminoacetone with acetylacetone, i.e. 2,4-dimethyl-3.acetyl- pyrrole; ALA-pyrrole, the pyrrole made bv the condensation of E-aminolevulinic acid with acetylacetone, i.e. 2-methyl-3-acetyl- 4-propionic acid-pyrrole; AIA, allylisopropylacetamide; DDC, 3,5-dicarbethoxy-1,4-dihydrocollidine; heme, iron protoporphyrin; mRNA, messenger RNA.

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Induction of ALA Xynthetase Vol. 241, No. 6

Lwoff (11). One is end product inhibition, the other end product repression. As applied to the heme-biosynthetic chain for the case of Rho&pseudomonas spheroides, a photosynthetic bacterium, Lascelles (8) has found that both mechanisms are active. The end product, heme (10e6 M), inhibited the first enzyme, ALA synthetase, allosterically (12). Also, heme when added to these bacterial cultures was found to repress the formation of ALA synthetase 3- to 4-fold.

With regard to the red cell, London, Bruns, and Karibian (13) reported evidence for end product inhibition; the addition of heme to rabbit reticulocytes decreased the incorporation of tracer glycine into the heme of hemoglobin. In the differentiating erythroblast, the control of hemoglobin synthesis appears to reside in a repressor for which no end product (i.e. heme) repression has yet been shown. Levere and Granick (9) have found that the colorless proerythroblasts of the chick embryo blasto- derm contain all of the enzymes for heme biosynthesis in nonlimiting amounts except the first enzyme; these precursor cells also contain mRNA and ribosomes for globin synthesis, yet they do not synthesize globin. It is only when the repression is removed that adequate amounts of ALA synthetase can be synthesized. Then ALA is made and heme is formed as end product. Once heme is made globin synthesis begins and hemoglobin is formed.

Concerning the liver cells, we present evidence in this paper that heme biosynthesis is not controlled by the allosteric inhibition of ALA synthetase but rather by a repressor mechanism that controls the synthesis of ALA synthetase.

The repressor mechanism, as proposed by the French school (ll), is considered to consist of a repressor protein which acts to block an operator gene. The function of an operator gene is to control the transcription of the operon (a short length of DNA) into mRNA. When the operator gene is blocked, no mRNA will form that can be translated into a polypeptide chain of ALA synthetase.

Because certain chemicals induce the formation of ALA syn- the&e in liver cells, the possibility was considered that they might affect the repressor mechanism. The following hypothesis was formulated for experimental testing. Consider the repressor to consist of an aporepressor which is a protein and a corepressor which is heme. Then there should be competition between heme and the inducing chemicals for the active site on the aporepressor. I f the chemical displaced the heme, the repressor action would be blocked and as a consequence more ALA synthetase would be synthesized and more porphyrin would be formed. Also, according to this hypothesis, the porphyrin-inducing activity of a chemical inducer should be prevented by heme. In this paper, experiments are presented which support this hypothesis.

METHODS AND PREPARATIONS

Culture of Chick Embryo Liver Cells 012 Cover Slips--With sterile technique, two or three livers are removed from 16- to 17day old chick embryos into a small Petri dish and washed with three changes of Earle’s salt-glucose solution (14) devoid of magnesium and calcium ions. The solution is removed and 8 ml of an enzyme solution are added. (The enzyme solution contains 75 mg of crystalline trypsin (Worthington) and 20 to 40 mg of Pangestin (Difco) dissolved in the Earle’s salt-glucose solution devoid of magnesium and calcium ions. For the preparation of Pangestin solution, 1 g is finely powdered in a mortar and sus- pended in 100 ml of the salt-glucose solution minus magnesium

and calcium. After 2 to 3 hours at 4”, it is filtered through paper, then through a sterile porcelain filter, adjusted to pH 7, and stored frozen. The Pangestin may be replaced by DNase, or Varidase may be used in place of trypsin and pancreatin.) The livers in the enzyme solution are chopped into tiny fragments with a sterile razor blade and placed at 38” for 15 to 30 min. During this period, the suspension is gently sucked in and out of a large bore pipette to separate the cell masses. The suspension of cells and small aggregates of cells is then transferred to a 15-m] conical centrifuge tube, and the heavier particles are allowed to settle. After 5 min, the supernatant solution is withdrawn and used as inoculum, or centrifuged at low speed, and the sediment is resuspended. The suspension contains liver cells, red cells, and cell fragments.

An inoculum containing lo5 liver cells in 0.05 ml of the suspension is added to a vial (18 X 60 mm) containing 1 ml of growth medium and a round 16-mm diameter cover slip that has been precleaned with aqua regia. The vial is covered with a loose aluminum cap. The composition of the growth medium is: 100 ml of Eagle’s basal medium & phenol red, 10 ml of fetal bovine serum, 1 ml of glutamine (0.2 M) (all purchased from Microbio- logical Associates, Inc.), and 0.2 ml of Mycostatin (2000 units), penicillin G (6 mg), and streptomycin (10 mg). After 24 hours of growth in an atmosphere of 5 y$, CO2 and air at 37”, the hepatic cells form monolayer colonies. The medium is replaced by 1 ml of fresh medium; this removes other cell types and cell fragments. The porphyrin-inducing chemical is added at this time as a solution either in 0.01 to 0.1 ml of 0.9% NaCl solution or in 1 to 3 ~1 of absolute ethanol. Observation of growth and of fluorescence is usually made 20 hours later with a Zeiss Ultraphot microscope with phase and fluorescence optics.

Determination of Porphyrins on Cover Slips by Fluorescenee- The cover slip is placed cell surface down on a glass slide, avoid- ing pressure; the excess liquid is drained away and the cover slip ringed with hot parafhn-Vaseline petroleum jelly. Then the surface of the cover slip is washed with water and dried, and the cells are examined with phase and with fluorescence optics. The amounts of porphyrins are estimated by scoring from +4 to 0 the relative fluorescence intensity of the monolayer of cells observed at a magnification of 25 times, with the arc of the 200 W high pressure mercury lamp focused on the specimen.

Quantitative fluorescence determinations have been made on extracts of these cells to obtain the following conversion factors: +4, all colonies fluoresce intensely; +3, most colonies fluoresce intensely; +2, most colonies fluoresce partially; +l, some colonies fluoresce partially. The fluorescence values of +4, +3, +2, and +l are equivalent, respectively, to 25 to 50, 10 to 25, 5 to 10, and <5 X 10-I’ mole of coproporphyrin per mg of protein. The amount of protein per cover slip after 48 hours of growth is 0.1 to 0.2 mg.

For determination of the porphyrins and protein in the cells, the cover slip is removed from the vial, gently washed by drip- ping 0.9% NaCl solution from a pipette, drained, and then placed into another vial containing 1 ml of a solution of 1 N

HCIO1-absolute ethanol (1: 1, by volume). After 5 mm, the clear solution containing the acidic porphyrins is decanted and its fluorescence is measured in a fluorometer. Calibration of the fluorescence curve was made with crystalline coproporphyrin III because the porphyrins which were isolated from the induced cells consisted of over 80% coproporphyrin with di- and tricarboxylic acid porphyrins as the remainder. The lower


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limit of fluorescence detection with the apparatus used is 1 x 10-l’ mole of coproporphyrin. (With the fluorescence microscope, porphyrins in the cells are readily detected below this limit.) The cover slip from the perchloric-ethanol extraction is washed with absolute ethanol and drained, and its protein is determined by the method of Lowry et al. (15) with crystalline bovine serum albumin as standard.

Analysis of Cover Slip for Porphyrins, Leucine or Orotic Acid Radioactivity, and Protein-The medium in the vial is removed, the cover slip washed with three 5-ml changes of a Dulbecco medium (16) containing, per liter, 40 mg of leucine and 20 mg of erotic acid. The wash medium is removed and to the cover slip in the vial is added 1 ml of perchloric acid-ethanol fixative (1 M HCIOcethanol, 1:l by volume). After 5 to 6 min, this solution containing the porphyrins is removed and the porphyrin content determined with a fluorometer. The cover slip is washed with 5 ml of ethanol and then with 5 ml of ethanol-ether (3 : 1, by volume) and dried in air. The radioactivity of the dry cover slip is counted in a gas flow counter. For r*C, the monolayer of cells on the cover slip represents an “infinitely thin” layer. Finally, the protein on the cover slip is determined by the Lowry method.

Analysis of Petri Dishes for Porphyrin and Protein-For studies with lo- to 20-fold the number of cells used in the vials, the chick embryo liver cells are grown in a monolayer on the surface of 5-cm diameter Petri dishes precleaned with aqua regia. The volume of the medium is 4 ml. After induction, the porphyrins in the medium are determined as follows. The medium is removed to a separatory funnel, the cells attached to the bottom of the dish are rinsed with 2 ml of Earle’s salt-glucose solution, and the solution is added to the separatory funnel. To the funnel are now added 10 ml of ether, 0.2 ml of glacial acetic acid, and 0.3 g of NaCl, and the porphyrins are extracted into the ether. The porphyrins in the ether layer are extracted with 1 ml of 1 M HClO,; this is diluted to 2 ml with ethanol and the fluorescence is determined in this solution.

The porphyrins in the cells are determined by extraction with 2 ml of a solution of 1 M HCIO1-ethanol (l:l, by volume) for 5 min at 4” and the fluorescence determined quantitatively as before.

