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Chemical induction of hepatic porphyria in inbred strains of mice
ARCHIVES
OF
BIOCHEMISTRY
Chemical
AND
141, 284-292 (1970)
BIOPHYSICS
Induction
of Hepatic Strains
JOHN
J. HUTTON
Roche Institute Received
of Molecular
Porphyria
in Inbred
of Mice
AND
STEPHEN
Biology,
Nutley,
June 10, 1970; accepted
July
R. GROSS New Jersey 07120 27, 1970
The activities of 6-aminolevulinic acid (ALA) synthetase,l ALA dehydratase, uroporphyrinogen synthetase, and the quantities of the metabolites, porphobilinogen (PBG), coproporphyrin, and protoporphyrin were serially assayed in the livers of inbred mice which had received repeated injections of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,&trimethylpyridine (DDC). During 48 hr of drug treatment, ALA synthetase activity increased 5- to 20-fold above a basal activity of 30-100 nmoles ALA formed/ hr/g liver. Hepatic uroporphyrinogen synthetase activity did not increase above a basal level of 3540 nmoles PBG consumed/hr/g liver and appeared to represent the rate-limiting step during most of the course of induction of porphyria. ALA dehydratase activity was dependent upon genotype at the Lv locus and was strain specific, but in all strains this enzyme was present in excess (7004800 nmoles/hr/g liver). The activity of the dehydratase increased 30% during 48 hr of drug treatment. PBG, proto- and, to a lesser extent, coproporphyrins accumulated, but much less porphyrin was found in livers of C57L/J mice than in those of A/HeJ, C57BL/6J, DBA/W, SWR/J or C3H/HeJ. Hepatic protoporphyrin accumulation was directly correlated with ALA synthetase activity provided that the protoporphyrin level was less than 50 nmoles/g liver. When hepatic protoporphyrin exceeded 100 nmoles/g liver, there was an inverse correlation between ALA synthetase activity and protoporphyrin content suggesting that either feedback inhibition or feedback repression of the synthetase had occurred.
The biosynthesis of heme from succinylCoA and glycine requires the participation of at least seven proteins. In the mammal the rate of synthesis of heme is tightly coordinated with demand, so that intermediates and final products of the pathway are not normally produced in excess (1). Many chemicals can induce the formation of excess intermediates of the heme pathway by liver (2), and several hereditary diseases are known which markedly affect the regulation of heme biosynthesis in liver and 1 The following ABBREVIATIONS are used in the text: ALA, &aminolevulinic acid; PBG, porphobilinogen; Uro, uroporphyrinogen; Copro, coproporphyrinogen; Proto, protoporphyrin; DDC, 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine; Tween 80, polyoxyethylene sorbitan monooleate; SEM, standard error of the mean.
bone marrow (3). The rate-limiting and regulatory step for the pathway appears to be ALA synthetase (4). Further understanding of the detailed mechanisms of regulation of heme biosynthesis in mammals would be facilitated if a variety of genetic variants affecting porphyrin synthesis were available in the same species of laboratory animal. At least one genetic variant affecting an enzyme of the porphyrin pathway has been described in inbred mice. Inbred strains have different levels of hepatic ALA dehydratase (5aminolevulinic hydrolyase, EC 4.2.1.24) (5). These differences are controlled by three alleles at the levulinate locus (Lv) on chromosome 8 (6) and are due to changes in the rate of synthesis of the dehydratase protein (7). Strains of inbred mice also differ in their response to the porphyria-inducing chemical,
PORPHYRIA
IN
3,5 - diethoxycarbonyl- 1,4 - dihydro -2,4,6trimethylpyridine (DDC) (8). When given a standard dose of DDC, strains such as C3H/HeJ or A/HeJ manifest three times higher levels of ALA synthetase than strains C57L/J and C57BR/cdJ (8). In the hope of identifying additional differences in response to DDC among inbred strains of mice, the biochemical changes occurring in chemically induced mouse porphyria have been further st,udied. The functional capacity of most of the enzymatic steps between the initial condensation of succinyl-CoA and glycine to yield ALA and the formation of protoporphyrin has been assessed either by enzyme assay or by assay of a substrate or product of the reaction in question (Fig. 1). Inbred strains have been found to differ markedly in their accumulat’ion of hepatic protoporphyrin. The differences in porphyrin accumulation appear to be correlated with known differences in inducibility of ALA
Glycine
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285
MICE
synthetase (8) and may not represent independent genetic or biochemical variants. MATERIALS
AND
METHODS
Unless otherwise stated, biochemicals were of the highest grade available from Sigma Chemical Company, St. Louis. DDC (Distillation Products Industries, Rochester) was extracted with 3 N HCI, dissolved in ethanol, and treated with neutral decolorieing carbon (Norit), and then recrystallized from ethanol-water. Chemically synthesized PBG (Protex Research and Consulting Reg’d, Montreal) was checked for homogeneity by thin-layer chromatography on silica-gel plates using methyl acetate:isopropyl alcohol:25% NHdOH (45:35:20 v/v) as solvent (9). Pyrroles were detected by spraying the dried plates with modified Ehrlich’s reagent (10). Ninety to ninetyfive per cent of the reactive material was PBG and remained near the origin. Two minor bands of Ehrlich-reactive material were present with Rp values of 0.7 and 1.0. Animals. Inbred and hybrid mice were obtained from The Jackson Laboratory. Granular
T
Ferrochelatase
+ Succinyl-CoA
Proto
ALA Synthetase"
1x*
T
I
Copro
ALA
Copro
III
Oxidase
III"
t Intramitochondrial Extramitochondrial Copro
ALA
I
ALA Dehydratase"
1
,
PBG*
FIG.
which
III*
Uro
I Synthetase*
Uro
III
uro
I
Uro Decarboxylase
III
Cosynthetase
1. The porphyrin-heme biosynthetic pathway (1, 2). Enzymes and metabolites are starred were assayed as described in Materials and Methods.