For determination of the protein in the cells by the Lowry method, after porphyrin extraction, the cells are rinsed with 5 ml of ethanol, then rinsed with 5 ml of ethanol-ether (3: 1, by volume), dried, and the protein determined.

Induction of Porphyria in VVhole Chick Embryo-An egg, incubated for 16 to 17 days, is inoculated sterilely onto the air sac with 0.5 ml of AIA (3 mg) through a pinhole made in the shell. The hole is covered with Scotch tape, and the egg is incubated in an upright position.

Determination of Mitochondrial ALA and Aminoacetone by Solvent Extraction of Their Pyrrole Derivatives-Because of the limited amount of mitochondria available from chick embryo liver, the determination of ALA generated by ALA synthetase activity in the mitochondria was not feasible with the chro- matographic resin methods previously described (17). There- fore, the following procedure was developed. After isolation and incubation of the liver mitochondria (“Experiments,” Section 3), the 2 ml of mitochondrial mixture are deproteinized with 1.0 ml of 0.3 M trichloracetic acid. After 10 min, the mixture is centrifuged to obtain a clear solution. Of this solution, 2.5 ml are removed to a glass-stoppered, conical 15-ml

tube and neutralized to pH 4.6 with 0.5 ml of 1.0 M sodium acetate; then 0.05 ml of acetylacetone is added, and the solution is heated in a boiling water bath for 10 min to convert ALA and aminoacetone to pyrrole derivatives.

After cooling, 1 ml of this solution is removed and to this is added 1 ml of Ehrlich mercury reagent (11). At 15 min after addition of the reagent, the optical density (W) at 552 rnM is determined in a cell of 1 cm optical length.

The 2 ml remaining are neutralized with 0.05 ml of Na2HP04 (0.5 M) and 0.15 ml of NaOH (1.0 M) to pH 7 to 7.5. Then the solution is shaken with 5 ml of equilibrated ether for 1 min. In the aqueous phase there remains 85% of the ALA-pyrrole and 5% of the aminoacetone pyrrole. Of the aqueous phase, 1 ml is removed and mixed with 1 ml of Ehrlich-mercury reagent and at 15 min after mixing the optical density (2) at 552 rnw is determined in a cell of 1 cm cpticsl length.

1Jnder the conditions used to determine the optical densities (W) and (Z), the em 552 rnp of the Ehrlich-color salt of ALA- pyrrole is 5.8 X lo* and of the aminoacetone pyrrole is 6.6 X lo*.

The equilibrated ether is prepared at room temperature by shaking 100 ml of peroxide-free ether with 100 ml of an aqueous solution containing (in millimoles) : trichloracetic acid, 7.7; sodium acetate, 15; NaOH, 0.68; and Na2HP04, 1.1.

The ALA and aminoacetone in the original 2 ml of mitochondrial mixture is given by the following equations.

Moles of ALA = 1.7 X 1OP [(Z) - 0.045 (IV)] Moles of aminoacetone = 1.5 X 1OW [0.77 (W) - (Z)]

Compounds Synthesized-The following compounds gave satis- factory analyses for carbon and hydrogen, and nitrogen by the Kjeldahl method. Dumas-determined nitrogen tended to be somewhat high. The melting points were made on a calibrated microscope hot stage.

Diethyl-1 ,4-dihydro-2,4,6-trimethylpyridine-3,5-dicarbosyl- ate was obtained from Eastman and recrystallized from ethanol-water. It was also synthesized (18, 19); m.p. 129-130”.

Diethyl-2,4,6-trimethylpyridine-3,5-dicarboxylate was prepared by the oxidation of the previous compound (18, 19). The liquid was distilled at 150”, 1 mm of pressure, and had a boiling point of 308-310”. Analysis was made on the picrate.

Dipotassium salt of 2,4,6-trimethylpyridine-3,5-dicarboxylic acid was prepared from the previous compound (18-20). It was dried over PzOr, under high vacuum at 100” for 3 hours to remove the solvent alcohol.

Diethyl-1 ,4-dihydro-2,6-dimethylpyridine -3,5-dicarboxylate was synthesized (21) and formed yellow crystals, m.p. 181.5- 182”. Diethyl-2,6-dimethylpyridine-3,5-dicarboxylate was obtained by oxidation of the previous compound (22); m.p. 72”.

Experiments

i. Induction of Porphyria in Chick Embryo Liver Cells in Culture in Vitro-The liver of the chick embryo consists primarily of parenchymal and endothelial cells. The liver cells and liver cell aggregates were prepared and grown on cover slips in vials containing 1 ml of medium as described under “Methods and Preparations.” During the first day, attachment to the cover slip occurred and the colonies began to spread out. According to Lovlie (23), mitotic activity in primary culture increases to a maximum in 24 hours and then declines. After a day of growth, the medium containing nonattached cells


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including erythrocytes and cell debris was removed and replaced by fresh medium. On the 2nd day, many of the colonies had coalesced to form a monolayer of parenchymal cells. In- between the colonies were relatively few flattened, fibroblast- like cells, possibly derived from endothelium.

The cells at the periphery of a healthy colony had filmy edges of protoplasm in which mitochondria could be seen readily (Fig. 1, D and F). Inhibition of growth, if it occurred, was recognized by retraction of the filmy edge, injury, by the rounding up of the parenchymal cells, and death by detachment of the cells from the cover slip.

When an inducing chemical, e.g. AIA, was added to the growth medium on the 2nd day, porphyrin formation was induced in the liver parenchymal cells (5) but not in the fibroblasts. The fluorescence was localized in the granules of the cytoplasm and ground cytoplasm, but was absent from the nuclei (Fig. 1, A and B). Only when excessive amounts of porphyrins were formed did the nucleus also become fluorescent. The cultures grown in the presence of the inducing chemical for 20 hours could not be distinguished from control cultures in appearance of colonies, cells, or rate of spread of the colonies on the cover slip (Fig. 1, D and E).

In the absence of chemical inducers, a trace of porphyrin fluorescence was observed occasionally in scattered cells during the 1st day of growth, which disappeared on the 2nd day.

In the absence of chemical inducers fluorescence could not be induced in the system in vitro by addition to the growth medium of the following compounds either individually or in various combinations: glycine (0.02 M), members of the citric acid cycle (1 mru), pyridoxal phosphate (0.05 mM), CoA (0.05 mM). These compounds did not appreciably enhance the amount of porphyrin induced by AIA. Fluorescence was not induced by proteolytic enzymes, DNase, antibiotics, or pancreatic lipase.

Chemical induction of fluorescence was not affected by the absence from the medium of glucose or glutamine or by the addition (per ml) of either glucagon (20 to 50 pg), insulin, (20 to 50 pg), or glycerol (1 to 2 mg) in the presence or absence of glucose. Ferrous iron (6 X lo+ M) did not decrease or enhance the fluorescence.

In the absence of serum in the medium, porphyrin fluorescence was inhibited in part because the proteolytic enzymes added for disaggregating cells were not completely inactivated and damaged the cells.

Multiplication of the chick embryo liver cells in vitro was not required for induction of porphyrin fluorescence, although protein synthesis was necessary. When beef serum or chicken serum was used in the culture medium in place of beef fetal serum, multiplication of the cells appeared to be inhibited but porphyrin fluorescence could be induced in these cells with AIA (30 pg per ml). Induction required protein synthesis, as shown by the inability of AIA (30 pg per ml) to induce fluorescence in the presence of actinomycin D (0.05 pg per ml) ; when the medium was changed on the 2nd day to include only AIA (i.e. without actinomycin D), an intense fluorescence was observed on the 3rd day. The inhibition by actinomycin D was therefore reversible.

There was some variability in the rate of development of porphyrin fluorescence. With AIA (30 pg per ml) as inducing chemical, the fluorescence was detected in the cells usually by 7 to 8 hours but occasionally as early as 4 and as late as 9 hours. The fluorescence first appeared in cytoplasmic granules in the

hepatic parenchymal cells. It then increased progressively in intensity in the cytoplasm, occasionally was seen in bile canaliculi, and by the 20th hour was observed in the nuclei.

The variability in the early appearance of fluorescence was probably related to the following factors: biological variation in the chick embryo livers; differences in the background level of ALA in the liver cells at the start; differences in the sample of beef fetal serum; differences in the length of time required to disaggregate the liver with enzymes (the longer the time the slower the rate of appearance of fluorescence).

For a determination of the minimal time for the detection of induction by the cover slip technique a series of concentrations of ALA were added to the medium together with AIA (30 kg per ml). In this particular experiment, the concentration of 0.5 pg per ml of ALA was found to be insufficient to cause detectable fluorescence by itself. However, at this concentration of ALA, the induced fluorescence due to the newly formed ALA could be detected in about 3 hours.

The property of induction was lost by liver cells grown for more than a week in culture. Likewise, there was a loss of other enzymes of the biosynthetic chain because these cells, when given ALA, formed relatively small amounts of porphyrin. Greater retention of the enzymes that convert ALA to porphyrins and possibly greater retention of ALA synthetase activity was observed when the liver tissue tended to grow in a spherical aggregate rather than as a monolayer. Fresh supernatant from the disaggregated liver cell suspension when added to the week- old cells in fresh medium did not cause the cells to recover the induction property possessed by fresh cells. The loss of special properties of organized tissues when cultured is well known and awaits understanding. Chemical induction leading to porphyrin formation may prove useful for a study of this phenomenon.

2. Induction of Porphyria in Chick Embryo as Specific Property of Liver Parenchymal Cells-Talman et al. (24, 25) found that when Sedormid or related chemicals were injected into the yolk sac of an 8-day-old chick embryo, a porphyria was produced in which the kidneys fluoresced intensely. The livers only rarely fluoresced.

We have found that induction of a chemical porphyria is a special property of the embryonic chick liver parenchymal cells. When liver, kidney, spleen, or brain tissue of a 16-day embryo was cultured in vitro, induction of porphyrin fluorescence by AIA, DDC, or griseofulvin was only observed in the liver tissue, even though kidney, like liver, could readily form porphyrin from added ALA. The property of the liver to respond to chemical induction had already appeared in the 3-mm diameter liver of a g-day-old chick embryo; these liver cells in primary culture formed porphyrin when induced with AIA.

Chick embryos which were given AIA via the air sac (see “Methods and Preparations”) were found to contain porphyrin in the kidney tubules which fluoresced intensely at a time when it was scarcely detectable in the liver, confirming the observa- tions of Talman et al. noted above. On the basis of the tissue culture experiments, it was concluded that the chemical induced the liver to form increased amounts of ALA synthetase; then the liver excreted ALA or other porphyrin precursors into the blood stream from which they were concentrated by the kidney tubules and converted to porphyrins. The induction could be detected as early as 2) hours after injection of a 0.5-ml NaCl solution of AIA (3 mg) onto the air sac of a 17-day-old embryo by the appearance of fluorescing porphyrins in the kidney tu-


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FIG. 1. Photomicrographs of chick embryo liver. A, porphyrin fluorescence is recorded as white in cells growing on a cover slip in the presence of the inducing chemical AIA (30 pg per ml) for 18 hours; magnification, 1000 diameters. A cell is outlined by the dashed line. The ffuorescence is localized in the ground cytoplasm and granules of the cytoplasm. The nucleus (N) is not fluorescent. R, same as A but at a magnification of 25 diameters. The dark dots seen best on the upper portion of the photograph represent nonfluorescent nuclei. C, a squash preparation of whole chick embryo liver to show the red fluorescence (recorded as white) in bile-canaliculi. The liver was from a 16-dav-old chick embrvo that had been treated with AIA via he air sic and incubated”for 20 hours. D, chick embryo hepatic

parenchymal cells cultured for 2 days on a cover slip and viewed with phase optics; magnification, 1000 diameters. The cells spread out as a monolayer. The spherical pale nuclei contain one or two dark nucleoli. Fatty droplets in the cytoplasm appear white. The tiny dark granules in the cytoplasm are mitochondria. E, similar to D except grown for 1 day in the presence of ATA (30 rg per ml); magnification, 1500 diameters. No changes from that of D were noted. F, chick embryo liver cells cultured for 2 days on a cover slip; magnification, 500 diameters. Cells at the edges of the colony of parenchymal cells form filmy proto- plasmic projections indicative of healthy growth. A fibroblast (F) lies between the two colonies.


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1364 Induction of ALA Synthetase Vol. 241, Ko. 6

bules; in the liver, porphyrins were localized to the bile canaliculi which fluoresced intensely after 4 to 24 hours (Fig. 1D).

S. ALA Synthetase Activity in Mitochondria of Chick Embryo Liver Increased When Induced with Allylisopropylacetamide- With guinea pigs fed DDC, it had been shown that a greater than 40-fold increase in ALA-synthetase activity occurred in the mitochondria isolated from the livers of these animals (3). To determine whether a similar increase in ALA synthetase act,ivity occurred in chick embryo liver, we induced a chemical porphyria in 17-day-old chick embryos by addition of 0.5 ml of -IL4 (3 mg) to the surface of the air sac and then incubation for 24 hours at 37” (see “Methods and Preparations”).

For the determination of ALA synthetase activity, the livers from five control and seven treated embryos were used. The mitochondria were isolated as previously described (17) and were incubated with succinate and glycine as substrates. In addition, mitochondria which had been frozen and thawed to increase their permeability were used with succinyl-Cob and glycine as substrates (3). The ALA formed was determined as ALA-pyrrole, as described in “Methods and Preparations.” As seen in Table I, the mitochondria from the livers of AIA- treated embryos formed over 8 times more ALA than the controls with either succinate or succinyl-CoA as substrate. Thus, an inducing chemical, AIA, brings about an increase in ALA synthetase activity in the mitochondria of livers of 17.day- old chick embryos.

4. Quantitative Determinations of Rate of Induced Formation of ALA Synthetase-In chick embryo liver, the rate-limiting enzyme for porphyrin formation is ALA synthetase (5). The rate of porphyrin formation is proportional to the amount of ALA synthetase and to the ALA formed. The rate of increase

TABLE I

Comparison of formation of b-aminolevulinic acid by liver mito-

chondria from normal 17-day-old chick embryos and from those treated in vivo for 5’3 hours with 3 mg of allylisopropylacetamide

The 2.ml mitochondrial suspension was incubated aerobically

at 38”. It contained either intact mitochondria or mitochondria

frozen and thawed four times (GO bl, packed volume) and (in micromoles) : phosphate (75) and Tris (75) buffer, pH 7.4; glycine, 150; MgC12 ,30; EDTA, 15; pyridoxal phosphate, 0.4; and sucrose, 375; with the additions noted.

. . . .

0 4 8 12 16 20

Hours

FIG. 2. Rate of porphyrin formation in chick embryo liver cells after induction with AIA, and after removal of AIA. CzLrve A, porphyrin in cells and medium; Curve B, porphyrin in cells. . . . . . , between A and l , increase in porphyrin after removal of AIA. About 5 X lo6 hepatic cells were inoculated into 4 ml of complete medium in a Petri dish, 5 cm diameter. After 20 hours of incubation at 38” in 5$& Cog-air, a monolayer of cells adhered firmly to the bottom. The medium was replaced by 4 ml of fresh medium, and AIA (0.3 mg) was added as the inducing chemical. Analysis for porphyrins was made separately on the medium and on the cell monolayer as described in “Methods and Prepara- tions.” In the study of porphyrin formation after removal of AIA, the medium containing the inducer was removed in the in- cubator room at 38”, replaced with 4 ml of fresh medium three times at 5-min intervals, and incubated until the 22nd hour.

of induced porphyrin formation is therefore a measure of the rate of synthesis of induced ALA synthetase.

To follow the induction of ALA synthetase quantitatively, especially at the early stages, we increased the number of cells per sample lo- to 20-fold (equivalent to 1 to 2 mg of protein) as compared to the cover slip technique. The chick embryo liver cells were grown in 5-cm diameter Petri dishes. After attachment of the cells to the bottom of the dish, AIA was added as inducing chemical. Porphyrins were found to increase at an almost logarithmic rate for the first 12 hours, and at 22 hours the concentration of porphyrins was loo-fold that of the control (Fig. 2, Curve A). The amount of porphyrin in the cells is given

by Curve B of Fig. 2. The amount of porphyrin that escaped from the cells into the medium during the first 12 hours was one-third or less of that in the cells, but at 22 hours was more than half of the total.

5. Reversible Inducing Action of Chemical Inducers-In guinea pigs, after the feeding of an inducing chemical for 2 days, there was an increased porphyrin excretion for several days which then decreased over the next several days to normal (3). Therefore, the action of the inducing chemical was reversible.

:nCU- ation time

min

120

120 120 120

120 15 15 15

15 15 15

-

.d Intact

mitochondria Frozen-thawed mitochondria Addition

ALA ALA found formed,

average AIA- .reated

AIA-

1 - S 1 Control ucci- Succinyl- late CoA

mgmJles

0 500

0

500 500

mpmoles

3.4; 5.0

6.2; 6.4 9.0 27; 26 22;a 2oa

3.0 3.2; 3.6

4.9; 6.0 14.0 16; 17.0 11”

+ + 2.1

17.0 11.08

0.4

8.6 11.0 5.6*

+ + +

+ + 350

350 700 700

n In NQ atmosphere. * With arsenite (2 pmoles).


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Similarly, the removal of AIA from cultures of chick embryo liver resulted in a decrease in rate of porphyrin formation. The ilIA was removed at the designated times noted by A in Fig. 2, Curve A, and the incubation was continued till the 22nd hour. The amount of porphyrin formed during this latter period was proportional to the length of time the cells had been preincu- bated with AIA. By the 22nd hour, that amount of porphyrin formed after removal of AIA was approximately equivalent to the porphyrin that would have been formed in 2 to 3 additional hours of induction, i.e. if the AIA had not been removed.

6. Half-life of AL.4 Synthetase Is 4 to 6 Hours-Acetoxycyclo-

heximide is considered to block protein synthesis at the ribosome level (26). We have determined that this compound does not inhibit the induced ALA synthetase of guinea pig liver mitochondria nor does it inhibit the conversion of added ALA to porphyrins by chick embryo liver cells.

When chick embryo liver cells were incubated with AIA (Fig. 3, Curve A) and at the 12th hour acetoxycycloheximide was added in excess (0.03 and 0.06 pg per ml), the rate of porphyrin formation diminished to half in 4 to 6 hours as determined from tan- gents to Curve B. The half-life of ALA synthetase may then be considered to be about 4 to 6 hours.