286
HUTTON
cellulose (San-I-Cel) was used as bedding and the mice were kept in a room with controlled lighting (12 hr light, 12 hr dark) and temperature (approximately 72°F). All animals were female and were between 7 weeks and 6 months of age when used in experiments. Enzyme assays. Hepatic ALA synthetase activity was essayed by a modification of the method of Marver et al. (11). Livers were weighed and then homogenized in a Potter-Elvehjem-type homogenizer in 3 vol of an 0.9% sodium chloride solution containing 0.5 mM EDTA and 10 mM Tris, pH 7.4. Erlenmeyer flasks (50 ml) for enzyme assays contained 440 pmoles glycine, 44 pmoles EDTA, 0.88 pmoles pyridoxal5’-phosphate, 330 rmoles Tris, pH 7.2, and 1.1 ml of liver homogenate in a final volume of 4.4 ml. Incubations were carried out aerobically at 37” with shaking. After 30 min of incubation, 4 ml of the reaction mixture were added to 1 ml of 25% TCA (w/v). After centrifugation, the ALA in the supernatant fluid was converted to the ALA-pyrrole by reaction with acetylacetone, chromatographed on Dowex-1 resin and quantitated by reaction with modified Ehrlich’s reagent. The production of ALA is a linear function of time for 30 min of in vitro incubation and for convenience activity is expressed as nmoles ALA formed/hi-/g liver. For assay of ALA dehydratase and uroporphyrinogen synthetase, 25yo (w/v) liver homogenates were prepared with a Potter-Elvehjemtype homogenizer in 0.15 M KCl, pH 7. ALA dehydratase was assayed as described by Hutton and Coleman (6). Uroporphyrinogen I synthetase was measured as the rate of disappearance of PBG (12). The mixture for assay of enzyme in crude spleen and liver homogenates and in 28,000g supernatant fluids contained 100 pmoles potassium phosphate buffer, pH 7.65, and 0.31 pmoles PBG, in a volume of 0.8 ml, and was incubated for 60 min at 37”. The reaction was stopped with 1 ml of 0.1 M HgCls in 10% TCA (w/v). After centrifugation, the PBG remaining was assayed as described by Levin and Coleman (12). Both zero time and 0” incubations were run in all experiments to measure the amount of PBG initially present. In the overall stoichiometry of the reaction, 4 moles of PBG are converted to 1 mole of uroporphyrinogen (12). Proteins were determined by the method of bovine serum Lowry et al. (13) using crystalline albumin as standard. Splenic erythropoiesis was induced by phenylhydrazine treatment of mice as described by Levin and Coleman (12). Assay of metabolites. For assay of tissue PBG aliquots of homogenates were deproteinized in 5% TCA. PBG in the supernatant fluid was quantitated by reaction with modified Ehrlich’s
AND
GROSS
reagent (10). For assay of copro- and protoporphyrin, livers were homogenized in 3 vol of 0.3 M phosphate buffer, pH 7.4, and then extracted with 5 vol of ethyl acetate:acetic acid (3:l v/v). Porphyrinogens were oxidized to porphyrins by placing extracts in front of an intense fluorescent light for 30 min. Porphyrins were subsequently fractionated by standard methods (14, 15) using successive extractions into diethyl ether, 2.5 N HCl, and diethyl ether. Coproporphyrins were extracted from ether into 0.05 N HCl and measured by fluorescence assay. One microgram of coproporphyrin-I (Sigma COP-1-5) was added to certain homogenates to serve as a fluorescence standard. Protoporphyrins were extracted into 1.5 N HCl and quantitated by measurement of optical absorption at the Soret maximum. E:& for protoporphyrin was assumed to be 4900 (15). Znduction of porphyria. DDC was added to isotonic saline containing O.Ol’% Tween 80 and sonicated to produce a uniform suspension of the desired concentration. In all cases the drug was administered intraperitoneally. When time courses were to be done, animals were caged six per box on granular cellulose bedding. In a typical experiment 30 female mice were weighed and caged in groups of six on the afternoon of the first day. At 9 AM on the second day, group 1 received 0.0125 ml/g body weight of the DDC suspension (4 mg/ml), which is a dose of 50 mg/kg body wt. At 9 PM group 1 was injected a second time and group 2 received its initial injection. This procedure was continued so that at 9 PM on day 3, groups l-4 received DDC, group 5 remained uninjected. Food was removed at this time, but water was provided ad libitum. At 9 AM on day 4, all animals were killed using COZ asphyxiation, and the livers were removed. Two livers were used for ALA synthetase assay and determination of tissue PBG, two for assay of ALA dehydratase and uroporphyrinogen synthetase, and two for assay of copro- and protoporphyrins. Livers were homogenized individually and were not pooled. Control experiments in which animals received injections of vehicle (saline plus 0.01% Tween 80) but no DDC, showed that vehicle alone did not induce porphyria. Modifications of the basic experimental design are described in the text, where appropriate. RESULTS
Uroporphyrinogen synthetase assay. In crude homogenates uroporphyrinogen synthetase can be assayed by measuring the rate of disappearance of porphobilinogen from an incubation mixture (12, 16). The amount of cosynthetase does not affect the
PORPHYRIA
IN
rate or the stoichiometry of the conversion of porphobilinogen into total uroporphyrinogen (isomers I and III) (12, 16). Uroporphyrinogen synthetase activity is very low in homogenates of adult mouse liver (7, 16). When homogenates of livers from normal and DDC-treated mice were assayed for uroporphyrinogen synthetase activity, very small quantities of PBG disappeared during the incubation, whereas, disappearance was readily detected in incubations containing homogenates of erythropoietic spleen (Table I). Liver homogenates do not contain significant amounts of inhibitors which could interfere with assay of the synthetase. For example, in one experiment 0.2 ml of liver homogenate was added to 0.2 ml of spleen homogenate. Incubation at 37” for 60 min resulted in the disappearance of 143 nmoles PBG, whereas, in control incubations containing spleen homogenate alone, 161 nmoles
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PBG disappeared (Table I). In the assay employed, a uroporphyrinogen synthetase activity of 60 nmoles PBG consumed/hr/g liver corresponds to a change of 0.01 optical absorbance units. Since the endogenous hepatic uroporphyrinogen synthetase activities were so low in normal and DDCtreated animals (35-60 nmoles PBG consumed/hr/g liver), it was not possible to obtain reliable estimates of the activity present in individual homogenates. In approximately 15 experiments which tested animals from six different inbred strains of mice, no differences were noted in the average synthetase activities present at 0, 12, 24, and 48 hr after DDC treatment. For this reason, synthetase activities are expressed as mean f SEM for each inbred strain and are calculated from PBG consumptions measured in at least 10 homogenates from normal and DDC-treated animals of that strain. In no case was an attempt made to quantit#ate the uroporphyrinogen formed, TABLE I but this has been done in incubations using UROPORPHYRINOGEN SYNTHETASE ACTIVITY IN homogenates of mouse spleen (12). Since HOMOGENATES OF PORPHYRIC LIVER AND PBG could be consumed by reactions other ERYTHROPOIETIC SPLEEN” than that catalyzed by uroporphyrinogen Homogenateadded IqbaPBG Incuba- consumed synthet,ase, our estimates of synthetase Incubate&&tion time (nmoles/ tion Liver spy (min) activity are likely to be too high rather than ture (“0 m (ml) hr) too low. 1 0.2 0 60 0 Changes in enzyme activities and precursor 2 0.2 0 60 15 concentrations during the induction of porphy3 0.2 37 60 161 ria. Mice from inbred strains A/HeJ, C3H/ 4 0.4 37 30 136 HeJ, C57BL/6J, C57L/J, DBA/2J, and 5 0.4 37 60 248 SWR/J were injected with DDC every 12 6 0.2 37 60 0 hr for a 48-hr period as described in Mate7 0.4 37 60 12 rials and Methods. At least four animals 8 0.2 0.2 37 60 143 from each strain were tested at each time a Porphyric livers were obtained from DBA/BJ point. Two distinctly different types of refemale mice which had received DDC (250 mg/ sponse were seen, t,he difference being most kg body wt) 24 and 18 hr before being killed. evident when hepatic protoporphyrin levels Erythropoietic spleens were obtained from DBA/ were compared after 48 hr of drug treatment. 25 female mice which had received three injections Livers from individual animals of strains of 0.4% phenylhydrazine (0.01 ml/g body wt) A/HeJ, C3H/HeJ, C57BL/BJ, DBA/BJ, at 12-hr intervals, 1 week before sacrifice. Homogand SWR/J contained 50-360 nmoles proenates were prepared in isotonic KC1 (3 v/g) and assayed as described in Methods. Protein toporphyrin per gram, whereas, livers from concentrations were: spleen, 29.7 mg/ml; porthe C57L strain contained less than 15 phyric liver, 28.4 mg/ml; normal liver, 23.4 mg/ nmoles protoporphyrin per gram. The time ml. No differences were noted between normal courses of hepatic enzyme activities and and porphyric liver, so data for porphyric liver metabolite concent’rations in SWR/J and alone are presented. At zero time each incubation C57L/J are shown in Figs. 2 and 3. In the contained 309 nmoles of PBG. Values in the table following discussion of strain differences, are the average of two determinations.