7. Lack of Effect of Induction of 9Ld Synthetase on Incorpora- tion of Tracers, Leucine and Orotic Acid, into Chick Embryo Liver Cells-In the guinea pig, it has been found that after induction of a chemical porphyria the ALA-synthetase activity was increased in the liver mitochondria (3). However, the activity of a similar enzyme, aminoacetone synthetase, was found to be relatively unchanged. This result suggested that the chemical might induce an increase in a specific enzyme rather than stimu- late a generalized protein synthesis.

To test this idea, chick embryo liver cells growing on cover slips were incubated with labeled leucine or labeled erotic acid, in the presence or absence of AIA as inducing chemical. Over a Z-hour period of incubation, no major differences were observed in the rate of labeling between the control cells and induced cells (Fig. 4, B and B). For leucine, the differences were within +lO%. During the first 10 hours, the ratio of leucine to erotic acid labeling was almost constant and then decreased (Fig. 4C).

The labeled cells on the cover slips were mainly of two kinds. During incubation, fibroblasts migrated out and grew between the colonies of hepatic parenchymal cells. For determination of the extent of labeling in these two kinds of cells, a radioauto- graph technique was used. On the basis of the area occupied by each cell type and the silver grain counts per unit area over each cell type, it was estimated that the parenchymal cells contained over 80% of the radioactivity. Therefore, the labeling repre- sented in Fig. 4, A and B, is due primarily to the incorporation of leucine and erotic acid into the hepatic parenchymal cells.

8. Chemicals that Induce Porphyria-In general, compounds that have been reported to induce a chemical porphyria in animals (1) were found to induce porphyrin fluorescence in a culture in vitro of chick embryo liver cells. The compounds which were tested and found active are listed in Table II. Related compounds which were inactive are listed in footnotes to this table.

The active chemicals consist of a wide variety of organic compounds of differing physiological activities. They include the central nervous depressants such as the barbiturates, sulfon- methanes, glutethimides; the anticonvulsant methsuximide; the central nervous stimulant bemegride; the fungicides griseofulvin and hexachlorobenzene; and the male and female sex steroids.

I I I I I

0 4 8 12 16 20 24

Hours

FIG. 3. Half-life of ALA synthetase. Chick embryo liver cells were incubated in small Petri dishes with 4 ml of complete growth medium (see legend to Fig. 2). The medium was changed after 24 hours and AIA (0.3 mg) was then added (zero time) which generated Curve A. After 12 hours of incubation, acetoxycycloheximide was added to inhibit protein synthesis which generated Curve B. The inhibition was maximum at a concentrat,ion of acetoxycycloheximide (0.03 rg per ml) as indicated by the same inhibition at O.OB rg per ml.

For the most part, these compounds are relatively insoluble. Therefore, steric and Van der Waal’s forces must be considered to be involved in the sites of their action.

A comparison of the known inducing compounds indicates that they may be divided into four classes: the barbiturate, collidine, sex steroid, and miscellaneous. In the barbiturate class, there are three chemical groups, A, B, and C (Fig. 5), any one of which may induce by itself. In the collidine class there are possibly two groups, D and E, (Fig. 5) capable of induction. The sex steroid class includes progest.erone, testosterone, and estradiol; none of the corticosteroids were active in induction. The miscellaneous class includes the compounds hexachlorobenzene, menthol, and chloretoro.

The most active inducing compounds were found in the barbiturate and collidine classes. The inducing activity was usually proportional to the concentration and increased progressively to +3 or +4 fluorescence (see “Methods and Preparations”). In contrast, in the sex steroid class, even at the highest concentrations tested, the fluorescence induced was never greater than +1

to + 1.5. The following compounds, which were among the most active, induced a $1 fluorescence in 22 hours at the concentrations noted: AIA (2 X 1O-5 M) which contained Group A; glutethimide (5 x lop6 M) which contained Groups A and B; DDC (1 X 1O-6 M) which contained Groups D and E; and progesterone (1 X 10e5 M). A fl fluorescence was equivalent to that produced by ALA (2 x lop5 M). The great insolubility of some compounds, e.g. griseofulvin and hexachlorobenzene, prevented an accurate comparison of their activities.

To what extent the chemical groups may be modified and still retain inducing activity cannot be predicted. For example, a high specificity was suggested, in the case of DDC, by the fact


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1366 Induction of ALA Synthetase Vol. 241, No. 6

80-

70 -

60 - c .- ar

‘0 & 50-

s \ H 40- 0. u

0 Control o with AIA

A

” “1 ’ ” “1 ” 4 8 12 16 20 24 28

Hours

c .- 0)

‘a L a

; 2.0-

0. u

2 * 0 0 .-

‘,

6 I.O-

0.8 -

0.6 -

0.4 -

0.2 -

0 Control

B

1 ” ” ” 1’ 1’ 1” 4 8 12 I6 20 24 28

Hours

4 8 12 I6 20 24 28

FIG. 4. Leucine or erotic acid incorporation into chick embryo liver cells on cover slips in the presence or absence of the inducing chemical AIA. A, uniformly labeled L-leucine-W (48.5 X 104 dpm) was added to 1 ml of medium containing 5 mg of leucine without or with AIA (30 fig). B, erotic acidW4C (25 X lo4 dpm)

that the removal of a methyl group from the 4 position caused a marked loss of activity. Marks et al. (28) have suggested that a methyl or other aliphatic group in this position is necessary to dis- tort the planar collidine ring out of the plane. In griseofulvin, the distance between and including the 2 oxygen atoms in Group D is about 7.5 A. It was possible to twist a Courtald model of DDC so that the 2 oxygen atoms of Group D were as close as they are in griseofulvin. In the barbiturate class, substitution in Groups I3 and C of a C=O for a C-OH or of an S=O for a C=O did not appreciably impair inducing activity.

9. Compounds That Inhibit Induction-For further study of the inducing mechanism, with the idea that one might find a compound that would block chemical porphyria, various compounds were tested. The procedure used was the cover slip technique of chick embryo liver cells (see “Methods and Preparations”). Inhibition of induced fluorescence was determined by addition of

Hours

was added to 1 ml of medium containing 5pg of erotic acid without or with AIA (30 pg). C, ratio of tracer leucine to tracer erotic acid incorporation. The methods of analysis are described in “Methods and Preparations.” Analyses were in triplicate. Average deviation from mean is indicated on the graphs.

the compound to the growth medium containing the inducing chemical, usually AIA (30 pg per ml). After the cells had been incubated in this medium for 20 hours, the degree of fluorescence was noted as compared to the control incubated with AIA alone. Compounds which inhibited induced fluorescence could be classi- fied into those that killed the cells, or prevented protein synthesis or activated the aporepressor (i.e. heme), or affected the cells in some unknown manner.

Cells which were injured usually rounded up and fell off the cover slip. They were not able to convert added ALA to porphyrins. Some antihist;amine drugs have been considered to prolong the action of barbiturates in animals. The following killed the cells at 100 pg per ml: chlorpheniramine maleate, tripelennamine citrate, and promethazine hydrochloride.2 The latter drug at 20

2 For the formulas of these compounds see the Merck Zndes c-w.


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TABLE 11

1367

Chemicals that induce porphyrin formation in primary cultures of chick embryo liver cells

Tests were made with the cover slip technique (see “Methods and Preparations”). The cells were grown for 1 day, the 1 ml of medium changed, and chemicals added; 22 hours later the fluorescence was observed.

Allylisopropylacetamide

Pyrazinamidea

AminopyrineQ BemegrideB

Theophyllinea

Caffeine”

Orinasee

2,2-Diethyl-1,3-propanediol

Meprobamate (Miltown)Q

Sulfonal” Ethyl aminomalonate Ethyl oximinomalonate

Sedormida Glutethimide (Doriden)=

Mesantoina

Celontina Milontin” Dilantin sodium”

Trimethadione”

Methyprylon (Noludar)”

Chloramphenicol

Diallylbarbituric acida

Phenobarbital sodiuma p-Methylglutarate diethyl ester

Griseofulvina

2,4,6-Trimethylpyridine Diethyl-1,4-dihydro-2,4,6-trimethylpyri-

dine-3,5-dicarboxylate (DDC)

Diethyl-2,4,6-trimethylpyridine-3,5-di-

carboxylate

ProgesteroneW

Amount added Intensity of fluorescence

Irg

300 30

3

500 125

45

50 25

500 250

50

500 100 500 125

125 50

200 20

45 200 200

4 3 1

1 0.5 1

2 1 1.5

1.0 Trace

1 1 3

0.5 1 0.5

3.5 1

3.5 1 1

20 4

8 3.5

1 1 20 3 50 4

50 2.5

40 0.5

20 Trace 200 0.5

60 Trace

loo 3 40 1 20 Trace

100 3 25 1 80 1.5

100 2 200 3.5 100 1.5

50 0.5 10 3

1 Trace

100 Trace 10 4

2 2

0.2 1 0.02 Trace

10 3

1 1 0.5 Trace 3 1

1 0.5 20 1.5 10 1

3 0.5

II nducing structures in the compound (Fig. 5)

A

B

B

C

C

C C C

A, B 4 B

A, B A, B

A, B A, B

A, B A, B

A, C

A, C

A, B, C A, B, C D

D

E

D, E

D, E

Sex steroids

Sex steroids

-

Footnote

b

c

d

c

f

f

h


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1368 Induction oj ALA Synthetase Vol. 241, No. 6

TABLE II-Continued

Testosterone”

Ethinyl estradiola Estradiola

NorethandroleneO Estrone”

Ethchlorvynola

Hexachlorobenzenea (alcohol-saturated)

MenthoP

Chloretonea

Amount added

Pg

20 10

3

3 20 10

20 40

50 20

<16 <4

40

20 350

- a See text, Footnote 2. * The following compounds containing structures related to

Structure B did not induce: allantoin, N-methylphthalimide (20

rg), Thalidomide. Thalidomide inhibited porphyrin induction

by AIA.

methylglutarate, o-methyl succinate, p-methyl succinate diethyl

ester, glutarate dimethyl ester, reserpine (5 to 100 pg), bishy- droxycoumarin (20 to 200 pg).

c One S=O replaces one C=O. d The following compounds containing structures related to

Structure C did not induce : diethyl ester of phenyl malonate, re- sorcinol, 2-methylresorcinol, acetyl salicylate, salicylate, phen- yibutazone.

i The following compounds related to Structure D, E did not

induce: 3,5-dicarbethoxylutidine, 3,5-dicarbethoxy-1,4-dihydrolutidine, potassium-2,4,6-trimethylpyridine-3,5-dicarboxylate.

i The following compounds relat,ed to the structures of t,he sex

steroids did not induce: cortisol, corticosterone, ll-dihydrocorti- costerone, deoxycorticosterone acetate, prednisone, diethylstil- besterol.

8 Two S=O groups replace two C=O. k The following compounds related to the structure of hexa- f Also effective in producing porphyrin fluorescence indirectly chlorobenzene did not induce: pentachlorophenol, p- or o-di-

in kidneys of whole egg embryos. chlorobenzene (1 to 50 rg) even when solubilized with sodium 0 Patchy distribution of fluorescence. deoxycholate. Rimington and Ziegler (27), however, reported h The following compounds containing structures related to experimental porphyria in rats with the lower chlorinated hy-

Structure D did not induce: p-methylglutarate, @-hydroxy-a- drocarbons.

Intensity of fluorescence I

1 1

0.5 0.5 1

0.5 0.5

0.5 0.5 0 2

1 2

1 1.5

nducing structures in the compound (Fig. 5)

Sex steroids

Sex steroids Sex steroids

Sex steroids

Sex steroids Sex steroids

Miscellaneous

Miscellaneous

Miscellaneous

Footnote

k

Fro. 5. A, B, and C, the three groups in the barbiturate class, any one of which may induce a chemical porphyria. D and E, the two groups in the collidine class, either of which may induce a chemical porphyria.

pg per ml decreased the fluorescence by one-fourth. 2,4-Di-

chloro-6-phenylphenoxyethylamine-HCl also has been reported

to prolong the hypnotic effect of barbiturates in animals by in-

hibiting their metabolic transformations (30). We have found

that this drug at 5 pg per ml decreased the induced porphyrin

fluorescence by half. Even at 1 pg per ml, this drug was in-

hibitory to cell growth. These results suggest that the com-

pounds that have been found to prolong barbiturate action

damage the liver cells and so prevent them from developing

mechanisms to detoxify the barbiturates. On the other hand,

chlorcyclizine was found by Conney, Michaelson, and Burns (31)

to be a potent stimulator of several drug-metabolizing enzymes

in the liver microsomes after only 24 hours of administration to

rats. With the cover slip technique, chlorcyclizine did not in-

duce, cells fell off the cover slip in the presence of 10 pg per ml

of this drug, and at 2 pg per ml it depressed fluorescence induc-

tion by AIA to one-third.

Compounds which inhibited induction probably by inhibition


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.of protein synthesis of ALA synthetase are listed below. The concentration in micrograms per ml is given which decreased by half the induction of porphyrin fluorescence: mitomycin C, 1; actinomycin D, 0.05 and 0.01; acetoxycycloheximide, 0.005 and 0.001; puromycin, 1; 5-fluorouridine deoxyribose, 5; %azaguanine, 8; 6-azauridine, 50; L-ethionine, 30; tyramine, 50. (Canavanine sulfate (50 to 500 pg per ml) did not inhibit induction.) Col- chicine or colcemide, which are considered to interfere with spin- dle formation and also inhibit uric acid-riboside phosphorylase, damaged the cells and inhibited induction when given in a concentration of 10 pg per ml. Because porphyrin formation was blocked by mit,omycin and actinomycin, it was concluded that the induction of ALA synthetase requires intact DNA.

2,4-Dinitrophenol (15 pg per ml) inhibited induced fluorescence by half. It was likewise shown to be inhibitory to protoporphyrin formation in chicken erythrocytes (32). Pentachloro- phenol (50 pg per ml) inhibited induced fluorescence by one-fourth. These phenols inhibit ATP synthesis which is required for protein synthesis.

Only a few compounds were found to inhibit the induction of porphyrin fluorescence without an apparent inhibitory effect on growth. Ascorbic acid (25 pg per ml) and TPNH (25 pg per ml) decreased the induction by AIA (30 pg per ml) by half, yet did not affect the rate of porphyrin formation from ALA. The induction by DDC or griseofulvin was also decreased by ascorbic acid. It required 5 times more isoascorbate than ascorbate to decrease the induction to the same degree. The reducing properties of ascorbate on TPNH did not prevent the autoxidation of porphyrinogens to porphyrins because there was no increased fluorescence of the cells after addition of IZ or phenanthrene- quinone sulfonate. It is of interest that both ascorbate and TPNH have been implicated in oxidase-catalyzed reactions with O2 that hydroxylate-inducing chemicals like the barbiturates (33-35). Perhaps the decrease in induction in the presence of these compounds is caused by a more rapid oxidation, and therefore inactivation, of the inducing chemicals.

IO. Factors That Do Not Prevent Chemical Porphyria in Chick Embryo Liver Cells in Vitro-In order to develop a chemical porphyria in the guinea pig, we had observed that it was necessary to starve the animals fc: a day. Even coprophagy caused a diminution in the degree to which the porphyria developed. De Matteis (36) and Tschudy et al. (37) observed that carbo- hydrates inhibited the induction of chemical porphyria. For example, De Matteis found that when 50 g of glucose per kg of rat per day were fed, no porphyrins or precursors were excreted in the urine.

We attempted to inhibit induction in chick embryo liver cells by affecting glucose metabolism. In these attempts, the glucose concentration of the medium was varied from 0 to 5000 pg per ml, additions (in micrograms per ml) were made of glucagon (20 to 50), of insulin (20 to 50), of glycerol (2000), and of sodium gluconate (1000). No inhibition of induced fluorescence by AIA (30 pg per ml) was observed. Ludwig, Scott, and Chaykin (38) had reported that when rats were given 0.3 to 2 mg per kg per day of malonate the urinary uroporphyrin and fecal protoporphyrin were increased lo-fold over the controls. We found no induction of porphyrins in chick embryo liver cells when they were incubated with the following compounds (in micrograms per ml): malonate, 30 to 500; malonate half ethyl ester, 30 to 500; malonate diethylester, 30 to 60.

The possibility was considered that inducing chemicals might

act indirectly to affect a dissociation of a DNA-histone complex and thus uncover genes. The following salts and bases (in micrograms per ml) did not induce or inhibit induction by AIA: KCI, 400 to 1200; histone, 200; putrescine, 50; protsmine, 200; or choline chloride, 2500. At 9000 pg per ml, NaCl did not induce; it decreased induced fluorescence by one-third and also inhibited growth. Oubain (2 to 5 pg per ml) did not induce; it inhibited growth.

Because there was a structural resemblance between the inducing compounds and pyrimidines, both pyrimidines and purines in the form of their nucleosides at concentrations of 90 to 300 pg per ml were tested for inducing ability. None were active. Some growth inhibition and decreased response to AIA (30 pg per ml) were observed with deoxyadenosine, guanosine, and adenosine; there was no effect with cytidine, deoxycytidine, or thymidine.

The addition of ferrous iron (20 to 50 pg per ml) as ferrous am- monium sulfate did not itself induce nor decrease induction of fluorescence by AIA. Therefore, its effect to remove porphyrin as heme or to catalyze ascorbate destruction was not a limiting condition.

One of the possible sources of succinyl-CoA is its formation from propionyl-CoA via a biotin enzyme. If this is a rate-limiting reaction in porphyrin biosynthesis then induced porphyrin formation should be inhibited in chick embryo liver cells by avidin (500 pg per ml) or by desthiobiotin (500 pg per ml). No inhibition was observed.

The inhibition of ALA dehydrase in chicken erythrocytes by 3- amino-l ,2,4-triazole and by &chlorolevulinic acid was reported by Tschudy and Collins (39). These compounds at 100 pg per ml did not inhibit the rate of porphyrin formation in chick embryo liver cells in vitro when induced with AIA (30 pg per ml), perhaps because even the partially inhibited enzyme was not limiting the rate of induced porphyrin synthesis.

11. An Inducing Chemical Does Not Protect ALA Synthetase from Destruction in Cell-The experiments in Sections 11 to 13 were made to seek an explanation for the increase in ALA synthetase activity brought about by inducing chemicals. Three hypotheses were tested: inhibition of enzyme breakdown; enhancement of enzyme activity; and enhancement of synthesis of the enzyme.

The hypothesis of enzyme breakdown assumes that ALA synthetase is being synthesized and destroyed at a given rate so that a steady state level of ALA synthetase is maintained. If the inducing chemical acts by merely decreasing the rate of destruction of the enzyme the steady state level of the enzyme will increase. Evidence against this hypothesis is as follows.