288
HUTTON
AND
12OOr
6000r
4’
II OO
1 12
1 24 Time (hours)
1 36
I 46
FIG. 2. Time course of enzyme activities in DDC-induced hepatic porphyria in the SWR/J (O--O) and C57L/J (a---@) strains of inbred mice. Animals were injected intraperitoneally with DDC (50 mg/kg body wt) at 0, 12, 24, and 36 hr. The results of a typical experiment are shown with points and bars indicating mean f range for paired animals. A. Hepatic ALA synthetase activity is indicated by interconnected points. The cross-hatched area indicates the mean uroporphyrinogen synthetase activity + SEM of 20 treated and normal animals from each strain. Average uroporphyrinogen synthetase activities were the same in the two mouse strains at all time points. B. Hepatic ALA-dehydratase activities.
SWR/J will serve as a typical example of strains which accumulate large amounts of protoporphyrin and, unless otherwise stated, characteristics of SWR/J are shared by this entire group. During 48 hr of DDC treat ment, ALA synthetase activity rises more rapidly and reaches higher values in the SWR/J than in the C57L/J inbred strain (Fig. 2A). After a 12-hr fast without drug treatment, the basal activity of ALA synthetase was 69 nmoles/hr/g in SWR/J and 34 nmoles/hr/g in C57L/J. Twelve hours after DDC treatment, ALA synthetase activity was 430 nmoles/hr/g in SWR/J and 230 nmoles/hr/g in C57L/J. At later t’ime periods, the average activity in SWRjJ is 1.5-2 times as great as that of C57L/J, but in both strains maximal act,ivity is at least 15 t,imes
GROSS
as great as basal act,ivity. In each strain ALA synthetase activity peaked after 24-36 hr of treatment (Fig. 2A). The level of ALA dehydratase activity varies among inbred strains of mice and is regulat,ed by a series of codominant alleles at the Lv locus (5,6). Although inbred strains C57L/J and SWR/J differ in their response to DDC, they do not differ in genotype at the Lv locus. Both of these strains manifest high dehydratase activity (250&35OOnmoles/ hr/g). A third strain, C57BL/6J, has low dehydratase activity (700-1000 nmoles/hr/ g), but responds to DDC in a manner similar to that of SWR/J. That is, animals of the C57BL/6 strain accumulate large amounts of hepatic protoporphyrin in response to DDC administration. The activity of ALA synthetase was generally higher in C57BL/6J than in SWR/J or C57L/J and increased from a basal level of 65 nmoles/hr/g to a level of 160 153 P
1501 c 3; % 40-
/’
Time
(hours)
3. Time course of proto- and coproporphyrin accumulations in DDC-induced hepatie porphyria in the SWR/J (O--O) and C57L/J (O-0) strains of inbred mice. Conditions and symbols are as described in the legend to Fig. 2. A. Hepatic protoporphyrin concentrations. B. Hepatic coproporphyrin concentrations. FIG.
PORPHYRIA
IN
600-1000 nmoles/hr/g 12 hr after DDC injection. In C57BL/6J, as in all strains tested, ALA dehydratase increased approximately 30% during 48 hr of DDC treatment (Fig. 2B). There is no evidence, therefore, that ALA dehydratase activity or genotype at the Lv locus affects the course of DDC-induced porphyria in the mouse. No changes in uroporphyrinogen synthetase activity were detected in t’he liver after DDC treatment. All six inbred strains tested had activities between 35 f 6 and 58 f 8 nmoles/hr/g. The mean value for each strain was based upon duplicate assay of between 13 and 20 homogenates. During the treatment period, the metabolites PBG, copro- and protoporphyrin appeared in homogenates of livers of animals from all strains. The minimal amount of PBG which our assay could detect was 20 nmoles/g liver. There were no consistent differences in PBG accumulation among the inbred strains. The amount of PBG appearing during 48 hr of DDC administration ranged from < 20 to 110 nmoles/g in SWR/J and from <20 to 73 nmoles/g in C57L/J. The largest amount of PBG observed in any strain was 139 nmoles/g in C3H/HeJ. Protoporphyrin levels showed strain-specific variations, as mentioned previously. Typical time courses of accumulation are shown in Fig. 3A. In all strains at all time points more protoporphyrin accumulated than coproporphyrin. Coproporphyrin levels were from 5-20% of the protoporphyrin levels; the ratio of copro- to protoporphyrin was generally higher during the first 24 hr of DDC treatment than at later times (Fig. 3B). The four inbred strains BALB/cJ, CBA/J, SEC/lReJ, and SJL/J, were tested using the protocol outlined in Materials and Methods and in Figs. 2 and 3 except that the experiment was terminated at 24 hr rather than 48 hr. All animals responded to DDC with at least a lo-fold increase in hepatic ALA synthetase activity. Uroporphyrinogen synthetase levels were low in all, and protoporphyrin was the major porphyrin accumulated. The basal level of ALA synthetase was higher in CBA/J (84 nmoles/hr/g liver) t,han in the other strains and larger amounts of protoporphyrin (76 nmoles/g liver) had accumulated by 24 hr in CBA/J.
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MICE
Response of individual animals to DDC. When analyzing the data obtained in experiments defining the time course of response to DDC, occasional animals were noted which had more hepatic protoporphyrin or ALA synthetase than would be expected for animals of that strain. To characterize this phenomenon in more detail, a simple experimental protocol was designed in which animals were given two doses of DDC, fasted, and the protoporphyrin content of their liver was measured 24 hr after the first dose (Fig. 4). The dose of DDC was increased to 250 mg/kg in order to obtain measurable porphyrin levels in all animals
k:
0 C57L/J
D 1
A
I
I
A C3H/HeJ A SWR/J
.
I
.
I
.
I . I ..OA I
I
I
(
0 20 40 60 60 100 120 140 160160200220 Protoporphyrin
(nmoles/g)
FIG. 4. Frequency distribution of the accumulation of hepatic protoporphyrin after DDC injection. Animals were weighed and injected with DDC, 250 mg/kg body wt, at 9 AM and 4 PM. Food was removed from the cages at the time of the initial injection. Animals were fasted until 9 AM the next day when they were asphyxiated with CO2 and the amount of protoporphyrin in livers from individual animals was determined. Each symbol in the figure represents an animal. The values obtained were grouped into classes encompassing 20 nmoles/g increments in hepatic protoporphyrin as indicated on the abscissa. LAF]/J were hybrid females obtained by mating C57L/J females with A/HeJ males.