(a) The rate of incorporation of tracer leucine into hepatic parenchymal cells was not affected by the presence or absence of the inducing chemical, ilIA (Fig. 4A). Therefore, no major change in protein synthesis or decrease in rate of protein destruction is caused by AIA.

(b) The removal of AIA from induced cells (Fig. 2) decreased the rate of porphyrin formation but this decrease was not greater than the decrease in rate of porphyrin formation when acetoxycycloheximide was added to cells incubated with AIA (Fig. 3). I f the action of AIA had been merely to stabilize the enzyme or to inhibit a proteolytic enzyme specific for ALA synthetase breakdown, then the ALA synthetase should have been destroyed more rapidly when the AIA had been removed than when, in the pres-


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ence of AIA, protein synthesis had been blocked with acetoxycycloheximide.

(c) Mitochondrial preparations with ALA synthetase activity were obtained from chick embryo liver induced with AIA (“Ex- periments,” Section 3). I f AIA merely had stabilized an active configuration of newly formed ALA synthetase, the washed mitochondria would have had negligible activity.

12. An Inducing Chemical Does Not Directly Enhance Ac- tivity of ALA Synthetase Nor Does Heme Inhibit Enzyme- Another hypothesis to explain an increased ALA synthetase activity is that the inducing chemical activates the enzyme, e.g. by a configurational change, or prevents an allosteric inhibitor such as heme from attaching to the enzyme.

No evidence for a direct activation of ALA synthetase by an inducing chemical was obtained. (a) Induction of normal liver mitochondria with DDC did not cause any increase in ALA formation greater than the control (3). (b) Inhibitors of protein synthesis such as actinomycin D, mitomycin C, puromycin (“Experiments,” Section 9) or acetoxycycloheximide (“Experi- ments,” Section 6) prevented an increase in ALA synthetase activity in chick embryo liver cells induced with AIA. These inhibitors of protein synthesis should have had no effect if AIA merely activated an inactive ALA synthetase.

No evidence was obtained for the inhibition of ALA synthetase activity by iron protoporphyrin, iron deuteroporphyrin, manganese protoporphyrin, nickel protoporphyrin, or bilirubin (Table

TABLE III

Effect of metalloporphyrins and inhibitors on ALA synthetase activity

Male guinea pigs, 500 g each, were kept in metabolism cages. Food was withheld for 24 hours; then DDC (40 mg) was given by stomach tube as a fine suspension in 4 ml of 0.25 M sucrose. After 1 day, the mitochondria were isolated (17). The 2-ml incubation mixture was incubated at 38” in 5% COZ-air for 24 hours. It contained DDC mitochondria (0.078 ml, packed volume) and (in micromoles): sucrose, 500; Tris buffer, pH 7.4, 100; glycine, 200; citrate, 100; MgClz, 40; EDTA, 20; pyridoxal-P, 1; and ATP, 1, with the additions noted. Two separate experiments are tabulated. For the determinations of ALA and aminoacetone, see “Methods and Preparations.”

Additions

Reagent control.. Zero hour control. Control after 24 hours..

Iron protoporphyrin. Iron protoporphyrin.. Iron deuteroporphyrin Manganese protoporphyrin. Nickel protoporphyrin. Nickel protoporphyrin. Protoporphyrin Bilirubin.

Control after 24 hours.. Iron protoporphyrin. Iron protoporphyrin.. Actinomycin D.. Puromycin................... Acetoxycycloheximide.

- Amount

/a

30 15

30 30 30 15

30 60

30 15

30 30 10

ALA Aminoacetone

m/Imoles

3 1 16, 17 3, 2 81, 82 20, 26

79 28

80 25 83 23 78 23

76 22 85 20 78 24 84 20

95 16 95 17 99 15 93 16 95 18

108 13

III). These compounds were added to liver mitochondria obtained from guinea pigs that had been given DDC; they did not decrease ALA production by the mitochondria.

From these experiments, it is concluded that inducing chemicals do not act to activate ALA synthetase of liver mitochondria and that there is no end product feedback inhibition by heme of ALA synthetase.

IS. An Inducing Chemical Causes an Increase in Synthesis of ALA Synthetase-The third hypothesis to explain induction is that the inducing chemical enhances the synthesis of ALA synthetase, perhaps by interfering with a repressor mechanism which inhibits the formation of the enzyme.

Evidence that a repressor mechanism is involved in the control of heme synthesis in the liver was suggested by the following findings. In isolated normal liver mitochondria, the activity of ALA synthetase is very low as measured by the rate of ALA production (3). When ALA was added to normal liver mitochondria it did not disappear, therefore the low rate of ALA production was not a result of a concomitant destruction of ALA or utiliza- tion to form porphobilinogen. (This is different from the action of mitochondria on aminoacetone which they oxidatively destroy (17).) The rate of ALA production in liver, even though low, must be sufficient to provide the heme required for the synthesis of the heme enzymes of liver cells such as catalase and the cyto- chromes. When an inducing chemical was given, the ALA synthetase activity increased over 40-fold in the guinea pig liver mitochondria (3) and over &fold in the chick embryo liver mitochondria (“Experiments,” Section 3). The guinea pig liver was now capable of making ALA at a rate equal to that of normal bone marrow. This increased ALA synthetase activity was abol- ished by agents that prevented protein synthesis (“Experiments,” Section 9). Therefore, the inducing chemical caused an increase in the synthesis of ALA synthetase rather than an activation of an inactive enzyme.

On the basis of these findings, it is surmised that the synthesis of ALA synthetase is repressed in normal liver and that the inducing chemical interferes with the repressor mechanism.

14. Increase in Synthesis of ALA Synthetase Caused by Induc- ing Chemicals Can Be Overcome by Certain Metalloporphyrins- One economical control method of heme biosynthesis would be a feedback control in which the repressor mechanism responded to heme. The simplest way to achieve this is to have a repressor that consists of a protein aporepressor and a heme corepressor. On the basis of this hypothesis, an inducing chemical might interfere with the repressor mechanism by displacing heme from the aporepressor; then more ALA synthetase would be made and more porphyrin would be formed. On the other hand, porphyrin formation should be found to decrease if heme displaced the inducing chemical from the aporepressor.

This hypothesis was tested by adding increasing concentrations of heme to chick embryo liver cells in the vials containing AIA (30 pg per ml) as inducing chemical; no inhibition of porphyrin formation was observed (5). However, with AIA at 15 pg per ml, inhibition was found. In the presence of iron protoporphyrin (1.5 kg per ml) or iron deuteroporphyrin (3 pg per ml) or manganese protoporphyrin (0.5 pg per ml), at the end of 18 hours of incubation the amount of porphyrin induced was 50% or less that of the controls (Table IV). Considering the low solubility of iron protoporphyrin as compared to the other two compounds, it is probable that iron protoporphyrin was the most effective inhibitor.


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Other interpretations of the conclusion that iron protoporphyrin (i.e. heme) caused a decrease in the formation of porphyrins in the cells were considered. The decrease in porphyrins formed was not caused by a general inhibition of cell metabolism because there was no concomitant inhibition of leucine or erotic acid incorporation into the cells (Table IV). Nor did the metalloporphyrins tested inhibit ALA synthetase (Table III). Nor did the metalloporphyrins inhibit the conversion by the cells of added ALA into porphyrins. In the above experiment (Table IV), only the porphyrins present in the cells were assayed. The possibility was considered that heme might decrease porphyrins in the cells by causing the porphyrins to leak out into the medium. This was tested by determining the effect of heme on the total porphyrins present both in the cells and the incubation medium. With the use of the small Petri dish incubation procedure (see “Methods and Preparations”), the cells were incubated with AIA (15 I.cg per ml) as inducer together with iron protoporphyrin. After 20 hours of incubation, the total porphyrins formed in the presence of 5, 2.5, and 0.5 pg of iron protoporphyrin per ml of medium were, respectively: 5,29, and 78 51. of the control. Thus, the decrease in porphyrins was a result of the inhibition by heme of porphyrin formation and not of leakage. Another possibility to explain the decrease in porphyrin by heme was that in the assay of porphyrins by fluorescence the presence of heme would quench the fluorescence. However, under the conditions of the assay, quenching by heme was found to be negligible.

From these experiments, it is concluded that the synthesis of ALA synthetase brought on by an inducing chemical can be inhibited by iron protoporphyrin. This may occur by displacing the chemical inducer from the aporepressor. Because iron deuteroporphyrin or manganese protoporphyrin also appear to act as corepressors to form functional repressor molecules the site on the aporepressor is not absolutely specific for the corepressor.

15. Additivity Effect of Inducing Chemicals on Porphyrin

8’ormattin-If it is assumed that the various inducing chemicals compete with heme for a corepressor site on the aporepressor, then there should be one kind of site, which should resemble that which would hold heme on the aporepressor. Tests of whether there is only one kind of site involved in the induction of porphyrin formation were made by adding AIA and DDC either alone or together to chick embryo liver cells :n culture. AIA is one of the inducing compounds from the barbiturate class and DDC is one of the inducing compounds from the collidine class (“Experi- ments,” Section 9). At low concentrations of each (6 pg of AIA and 0.6 I.rg of DDC per ml), their simultaneous addition resulted in a greater fluorescence, i.e. their effect was additive. When both compounds were given at concentrations that would induce a maximum fluorescence by each alone (60 pg of AIA and 6 pg of DDC per ml), the rate of increase in fluorescence was no faster than when given singly. This result suggests that only one kind of active site is involved for both classes of compounds.

If the inducing activity is related to binding affinity at an active site then a compound with greater inducing activity should not readily be displaced by one of lower inducing activity. This was tested by giving DDC (2 I.cg per ml) alone or simultaneously with 3,5-dicarbethoxy-l , 4-dihydrolutidine (30 pg per ml). The latter compound differs from DDC in lacking a methyl group at the 4 position; it has only a trace of inducing activity by itself. In the presence of 15 times the concentration of this compound, there was no decrease in the inducing activity caused by DDC, a result which suggests that DDC was rather firmly bound to the

TABLE IV

Inhibitory effect of metalloporphyrins on porphyrin synthesis induced by allylisopropylacetamide, but not on leucine or erotic

acid incorporation

The cover slip technique was used (see “Methods and Prepara- tions”). After the cells had grown on the cover slip for 20 hours, the medium was replaced with 1 ml of fresh medium. To this were added AIA (15 rg), metalloporphyrins dissolved in 10m3 M KOH, and leucine (5 X lo6 dpm) or erotic acid (2.5 X

lo5 dpm). The final pH was 7.2 to 7.4. After 18 hours of incubation, the porphyrins were extracted, the radioactivity on the cover slip was determined in a gas flow counter, and the protein

on the cover slip was then determined as described in “Methods and Preparations.” Analyses were run in triplicate and the average deviation from the mean is given.

Compound

Control minus AIA

Control with AIA

Manganese protoporphyrin

Iron protopor-

phyrin

Iron deuteroporphyrin

-

C t

1

:oncen .ratior

u&-/ml

5.0 1.5

0.5

5.0

1.5 0.5

10.0 3.0 1.0

Coproporphyrin

x 10” moles/m.g protein

1.5 f 0.1

14.0 f 2.0

1.5 f 0.1 2.8 f 1.6

6.1 f 1.6

1.4 f 0.1

6.1 f 2.3 10.1 f 4.6

2.4 f 1.0 3.9 f 1.6

17.0 f 6.4

-

-

Radioactivity

Leucine Orotic acid

cpm/,ug protein

106 f 2

106 f 2

108 f 2

102 f 5

113 f 6

104 f 8

113 f 4 108 f 4

104 f 2 105 f 1 99 f 5

3.20 f 0.40

2.85 f 0.28

2.67 f 0.29

2.30 f 0.11

2.88 f 0.5

site and not readily displaced by a rather similar but relatively inactive compound.

DISCUSSION

Basis of Method for Following Synthesis of ALA Synthetase-

The effect of certain chemicals to bring about an increase in the rate of synthesis of ALA synthetase has been investigated. This investigation has been made feasible by the finding of a simple technique for the recognition of an increase in this enzyme. In this technique, chick embryo liver cells are cultured for 1 day. During this time, they attach themselves to a cover slip as a monolayer of cells. On the 2nd day they are treated with an inducing chemical; after 20 hours, the individual cells on the cover slip are examined for porphyrin fluorescence with a fluorescence microscope, or the porphyrin is isolated from the cover slip and determined quantitatively in a fluorometer. This method is used with the implied assumption that porphyrin fluorescence can be equated with the synthesis of ALA synthetase. This assumption has been shown to be reasonable for the following reasons: (a) chick embryos treated with an inducing chemical (AIA) have been found to have an &fold greater activity of ALA synthetase in the mitochondria isolated from their livers than controls (“Experiments,” Section 3) ; (b) the enzyme ALA synthetase is the limiting one in the heme biosynthetic chain. When ALA is added directly to the chick embryo liver cells in culture, the ALA is converted within 1 to 3 hours into excessively large


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1372 Induction of ALA Synthetase Vol. 241, X0. 0

amounts of porphyrins which fluoresce intensely in a fluorescence microscope. Under these conditions, other enzymes of the biosynthetic chain become rate-limiting so that mainly coproporphyrin, but also some protoporphyrin, accumulates in the hepatic cells.

The method does not detect an increase in heme which should also be formed under these conditions. Evidence that excess heme is also formed is suggested by the work of others. Green- gard and Feigelson (40) have reported that when rats are given the inducing chemical, Sedormid, an increase is found in liver tryptophan pyrrolase, a heme enzyme. Studies of Israels et al. (41) likewise suggest on the basis of animal experiments that the normal liver can readily convert labeled ALA to heme and thence to bilirubin within a few hours; with Sedormid-induced porphyria, the conversion of labeled glycine to bilirubin also occurs within a few hours.

Hypotheses on induction--With this method of following the activity of the inducing chemicals, we have attempted to determine the properties of the inducing mechanism. Because the inducing mechanism involves an increase of ,4LA synthetase activity, three possible explanations of this increase have been examined. An increase in the activity of ALA synthetase would appear if the rate of destruction of the enzyme were decreased by the chemical. No evidence was found for this possibility (“Ex- periments,” Section 11).

Another possibility was investigated that the inducing chemical activated ALA synthetase directly, perhaps by causing some configurational change on the enzyme or by blocking a hypothetical allosteric inhibitor such as heme. No evidence was found for an increase in the activity of the enzyme by the addition of an inducing chemical to isolated liver mitochondria (“Experiments,” Section 12). No evidence was found that heme inhibited the ALA synthetase of isolated liver mitochondria (Table III). The latter experiment also ruled out a feedback inhibition type of control that has been reported to occur in R. spheroides (12) and in reticulocytes (13) in which heme directly inhibits ALA synthetase.

The third possibility to explain the increase in ALA synthetase activity by inducing chemicals is that the chemicals interfere with a repressor mechanism that normally controls the synthesis of ALA synthetase. The evidence that there is such a repressor mechanism is indirect but reasonable. Normally, the activity of ALA synthetase in liver mitochondria is very low, yet it must be sufficient to provide the heme required for t,he synthesis of the heme enzymes of liver cells. When an inducing chemical was given to a guinea pig the ALA synthetase activity increased 40-fold in the liver mitochondria; when the inducing chemical was given to chick embryos, the activity of this enzyme increased over &fold in the liver mitochondria (Table I). That this increase in activity was a result of an increased synthesis of ALA synthetase was shown by preventing the increase by agents that prevented protein synthesis, such as actinomycin D, mitomycin C, puromycin, and acetoxycycloheximide (“Experiments,” Sec- tions 6 and 9). These facts suggest that there is a repressor mechanism in the liver which normally maintains the synthesis of ALA synthetase at a very low level; the repressor mechanism may be interfered with in some way by inducing chemicals so that larger amounts of ALA synthetase may be formed.

Repressor-A mechanism of control termed feedback repression or end product repression has been proposed by the French school of Jacob, Monod, and Wollman (11) to explain the control of

enzyme formation. In this mechanism, a specific repressor molecule is considered to inhibit a specific DNA region (operon) from being decoded into mRNA, thus preventing the synthesis of an enzyme. In the case we consider here, the repressor is considered to be a protein, the aporepressor, to which is attached the corepressor, heme.

In explanation of the inducing action of certain chemicals, the tentative hypothesis is proposed that the inducing chemical com- petes with and displaces heme from the corepressor site resulting in an inactive repressor. This condition permits more ALA synthetase to be made. This hypothesis was tested by an experiment to show the competition between AIA, an inducing chemical, and heme. When AIA was used at a concentration of 15 I.cg per ml, it was found that heme (1.5 pg per ml) decreased the porphyrin-inducing activity of AIA by half (Table IV). This experiment is suggestive of a competition between AIA and heme for the corepressor site. Because iron deuteroporphyrin and manganese protoporphyrin also were found to be inhibitory, the corepressor site is not absolutely specific for heme.

If the above hypothesis is correct, then there should be on13 one site to which the inducing chemicals can attach (i.e. the corepressor site), the attachment should be reversible, and the site should resemble that which would hold the heme. Although a wide variety of organic compounds of differing physiological activities have been found to induce (Table II), most of the inducing chemicals belong to two classes of compounds, the barbiturates and collidines. That both classes of compounds were active at the same site was suggested by the experiment in which representatives of both classes, i.e. AIA and DDC, were added to chick embryo liver cells (“Experiments,” Section 15). When both compounds were given simultaneously at concentrations which could induce a maximum fluorescence by each alone, the rate of increase in fluorescence was no faster than when given singly. In Fig. 6, a hypothetical corepressor site is pictured which illustrates how this site might be occupied by a barbiturate or collidine type molecule.

In the barbiturate class there are three chemical groups which have been found individually to be capable of inducing. In the collidine class there are possibly two other groups (Table II and Fig. 5). These groups could attach to subsites on the corepressor site. Occupation of only one of the subsites would appear to be sufficient for induction.

The attachment of the inducing chemical is reversible as shown in Fig. 2. When AIA was removed from the medium containing chick embryo liver cells, a rapid decrease in the rate of porphyrin formation occurred.

The above experiments therefore suggest that there is one site as pictured in Fig. 6. This corepressor site or inducing site is relatively large; it has different regions, each more or less specific for one of the five inducing groups; these groups can attach re- versibly to the site and thus block the attachment of heme.

Repressor Mechanism-This mechanism includes the concepts of DNA activation to permit a specific mRNA to be made which then is translated into the polypeptide chain representing ALA synthetase. Support for the idea that protein synthesis of ALA synthetase is required for induction was obtained by the use of specific inhibitors. Mitomycin C inhibited the synthesis of ALA synthetase by damaging DNA and, actinomycin D inhibited by attaching to the double stranded DNA thus preventing transcription of the DNA code to mRNA. Puromycin and acetoxycyclo-


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Issue of March 25, 1966 S. Granick 1373

HOOC- H,C -

CH3

CH=CH;!