290
HUTTON
AND GROSS
within the 24-hr time period. The distribution of individual responses to DDC does not appear to be Gaussian. In testing 19 mice of the C57L/J strain, 12 had less than 20 nmoles protoporphyrin/g (mean value 11.9), but 7 mice had more than 20 nmoles/g (Fig. 4A). These seven animals seemed to fall in a nonrandom distribution with most having between 40 and 60 nmoles protoporphyrin/g. A similar result was obtained when animals of the A/HeJ strain were tested except that most had between 20 and 80 nmoles protoporphyrin/g liver with a single animal falling below 20 and four above 80 nmoles/g (Fig. 4B). The distribution of hybrid animals obtained by mating C57L/J females with A/HeJ males could not be distinguished from the A/HeJ distribution except that fewer sporadic high protoporphyrin levels were observed (Fig. 4C). A few females from the AKR/J, C3H/HeJ, and SWR/J inbred strains were tested. In most cases the protoporphyrin levels were higher than those found in A/HeJ, but there was wide variation in individual response (Fig. 4D). Since animals of the same inbred strain are genetically identical, t,he intrastrain variation in response cannot be attributed to genetic heterogeneity but must be attributed to physiological or environmental differences (17). Relationships between ALA synthetase and protoporphyrin accumulation. ALA synthetase is assumed to be the rate-controlling enzyme in the porphyrin pathway (4). Porphyrin accumulation in chick hepatocytes has been used by Granick and others as an indirect, but semiquantitative, measure of the induction of ALA synthetase (18). In the mouse a correlation exists between hepatic protoporphyrin accumulation in vivo and ALA synthetase activity assayed in vitro (Fig. 5). In the experiment shown in Fig. 5, mice from three inbred and one hybrid strain received two doses of either 50 mg/kg or 250 mg/kg body wt of DDC in order to generate a spectrum of protoporphyrin values. At protoporphyrin levels of 1.7-20 nmoles/g the relationship between porphyrin which had accumulated and synthetase activity at that time is approximately linear. Extrapolation of the curve to zero protoporphyrin suggests that ALA
z
X 0 0 X-
E E 2
0
40
80
C3H/HeJ C57L/J LAF,/J
120
Protoporphyrin
160
200
(nmoles/g)
FIG. 5. Correlation of ALA synthetase activity and protoporphyrin content of liver. Animals were weighed, injected with DDC, and fasted as described in the legend to Fig. 4 except that some animals received two doses of 50 mg/kg and others two doses of 250 mg/kg body wt. At 9 AM, 24 hr after initial injection, livers were removed for assay of both ALA synthetase activity and protoporphyrin content. Each symbol in the figure represents an individual animal.
synthetase levels of 100 nmoles/hr/g liver must be present at 24 hr before porphyrin will accumulate. Such a synthetase activity would be two to three times the basal activity normally observed in mouse liver. Since ALA synthetase activity usually is as high at 24 hr as at any time during the preceding 24-hr period (Fig. 2A), it is likely that average synthetase activities which are even lower than 100 nmoles/hr/g can produce hepatic porphyria in mice. Increases in protoporphyrin above 100 nmoles/g liver are associated with a decrease in in vitro ALA synthetase act,ivity. This could be due to feedback inhibition of the synthetase in vitro ( and in vivo) or to feedback repression in vivo by protoporphyrin, heme, or some other metabolite. Individuals from each strain fell on the same curve (Fig. 5), but no C57L/J animals had more than 50 nmoles protoporphyrin/g liver so this strain could not be tested for inhibitory effects. DISCUSSION
Elucidation of the specific function of individual genes in the regulation of metabolic processes requires the identification and characterization of genet)ic mutants. Many
PORPHYRIA
IN
mutations have been reported in the inbred mouse, but few of these affect the regulation of activity of a specific enzyme (7, 19). In no case has a series of quantitative and qualitative variants been reported in a laboratory mammal which affect different enzymatic steps within a single multistepped pathway. We have been seeking such a series of mutants in the mouse and have chosen to study the porphyrin-heme biosynthetic pathway in liver. This pathway was originally selected because genetic variants affecting the activity and rate of synthesis of hepatic ALA dehydratase were already known (5-7); the regulat,ory step of the porphyrin-heme pathway in liver had been identified as ALA synthetase (4) ; the hepatic synthetase could be induced by many different agents (2) ; and a theory of the genetic mechanism of regulation had been proposed (18). We subsequently found that inbred mouse strains responded differently to the ALA synthetase inducer, DDC (8). We have now systematically examined the porphyrin-heme pathway of inbred mice for strain-specific variations in basal enzyme levels and effects of chemical induction of ALA synthetase both upon the activity of other enzymes of the pathway and upon the accumulation of metabolites (Fig. 1). Chemical porphyria in the mouse does not differ qualitatively from that described in the guinea pig (4). ALA dehydratase activity is always high and increases 30 % with DDC treatment. Differences in basal ALA dehydratase levels were observed, but these were due to strain differences in genotype at the levulinat,e dehydratase locus (6) and did not affect the response of ALA synthetase to DDC treatment. Uroporphyrinogen synthetase activity is low in all inbred strains (Fig. 2A) and does not increase with DDC treatment. Within hours after the administration of 50 mg DDC/kg body wt, the activity of ALA synthetase in liver greatly exceeded the activity of uroporphyrinogen synthetase, so that PBG accumulated and the conversion of PBG to uroporphyrinogen became the rate-limiting step in the porphyrin-heme pathway. There was no evidence that induction of ALA synthetase affected the uroporphyrinogen synthetase. These results in liver
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291
contrast with those obtained in erythropoietic tissue. In erythropoietic spleen, both uroporphyrinogen (Table I) and ALA (20) synthetase activities are high. ALA synthetase cannot be induced with DDC (20) and metabolites are not produced in excess in erythropoietic tissue. Coproporphyrin accumulates in the DDCtreated chick hepatocyte and is thought to be related quantitatively to increased ALA synthetase activity (18). Protoporphyrin accumulates in the livers of DDC-treated mice and is correlated with ALA synthetase activity, although the correlation becomes inverse at protoporphyrin levels above 100 nmoles/g liver (Fig. 5). This amount of protoporphyrin corresponds to a tissue concentration of 104;1.1.Both heme and protoporphyrin IX inhibit partially purified rat ALA synthetase at 10-4~ or below, although heme is a more effective inhibitor than protoporphyrin (21). The decreased activity of ALA synthetase in mouse livers which contain large amounts of protoporphyrin could be due to feedback inhibition by t’his compound or some other metabolite. On the other hand, the decreased activity could also be the result of feedback repression in viva with a reduction in the rate of synthesis of ALA synthetase. Since heme and protoporphyrin can act as feedback inhibitors, contrary to prior assumptions (18), the postulated role of these compounds as repressors in viva (18) requires further study. Interstrain variation in protoporphyrin accumulation and ALA synthetase activity in response to DDC treatment can be attributed to genetic differences among the inbred strains of mice (17). Mice of the same inbred strain are genetically identical to one another but different from mice of other inbred strains. By genetic breeding experiments one can ascertain whether genes at one or more than one locus are responsible for the differences in response to DDC. In addition the correlation between ALA synthetase activity and protoporphyrin accumulation can be examined more closely in order to determine whet’her strain variations in protoporphyrin accumulation are caused by the same genetic differences as those which cause variations in ALA synthetase act,ivity.
292
HUTTON
AND
ACKNOWLEDGMENTS The authors expert technical
thank Miss assistance.
Gloria
Semenuk
for
REFERENCES 1. KAPPAS, A., LEVERE, R. D., AND GRANICK, S., Semin. Hematol. 6, 323 (1968). 2. DE MATTEIS, F., Semin. Hematol. 6,409 (1968). 3. SCHMID, R., in “The Metabolic Basis of Inherited Disease” (J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson, eds), 2nd edition. p. 813. McGraw-Hill, New York, 1966. 4. GRANICK, S., AND URATA, G., J. Biol. Chem. 238, 821 (1963). 5. RUSSELL, R. L., AND COLEMAN, D. L., Genetics 48, 1033 (1963). 6. HUTTON, J. J., AND COLEMAN, D. L., Biochem. Genet. 3, 517 (1969). 7. DOYLE, D., .~ND SCHIMKE, R. T., J. Biol. Chem. 244, 5449 (1969). 8. GROSS, S. R., AND HUTTON, J. J., J. Biol. Chem. (accepted for publication). 9. IRVING, E. A., AND ELLIOTT, W. H., J. Biol. Chem. 244, 60 (1969). 10. MAUZERALL, D., AND GRANICK, S., J. Biol. Chem. 219, 435 (1956).
GROSS
11. MARVER, H. S., TSCHCDY, D. P., PERLROTH, M. G., AND COLLINS, A., J. Biol. Chem. 241, 2803 (1966). 12. LEVIN, E. Y., AND COLEMAN, D. L., J. Biol. Chem. 242, 4248 (1967). 13. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDILL, R. J., J. Biol. Chem. 193, 265 (1951). 14. SANO, S., AND GRANICK, S., J. Biol. Chem. 236, 1173 (1961). 15. SCHWARTZ, S., BERG, M. H., BOSSENMAIER, I., AND DINSMORE, H., Methods Biochem. Anal. 8, 221 (1960). 7, 3781 (1968). 16. LEVIN, E. Y., Biochemistry 17. MCLAREN, A., AND MICHIE, D. J., J. Genetics 64, 440 (1956). 18. GRANICK, S., J. Biol. Chem. 241, 1359 (1966). of the Laboratory 19. GREEN, M. C., in “Biology Mouse” (E. L. Green, ed.), 2nd edition, p. 87. McGraw-Hill, New York, 1966. 20. WADA, O., SASSA, S., TAKaKU, F., YANO, Y., URATA, G., AND NAK~O, K., Biochim. Biophys. Acta 148, 585 (1967). 21. SCFIOLNICK, P. L., HAMMAKER, L. E., AND MARVER, H. S., Proc. Nat. Acad. Sci. U.S.A. 63, 65 (1969).
OF
BIOCHEMISTRY
Chemical
AND
141, 284-292 (1970)
BIOPHYSICS
Induction
of Hepatic Strains
JOHN
J. HUTTON
Roche Institute Received
of Molecular
Porphyria
in Inbred
of Mice
AND
STEPHEN
Biology,
Nutley,
June 10, 1970; accepted
July
R. GROSS New Jersey 07120 27, 1970
The activities of 6-aminolevulinic acid (ALA) synthetase,l ALA dehydratase, uroporphyrinogen synthetase, and the quantities of the metabolites, porphobilinogen (PBG), coproporphyrin, and protoporphyrin were serially assayed in the livers of inbred mice which had received repeated injections of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,&trimethylpyridine (DDC). During 48 hr of drug treatment, ALA synthetase activity increased 5- to 20-fold above a basal activity of 30-100 nmoles ALA formed/ hr/g liver. Hepatic uroporphyrinogen synthetase activity did not increase above a basal level of 3540 nmoles PBG consumed/hr/g liver and appeared to represent the rate-limiting step during most of the course of induction of porphyria. ALA dehydratase activity was dependent upon genotype at the Lv locus and was strain specific, but in all strains this enzyme was present in excess (7004800 nmoles/hr/g liver). The activity of the dehydratase increased 30% during 48 hr of drug treatment. PBG, proto- and, to a lesser extent, coproporphyrins accumulated, but much less porphyrin was found in livers of C57L/J mice than in those of A/HeJ, C57BL/6J, DBA/W, SWR/J or C3H/HeJ. Hepatic protoporphyrin accumulation was directly correlated with ALA synthetase activity provided that the protoporphyrin level was less than 50 nmoles/g liver. When hepatic protoporphyrin exceeded 100 nmoles/g liver, there was an inverse correlation between ALA synthetase activity and protoporphyrin content suggesting that either feedback inhibition or feedback repression of the synthetase had occurred.
The biosynthesis of heme from succinylCoA and glycine requires the participation of at least seven proteins. In the mammal the rate of synthesis of heme is tightly coordinated with demand, so that intermediates and final products of the pathway are not normally produced in excess (1). Many chemicals can induce the formation of excess intermediates of the heme pathway by liver (2), and several hereditary diseases are known which markedly affect the regulation of heme biosynthesis in liver and 1 The following ABBREVIATIONS are used in the text: ALA, &aminolevulinic acid; PBG, porphobilinogen; Uro, uroporphyrinogen; Copro, coproporphyrinogen; Proto, protoporphyrin; DDC, 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine; Tween 80, polyoxyethylene sorbitan monooleate; SEM, standard error of the mean.
bone marrow (3). The rate-limiting and regulatory step for the pathway appears to be ALA synthetase (4). Further understanding of the detailed mechanisms of regulation of heme biosynthesis in mammals would be facilitated if a variety of genetic variants affecting porphyrin synthesis were available in the same species of laboratory animal. At least one genetic variant affecting an enzyme of the porphyrin pathway has been described in inbred mice. Inbred strains have different levels of hepatic ALA dehydratase (5aminolevulinic hydrolyase, EC 4.2.1.24) (5). These differences are controlled by three alleles at the levulinate locus (Lv) on chromosome 8 (6) and are due to changes in the rate of synthesis of the dehydratase protein (7). Strains of inbred mice also differ in their response to the porphyria-inducing chemical,
PORPHYRIA
IN
3,5 - diethoxycarbonyl- 1,4 - dihydro -2,4,6trimethylpyridine (DDC) (8). When given a standard dose of DDC, strains such as C3H/HeJ or A/HeJ manifest three times higher levels of ALA synthetase than strains C57L/J and C57BR/cdJ (8). In the hope of identifying additional differences in response to DDC among inbred strains of mice, the biochemical changes occurring in chemically induced mouse porphyria have been further st,udied. The functional capacity of most of the enzymatic steps between the initial condensation of succinyl-CoA and glycine to yield ALA and the formation of protoporphyrin has been assessed either by enzyme assay or by assay of a substrate or product of the reaction in question (Fig. 1). Inbred strains have been found to differ markedly in their accumulat’ion of hepatic protoporphyrin. The differences in porphyrin accumulation appear to be correlated with known differences in inducibility of ALA
Glycine
INBRED
285
MICE
synthetase (8) and may not represent independent genetic or biochemical variants. MATERIALS
AND
METHODS
Unless otherwise stated, biochemicals were of the highest grade available from Sigma Chemical Company, St. Louis. DDC (Distillation Products Industries, Rochester) was extracted with 3 N HCI, dissolved in ethanol, and treated with neutral decolorieing carbon (Norit), and then recrystallized from ethanol-water. Chemically synthesized PBG (Protex Research and Consulting Reg’d, Montreal) was checked for homogeneity by thin-layer chromatography on silica-gel plates using methyl acetate:isopropyl alcohol:25% NHdOH (45:35:20 v/v) as solvent (9). Pyrroles were detected by spraying the dried plates with modified Ehrlich’s reagent (10). Ninety to ninetyfive per cent of the reactive material was PBG and remained near the origin. Two minor bands of Ehrlich-reactive material were present with Rp values of 0.7 and 1.0. Animals. Inbred and hybrid mice were obtained from The Jackson Laboratory. Granular
T
Ferrochelatase
+ Succinyl-CoA
Proto
ALA Synthetase"
1x*
T
I
Copro
ALA
Copro
III
Oxidase
III"
t Intramitochondrial Extramitochondrial Copro
ALA
I
ALA Dehydratase"
1
,
PBG*
FIG.
which
III*
Uro
I Synthetase*
Uro
III
uro
I
Uro Decarboxylase
III
Cosynthetase
1. The porphyrin-heme biosynthetic pathway (1, 2). Enzymes and metabolites are starred were assayed as described in Materials and Methods.