H2F CH,

H2C’ CH II *

CH HOOC II 2

H3C H3C

HOOC-H2C-H2C HOOC-H,C-H,C

H2y CH3

H27

HOOC

3,5-dicarbethaxy-1,4-dihydfa callidine Diallyl barbituric acid

an heme template an heme template

FIG. 6. Hypothesis on the competition of the inducing chemicals DDC and diallylbarbit,urate for a portion of a site on the aporepressor normally occupied by heme, the corepressor.

heximide inhibited at the ribosome level where the polypeptide chain is assembled.

The rate of porphyrin formation induced by the inducing chemical, also lends support to the involvement of a mRNA giving rise to the enzyme ALA synthetase. When AIA was added to the culture in vitro of chick embryo liver cells, the rate of porphyrin formation was found to increase almost logarith- mically (Fig. 2). This result is compatible with the assumption that a number of mRNA molecules were being formed at a steady rate, and that each mRNA molecule gave rise to a number of ALA synthetase molecules.

To obtain information on the half-life of ALA synthetase, the chick embryo liver cells were induced with AIA to form porphyrins for 12 hours. Then acetoxycycloheximide, which blocked protein synthesis at the ribosome level, was added. The rate of porphyrin formation diminished to half in 4 to 6 hours (Fig. 3). Thus, the half-life of ALA synthetase may be considered to be about 4 to 6 hours.

An idea of the lifetime of mRNA and ALA synthetase may also be obtained from another experiment. When AIA was used to induce porphyrin formation in the cells and later removed, the rate of porphyrin formation diminished rapidly (Fig. 2). This rapid decrease in rate suggests that both mRNA and ALA synthetase have a half-life measured in hours rather than in days. Bloom, Goldberg, and Green (42) have noted that for a number of enzymes in cultured HeLa and fibroblast cells the half-lives of these mRNA molecules were about 2 to 3 hours.

Detoxication and Porphyria-inducing Chemicals-The response of the liver to drugs such as the barbiturates is to synthesize enzymes that will “detoxify” the drugs (33, 43, 44). One of the mechanisms for detoxication is that of hydroxylation of aliphatic

groups or aromatic rings, performed by mixed function oxidases which require heme, iron, flavin, and TPNH or ascorbic acid for activity. In this way, the drugs which are usually rather insoluble are rendered more soluble and can be more readily excreted in the urine. A cytochrome which resides in the endoplasmic reticulum of the liver (45) partakes in the oxidation, possibly serving as a terminal oxidase. It has been reported by Orrenius and Ernster (34) and by Remmer and Merker (35) that during the first few days of barbiturate administration to an animal cytochrome increases. Thus, it is reasonable to postulate a detoxifying mechanism by which the inducing chemical, e.g. barbiturate, induces the synthesis of ALA synthetase, the limiting enzyme for heme biosynthesis. More heme is then made which can be used to make more heme enzymes to hydroxylate the barbiturate and thus aid in its elimination from the body.

Summary qf Hypothesis on Induction of Chemical Porphyria in Liver Cell-The various hypotheses on induction of porphyrins by certain chemicals are incorporated into the schema of Fig. 7. The inducing chemical induces by virtue of its affinity for a corepressor site. The chemical displaces the heme from this site and renders the repressor ineffective. Then mRNA can be made and ALA synthetase is formed. Because ALA synthetase is the limiting enzyme of the heme biosynthetic chain, excessive amounts of porphyrin and heme are made. The excessive heme is used for the formation of heme enzymes in the endoplasmic reticulum that act as mixed function oxygenases to hydroxylate and thus detoxify the inducing chemical.

Relation of Chemical Porphyria to Hepatic Porphyria-In the human metabolic disease of acute intermittent hepatic porphyria it has recently been found by Tschudy et al. (46) that a post mortem porphyric liver homogenate had a 7- to 14-fold greater


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1374 Induction of ALA Synthetase Vol. 241, A-o. 6

VII I

OP S.G.

mRNA

+ 3 d

Aporepressor VI mRNA II

’ I

Detoxication

TPNH + O2 - dALA

Apocytochrome

+ HEME +

LP,so-CO cytachrome IX

HEME IV

I t

Endoplasm!c Bile retlculbm pigment V

FIG. 7. Hypothetical schema for the detoxication of chemical inducers in liver by derepression of the repressor control on the synthesis of ALA synthetase. The control of heme biosynthesis in the liver is pictured as a competition between heme and a chemical inducer, such as a barbiturate, for a site on the aporepressor that governs the synthesis of ALA synthetase. The effect of the chemical inducer is to permit more heme to be made. The extra heme is then used for oxvzenase reactions to “detoxifv” the chemical.

The schema shows the interaction of the nucleus, mitochondria, and endonlasmic reticulum in this control. In the nucleus at I. there is a structural gene (S.G.) that codes for ALA synthetase: This code is transcribed into a messenger RNA at ZZ. The mRNA is translated into the polypeptide chain of ALA synthetase which comes to reside in the mitochondrion at ZZZ.

The ALA synthetase is the limiting enzyme in heme biosynthesis. When it is increased, more ALA is formed. The ALA is converted to heme in the mitochondria by enzymes of the biosynthetic chain, several of which reside outside the mitochondria. (In the liver, heme does not inhibit ALA synthetase, i.e. there is no end product inhibition.) The heme thus formed can enter the endoplasmic reticulum to become part of the oxygenase enzymes (IX) that detoxicate chemical inducers with the aid of TPNH and 02, or the heme may be converted to bile pigment (V), or the heme may enter the nucleus to form part of the repressor (VI) that controls the synthesis of ALA synthetase. When heme sits on the aporepressor, then the operator gene (0~) is inactive and no mRNA can be formed. When the chemical inducer (VZZZ) displaces the heme from the aporepressor, the operator becomes active, ALA synthetase can now be made, and porphyrins and heme are formed.

activity of ALA synthetase than normal. This fact supports the idea that the immediate underlying cause of the excretion of porphyrin precursors in both chemical porphyria and hepatic porphyria is elevated liver ALA synthetase.

Hepatic porphyria is inherited as a Mendelian dominant trait. This is most readily explained on the assumption that the defec- tive gene is an operator gene (Op, VZZ, Fig. 7) which is mutated so that it is repressed with difficulty by the repressor (6, 10). For this reason, small amounts of inducing chemicals, e.g. barbiturates, which would cause no detectable porphyria in normal individuals, bring about a porphyria in individuals with hepatic porphyria.

In hepatic porphyria, the symptoms appear only after puberty (47). A number of investigators have called attention to the possibility that sex steroids might occasionally precipitate an

acute attack in these individuals (1). Welland et al. (48) have administered ethinyl estradiol orally to seven patients and reported an elevation of the urinary excretion of ALA and porphobilinogen. With the cover slip technique, it was found that sex steroids but not corticosteroids could induce a low degree of porphyria in chick embryo liver cells (“Experiments,” Section 8). It is therefore probable that the porphyria-inducing effect of sex steroids is mediated directly on the liver cells rather than by an indirect mechanism.

There are two main types of hepatic porphyria with focal centers in Sweden and in South Africa. In the Swedish type, early intermediates such as ALA and porphobilinogen are excreted, whereas in the South African type coproporphyrin and protoporphyrin are excreted. An explanation of these types may be that they represent the identical genetic lesion expressed on a different genetic background. Under the stress condition of excessive ALA formation in the liver, other enzymes of the heme biosynthetic chain become limiting. The limiting enzyme in the Swedish type may become the one that condenses porphobilinogen to uroporphyrinogen, whereas in the South African type the limiting enzyme may become the one that converts coproporphy- rinogen to protoporphyrinogen.

Not only is the synthesis of ALA synthetase increased in hepatic porphyrias but it is also increased in erythropoietic porphyrias (10, 49). Consideration of these inheritable diseases leads to the following implication concerning the relation of cell differentiation to the synthesis and control of ALA synthetase. In the genome, there may be at least three operons for ALA synthetase, each operon having its ownoperator, repressor, andstruc- tural gene. The first operon would be active in liver parenchy- ma1 cells, the second in erythroid cells, and the third in other cells.

Alternative Hypothesis-The hypothesis schematized in Fig. 7 to explain chemical porphyria does not uniquely explain the data. An alternative hypothesis with other assumptions may be formulated, e.g. that the ALA synthetase of the mitochondrion is synthesized in the mitochondrion, coded by the structural gene associated with the DNA of the mitochondrion and controlled by some nuclear genes.

None of the schemes explains the decrease in liver catalase activity that occurs in chemically induced porphyria, or the nervous symptoms and abdominal pains that occur in acute hepatic porphyria (1, 2, 47).

Acknowledgments-We gratefully acknowledge the helpful sug- gestions and criticisms made by Dr. David Mauzerall and Dr. Richard Levere. We desire also to thank the various pharma- ceutical companies for their generosity in providing us with pure samples of their compounds which we have used in our tests.

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S. Granick and With the technical assistance of William Cumming and Rita LauForeign Chemicals

Chemical Porphyria: A Response to Certain Drugs, Sex Hormones, and -Aminolevulinic Acid Synthetase inδ of the Synthesis of in VitroThe Induction

1966, 241:1359-1375.J. Biol. Chem.

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