286
HUTTON
cellulose (San-I-Cel) was used as bedding and the mice were kept in a room with controlled lighting (12 hr light, 12 hr dark) and temperature (approximately 72°F). All animals were female and were between 7 weeks and 6 months of age when used in experiments. Enzyme assays. Hepatic ALA synthetase activity was essayed by a modification of the method of Marver et al. (11). Livers were weighed and then homogenized in a Potter-Elvehjem-type homogenizer in 3 vol of an 0.9% sodium chloride solution containing 0.5 mM EDTA and 10 mM Tris, pH 7.4. Erlenmeyer flasks (50 ml) for enzyme assays contained 440 pmoles glycine, 44 pmoles EDTA, 0.88 pmoles pyridoxal5’-phosphate, 330 rmoles Tris, pH 7.2, and 1.1 ml of liver homogenate in a final volume of 4.4 ml. Incubations were carried out aerobically at 37” with shaking. After 30 min of incubation, 4 ml of the reaction mixture were added to 1 ml of 25% TCA (w/v). After centrifugation, the ALA in the supernatant fluid was converted to the ALA-pyrrole by reaction with acetylacetone, chromatographed on Dowex-1 resin and quantitated by reaction with modified Ehrlich’s reagent. The production of ALA is a linear function of time for 30 min of in vitro incubation and for convenience activity is expressed as nmoles ALA formed/hi-/g liver. For assay of ALA dehydratase and uroporphyrinogen synthetase, 25yo (w/v) liver homogenates were prepared with a Potter-Elvehjemtype homogenizer in 0.15 M KCl, pH 7. ALA dehydratase was assayed as described by Hutton and Coleman (6). Uroporphyrinogen I synthetase was measured as the rate of disappearance of PBG (12). The mixture for assay of enzyme in crude spleen and liver homogenates and in 28,000g supernatant fluids contained 100 pmoles potassium phosphate buffer, pH 7.65, and 0.31 pmoles PBG, in a volume of 0.8 ml, and was incubated for 60 min at 37”. The reaction was stopped with 1 ml of 0.1 M HgCls in 10% TCA (w/v). After centrifugation, the PBG remaining was assayed as described by Levin and Coleman (12). Both zero time and 0” incubations were run in all experiments to measure the amount of PBG initially present. In the overall stoichiometry of the reaction, 4 moles of PBG are converted to 1 mole of uroporphyrinogen (12). Proteins were determined by the method of bovine serum Lowry et al. (13) using crystalline albumin as standard. Splenic erythropoiesis was induced by phenylhydrazine treatment of mice as described by Levin and Coleman (12). Assay of metabolites. For assay of tissue PBG aliquots of homogenates were deproteinized in 5% TCA. PBG in the supernatant fluid was quantitated by reaction with modified Ehrlich’s
AND
GROSS
reagent (10). For assay of copro- and protoporphyrin, livers were homogenized in 3 vol of 0.3 M phosphate buffer, pH 7.4, and then extracted with 5 vol of ethyl acetate:acetic acid (3:l v/v). Porphyrinogens were oxidized to porphyrins by placing extracts in front of an intense fluorescent light for 30 min. Porphyrins were subsequently fractionated by standard methods (14, 15) using successive extractions into diethyl ether, 2.5 N HCl, and diethyl ether. Coproporphyrins were extracted from ether into 0.05 N HCl and measured by fluorescence assay. One microgram of coproporphyrin-I (Sigma COP-1-5) was added to certain homogenates to serve as a fluorescence standard. Protoporphyrins were extracted into 1.5 N HCl and quantitated by measurement of optical absorption at the Soret maximum. E:& for protoporphyrin was assumed to be 4900 (15). Znduction of porphyria. DDC was added to isotonic saline containing O.Ol’% Tween 80 and sonicated to produce a uniform suspension of the desired concentration. In all cases the drug was administered intraperitoneally. When time courses were to be done, animals were caged six per box on granular cellulose bedding. In a typical experiment 30 female mice were weighed and caged in groups of six on the afternoon of the first day. At 9 AM on the second day, group 1 received 0.0125 ml/g body weight of the DDC suspension (4 mg/ml), which is a dose of 50 mg/kg body wt. At 9 PM group 1 was injected a second time and group 2 received its initial injection. This procedure was continued so that at 9 PM on day 3, groups l-4 received DDC, group 5 remained uninjected. Food was removed at this time, but water was provided ad libitum. At 9 AM on day 4, all animals were killed using COZ asphyxiation, and the livers were removed. Two livers were used for ALA synthetase assay and determination of tissue PBG, two for assay of ALA dehydratase and uroporphyrinogen synthetase, and two for assay of copro- and protoporphyrins. Livers were homogenized individually and were not pooled. Control experiments in which animals received injections of vehicle (saline plus 0.01% Tween 80) but no DDC, showed that vehicle alone did not induce porphyria. Modifications of the basic experimental design are described in the text, where appropriate. RESULTS
Uroporphyrinogen synthetase assay. In crude homogenates uroporphyrinogen synthetase can be assayed by measuring the rate of disappearance of porphobilinogen from an incubation mixture (12, 16). The amount of cosynthetase does not affect the
PORPHYRIA
IN
rate or the stoichiometry of the conversion of porphobilinogen into total uroporphyrinogen (isomers I and III) (12, 16). Uroporphyrinogen synthetase activity is very low in homogenates of adult mouse liver (7, 16). When homogenates of livers from normal and DDC-treated mice were assayed for uroporphyrinogen synthetase activity, very small quantities of PBG disappeared during the incubation, whereas, disappearance was readily detected in incubations containing homogenates of erythropoietic spleen (Table I). Liver homogenates do not contain significant amounts of inhibitors which could interfere with assay of the synthetase. For example, in one experiment 0.2 ml of liver homogenate was added to 0.2 ml of spleen homogenate. Incubation at 37” for 60 min resulted in the disappearance of 143 nmoles PBG, whereas, in control incubations containing spleen homogenate alone, 161 nmoles
INBRED
MICE
287
PBG disappeared (Table I). In the assay employed, a uroporphyrinogen synthetase activity of 60 nmoles PBG consumed/hr/g liver corresponds to a change of 0.01 optical absorbance units. Since the endogenous hepatic uroporphyrinogen synthetase activities were so low in normal and DDCtreated animals (35-60 nmoles PBG consumed/hr/g liver), it was not possible to obtain reliable estimates of the activity present in individual homogenates. In approximately 15 experiments which tested animals from six different inbred strains of mice, no differences were noted in the average synthetase activities present at 0, 12, 24, and 48 hr after DDC treatment. For this reason, synthetase activities are expressed as mean f SEM for each inbred strain and are calculated from PBG consumptions measured in at least 10 homogenates from normal and DDC-treated animals of that strain. In no case was an attempt made to quantit#ate the uroporphyrinogen formed, TABLE I but this has been done in incubations using UROPORPHYRINOGEN SYNTHETASE ACTIVITY IN homogenates of mouse spleen (12). Since HOMOGENATES OF PORPHYRIC LIVER AND PBG could be consumed by reactions other ERYTHROPOIETIC SPLEEN” than that catalyzed by uroporphyrinogen Homogenateadded IqbaPBG Incuba- consumed synthet,ase, our estimates of synthetase Incubate&&tion time (nmoles/ tion Liver spy (min) activity are likely to be too high rather than ture (“0 m (ml) hr) too low. 1 0.2 0 60 0 Changes in enzyme activities and precursor 2 0.2 0 60 15 concentrations during the induction of porphy3 0.2 37 60 161 ria. Mice from inbred strains A/HeJ, C3H/ 4 0.4 37 30 136 HeJ, C57BL/6J, C57L/J, DBA/2J, and 5 0.4 37 60 248 SWR/J were injected with DDC every 12 6 0.2 37 60 0 hr for a 48-hr period as described in Mate7 0.4 37 60 12 rials and Methods. At least four animals 8 0.2 0.2 37 60 143 from each strain were tested at each time a Porphyric livers were obtained from DBA/BJ point. Two distinctly different types of refemale mice which had received DDC (250 mg/ sponse were seen, t,he difference being most kg body wt) 24 and 18 hr before being killed. evident when hepatic protoporphyrin levels Erythropoietic spleens were obtained from DBA/ were compared after 48 hr of drug treatment. 25 female mice which had received three injections Livers from individual animals of strains of 0.4% phenylhydrazine (0.01 ml/g body wt) A/HeJ, C3H/HeJ, C57BL/BJ, DBA/BJ, at 12-hr intervals, 1 week before sacrifice. Homogand SWR/J contained 50-360 nmoles proenates were prepared in isotonic KC1 (3 v/g) and assayed as described in Methods. Protein toporphyrin per gram, whereas, livers from concentrations were: spleen, 29.7 mg/ml; porthe C57L strain contained less than 15 phyric liver, 28.4 mg/ml; normal liver, 23.4 mg/ nmoles protoporphyrin per gram. The time ml. No differences were noted between normal courses of hepatic enzyme activities and and porphyric liver, so data for porphyric liver metabolite concent’rations in SWR/J and alone are presented. At zero time each incubation C57L/J are shown in Figs. 2 and 3. In the contained 309 nmoles of PBG. Values in the table following discussion of strain differences, are the average of two determinations.
288
HUTTON
AND
12OOr
6000r
4’
II OO
1 12
1 24 Time (hours)
1 36
I 46
FIG. 2. Time course of enzyme activities in DDC-induced hepatic porphyria in the SWR/J (O--O) and C57L/J (a---@) strains of inbred mice. Animals were injected intraperitoneally with DDC (50 mg/kg body wt) at 0, 12, 24, and 36 hr. The results of a typical experiment are shown with points and bars indicating mean f range for paired animals. A. Hepatic ALA synthetase activity is indicated by interconnected points. The cross-hatched area indicates the mean uroporphyrinogen synthetase activity + SEM of 20 treated and normal animals from each strain. Average uroporphyrinogen synthetase activities were the same in the two mouse strains at all time points. B. Hepatic ALA-dehydratase activities.
SWR/J will serve as a typical example of strains which accumulate large amounts of protoporphyrin and, unless otherwise stated, characteristics of SWR/J are shared by this entire group. During 48 hr of DDC treat ment, ALA synthetase activity rises more rapidly and reaches higher values in the SWR/J than in the C57L/J inbred strain (Fig. 2A). After a 12-hr fast without drug treatment, the basal activity of ALA synthetase was 69 nmoles/hr/g in SWR/J and 34 nmoles/hr/g in C57L/J. Twelve hours after DDC treatment, ALA synthetase activity was 430 nmoles/hr/g in SWR/J and 230 nmoles/hr/g in C57L/J. At later t’ime periods, the average activity in SWRjJ is 1.5-2 times as great as that of C57L/J, but in both strains maximal act,ivity is at least 15 t,imes
GROSS
as great as basal act,ivity. In each strain ALA synthetase activity peaked after 24-36 hr of treatment (Fig. 2A). The level of ALA dehydratase activity varies among inbred strains of mice and is regulat,ed by a series of codominant alleles at the Lv locus (5,6). Although inbred strains C57L/J and SWR/J differ in their response to DDC, they do not differ in genotype at the Lv locus. Both of these strains manifest high dehydratase activity (250&35OOnmoles/ hr/g). A third strain, C57BL/6J, has low dehydratase activity (700-1000 nmoles/hr/ g), but responds to DDC in a manner similar to that of SWR/J. That is, animals of the C57BL/6 strain accumulate large amounts of hepatic protoporphyrin in response to DDC administration. The activity of ALA synthetase was generally higher in C57BL/6J than in SWR/J or C57L/J and increased from a basal level of 65 nmoles/hr/g to a level of 160 153 P
1501 c 3; % 40-
/’
Time
(hours)
3. Time course of proto- and coproporphyrin accumulations in DDC-induced hepatie porphyria in the SWR/J (O--O) and C57L/J (O-0) strains of inbred mice. Conditions and symbols are as described in the legend to Fig. 2. A. Hepatic protoporphyrin concentrations. B. Hepatic coproporphyrin concentrations. FIG.
PORPHYRIA
IN
600-1000 nmoles/hr/g 12 hr after DDC injection. In C57BL/6J, as in all strains tested, ALA dehydratase increased approximately 30% during 48 hr of DDC treatment (Fig. 2B). There is no evidence, therefore, that ALA dehydratase activity or genotype at the Lv locus affects the course of DDC-induced porphyria in the mouse. No changes in uroporphyrinogen synthetase activity were detected in t’he liver after DDC treatment. All six inbred strains tested had activities between 35 f 6 and 58 f 8 nmoles/hr/g. The mean value for each strain was based upon duplicate assay of between 13 and 20 homogenates. During the treatment period, the metabolites PBG, copro- and protoporphyrin appeared in homogenates of livers of animals from all strains. The minimal amount of PBG which our assay could detect was 20 nmoles/g liver. There were no consistent differences in PBG accumulation among the inbred strains. The amount of PBG appearing during 48 hr of DDC administration ranged from < 20 to 110 nmoles/g in SWR/J and from <20 to 73 nmoles/g in C57L/J. The largest amount of PBG observed in any strain was 139 nmoles/g in C3H/HeJ. Protoporphyrin levels showed strain-specific variations, as mentioned previously. Typical time courses of accumulation are shown in Fig. 3A. In all strains at all time points more protoporphyrin accumulated than coproporphyrin. Coproporphyrin levels were from 5-20% of the protoporphyrin levels; the ratio of copro- to protoporphyrin was generally higher during the first 24 hr of DDC treatment than at later times (Fig. 3B). The four inbred strains BALB/cJ, CBA/J, SEC/lReJ, and SJL/J, were tested using the protocol outlined in Materials and Methods and in Figs. 2 and 3 except that the experiment was terminated at 24 hr rather than 48 hr. All animals responded to DDC with at least a lo-fold increase in hepatic ALA synthetase activity. Uroporphyrinogen synthetase levels were low in all, and protoporphyrin was the major porphyrin accumulated. The basal level of ALA synthetase was higher in CBA/J (84 nmoles/hr/g liver) t,han in the other strains and larger amounts of protoporphyrin (76 nmoles/g liver) had accumulated by 24 hr in CBA/J.
INBRED
259
MICE
Response of individual animals to DDC. When analyzing the data obtained in experiments defining the time course of response to DDC, occasional animals were noted which had more hepatic protoporphyrin or ALA synthetase than would be expected for animals of that strain. To characterize this phenomenon in more detail, a simple experimental protocol was designed in which animals were given two doses of DDC, fasted, and the protoporphyrin content of their liver was measured 24 hr after the first dose (Fig. 4). The dose of DDC was increased to 250 mg/kg in order to obtain measurable porphyrin levels in all animals
k:
0 C57L/J
D 1
A
I
I
A C3H/HeJ A SWR/J
.
I
.
I
.
I . I ..OA I
I
I
(
0 20 40 60 60 100 120 140 160160200220 Protoporphyrin
(nmoles/g)
FIG. 4. Frequency distribution of the accumulation of hepatic protoporphyrin after DDC injection. Animals were weighed and injected with DDC, 250 mg/kg body wt, at 9 AM and 4 PM. Food was removed from the cages at the time of the initial injection. Animals were fasted until 9 AM the next day when they were asphyxiated with CO2 and the amount of protoporphyrin in livers from individual animals was determined. Each symbol in the figure represents an animal. The values obtained were grouped into classes encompassing 20 nmoles/g increments in hepatic protoporphyrin as indicated on the abscissa. LAF]/J were hybrid females obtained by mating C57L/J females with A/HeJ males.
290
HUTTON
AND GROSS
within the 24-hr time period. The distribution of individual responses to DDC does not appear to be Gaussian. In testing 19 mice of the C57L/J strain, 12 had less than 20 nmoles protoporphyrin/g (mean value 11.9), but 7 mice had more than 20 nmoles/g (Fig. 4A). These seven animals seemed to fall in a nonrandom distribution with most having between 40 and 60 nmoles protoporphyrin/g. A similar result was obtained when animals of the A/HeJ strain were tested except that most had between 20 and 80 nmoles protoporphyrin/g liver with a single animal falling below 20 and four above 80 nmoles/g (Fig. 4B). The distribution of hybrid animals obtained by mating C57L/J females with A/HeJ males could not be distinguished from the A/HeJ distribution except that fewer sporadic high protoporphyrin levels were observed (Fig. 4C). A few females from the AKR/J, C3H/HeJ, and SWR/J inbred strains were tested. In most cases the protoporphyrin levels were higher than those found in A/HeJ, but there was wide variation in individual response (Fig. 4D). Since animals of the same inbred strain are genetically identical, t,he intrastrain variation in response cannot be attributed to genetic heterogeneity but must be attributed to physiological or environmental differences (17). Relationships between ALA synthetase and protoporphyrin accumulation. ALA synthetase is assumed to be the rate-controlling enzyme in the porphyrin pathway (4). Porphyrin accumulation in chick hepatocytes has been used by Granick and others as an indirect, but semiquantitative, measure of the induction of ALA synthetase (18). In the mouse a correlation exists between hepatic protoporphyrin accumulation in vivo and ALA synthetase activity assayed in vitro (Fig. 5). In the experiment shown in Fig. 5, mice from three inbred and one hybrid strain received two doses of either 50 mg/kg or 250 mg/kg body wt of DDC in order to generate a spectrum of protoporphyrin values. At protoporphyrin levels of 1.7-20 nmoles/g the relationship between porphyrin which had accumulated and synthetase activity at that time is approximately linear. Extrapolation of the curve to zero protoporphyrin suggests that ALA
z
X 0 0 X-
E E 2
0
40
80
C3H/HeJ C57L/J LAF,/J
120
Protoporphyrin
160
200
(nmoles/g)
FIG. 5. Correlation of ALA synthetase activity and protoporphyrin content of liver. Animals were weighed, injected with DDC, and fasted as described in the legend to Fig. 4 except that some animals received two doses of 50 mg/kg and others two doses of 250 mg/kg body wt. At 9 AM, 24 hr after initial injection, livers were removed for assay of both ALA synthetase activity and protoporphyrin content. Each symbol in the figure represents an individual animal.
synthetase levels of 100 nmoles/hr/g liver must be present at 24 hr before porphyrin will accumulate. Such a synthetase activity would be two to three times the basal activity normally observed in mouse liver. Since ALA synthetase activity usually is as high at 24 hr as at any time during the preceding 24-hr period (Fig. 2A), it is likely that average synthetase activities which are even lower than 100 nmoles/hr/g can produce hepatic porphyria in mice. Increases in protoporphyrin above 100 nmoles/g liver are associated with a decrease in in vitro ALA synthetase act,ivity. This could be due to feedback inhibition of the synthetase in vitro ( and in vivo) or to feedback repression in vivo by protoporphyrin, heme, or some other metabolite. Individuals from each strain fell on the same curve (Fig. 5), but no C57L/J animals had more than 50 nmoles protoporphyrin/g liver so this strain could not be tested for inhibitory effects. DISCUSSION
Elucidation of the specific function of individual genes in the regulation of metabolic processes requires the identification and characterization of genet)ic mutants. Many
PORPHYRIA
IN
mutations have been reported in the inbred mouse, but few of these affect the regulation of activity of a specific enzyme (7, 19). In no case has a series of quantitative and qualitative variants been reported in a laboratory mammal which affect different enzymatic steps within a single multistepped pathway. We have been seeking such a series of mutants in the mouse and have chosen to study the porphyrin-heme biosynthetic pathway in liver. This pathway was originally selected because genetic variants affecting the activity and rate of synthesis of hepatic ALA dehydratase were already known (5-7); the regulat,ory step of the porphyrin-heme pathway in liver had been identified as ALA synthetase (4) ; the hepatic synthetase could be induced by many different agents (2) ; and a theory of the genetic mechanism of regulation had been proposed (18). We subsequently found that inbred mouse strains responded differently to the ALA synthetase inducer, DDC (8). We have now systematically examined the porphyrin-heme pathway of inbred mice for strain-specific variations in basal enzyme levels and effects of chemical induction of ALA synthetase both upon the activity of other enzymes of the pathway and upon the accumulation of metabolites (Fig. 1). Chemical porphyria in the mouse does not differ qualitatively from that described in the guinea pig (4). ALA dehydratase activity is always high and increases 30 % with DDC treatment. Differences in basal ALA dehydratase levels were observed, but these were due to strain differences in genotype at the levulinat,e dehydratase locus (6) and did not affect the response of ALA synthetase to DDC treatment. Uroporphyrinogen synthetase activity is low in all inbred strains (Fig. 2A) and does not increase with DDC treatment. Within hours after the administration of 50 mg DDC/kg body wt, the activity of ALA synthetase in liver greatly exceeded the activity of uroporphyrinogen synthetase, so that PBG accumulated and the conversion of PBG to uroporphyrinogen became the rate-limiting step in the porphyrin-heme pathway. There was no evidence that induction of ALA synthetase affected the uroporphyrinogen synthetase. These results in liver
INBRED
MICE
291
contrast with those obtained in erythropoietic tissue. In erythropoietic spleen, both uroporphyrinogen (Table I) and ALA (20) synthetase activities are high. ALA synthetase cannot be induced with DDC (20) and metabolites are not produced in excess in erythropoietic tissue. Coproporphyrin accumulates in the DDCtreated chick hepatocyte and is thought to be related quantitatively to increased ALA synthetase activity (18). Protoporphyrin accumulates in the livers of DDC-treated mice and is correlated with ALA synthetase activity, although the correlation becomes inverse at protoporphyrin levels above 100 nmoles/g liver (Fig. 5). This amount of protoporphyrin corresponds to a tissue concentration of 104;1.1.Both heme and protoporphyrin IX inhibit partially purified rat ALA synthetase at 10-4~ or below, although heme is a more effective inhibitor than protoporphyrin (21). The decreased activity of ALA synthetase in mouse livers which contain large amounts of protoporphyrin could be due to feedback inhibition by t’his compound or some other metabolite. On the other hand, the decreased activity could also be the result of feedback repression in viva with a reduction in the rate of synthesis of ALA synthetase. Since heme and protoporphyrin can act as feedback inhibitors, contrary to prior assumptions (18), the postulated role of these compounds as repressors in viva (18) requires further study. Interstrain variation in protoporphyrin accumulation and ALA synthetase activity in response to DDC treatment can be attributed to genetic differences among the inbred strains of mice (17). Mice of the same inbred strain are genetically identical to one another but different from mice of other inbred strains. By genetic breeding experiments one can ascertain whether genes at one or more than one locus are responsible for the differences in response to DDC. In addition the correlation between ALA synthetase activity and protoporphyrin accumulation can be examined more closely in order to determine whet’her strain variations in protoporphyrin accumulation are caused by the same genetic differences as those which cause variations in ALA synthetase act,ivity.
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AND
ACKNOWLEDGMENTS The authors expert technical
thank Miss assistance.
Gloria
Semenuk
for
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