Δ-aminolevulinic Acid Synthetase Activity Research Articles

Effects of lead on the heme biosynthetic pathway in rat kidney

Exposure of rats to lead in drinking water at concentrations of 500 ppm and 1000 ppm for 3 and 6 months resulted in elevated blood lead levels, formation of kidney intranuclear inclusion bodies, and increased urinary excretion of uroporphyrin and coproporphyrin. The erythrocytic Zn-protoporphyrin was increased in the highest dose group. No significant effects on body weight gain or kidney weight were observed. Renal activity of δ-aminolevulinic acid synthetase (ALAS) was not significantly affected by lead treatment. The renal activity of δ-aminolevulinic acid dehydrase (ALAD) was moderately increased and ferrochelatase activity was significantly decreased. The relatively small effects of chronic lead exposure on renal heme biosynthesis suggests that intracellular complexation of lead with high-affinity renal lead binding proteins (PbBP) and formation of intranuclear inclusions in proximal tubule cells protects this highly sensitive pathway in kidney from lead inhibition in vivo. These data also suggest that the observed increases in urinary porphyrin excretion are primarily due to lead effects on the erythropoietic system.

Delta-Aminolevulinic acid synthetase and heme oxygenase activity in lung and liver of rats given DDT and endosulfan intratracheally.

delta-Aminolevulinic acid synthetase and heme oxygenase activity in lung and liver of rats given DDT and endosulfan intratracheally.

Heme metabolism in liver and spleen of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU)-treated rats

The effect of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), an anticancer alkylating agent of the nitrosourea group, on liver and spleen enzymes involved in the control of heme metabolism was studied. A single oral dose of 50 or 100 mg/kg CCNU caused a time-dependent loss in weight of both spleen and liver. Seven days after CCNU treatment (lOO mg/kg) the weights were at 45 and 65% of controls respectively. The activity of δ-aminolevulinic acid synthetase (ALA-S), the rate-limiting enzyme in heme biosynthesis, declined in spleen and liver to 11 and 24% of control values, respectively, 7 days after CCNU treatment. Heme oxygenase activity, the rate-limiting enzyme of heme breakdown, was moderately increased in liver and spleen following CCNU administration. In liver, heme oxygenase activity was 142% of control values at 24 hr, and in spleen the activity was 180% of controls at 1 week. Pretreatment of the animals with phenobarbital (PB) (40 mg/kg/day, i.p.) for 4 days caused a reversal in the decline of liver weight with no effect on the decline in spleen weight following CCNU treatment. Similarly, PB pretreatment reversed the decline in hepatic ALA-S activity after CCNU administration but had no effect on the decline in splenic ALA-S activity. This study indicates that CCNU causes significant decreases in the activity of enzymes of heme biosynthesis in spleen and liver. The CCNU hemotoxicity in the liver was reversed by PB pretreatment whereas the splenic hemotoxicity was unchanged.

Regulation of heme metabolism and cytochrome p-450 levels in primary culture of rat hepatocytes in a defined medium

Liver cells were prepared from adult Sprague-Dawley rats and used for the determination of δ-aminolevulinic acid synthetase (ALAS) activity and cytochrome P-450 concentrations at different time intervals in tissue culture in a serum-free synthetic medium. During the first 24 hr in culture, the level of cytochrome P-450 decreased to 30–40% of the level in isolated liver cells from untreated animals. The disappearance of cytochrome P-450 was especially fast in hepatocytes obtained from female phenobarbital-treated rats where only 40% of the original cytochrome P-450 was present after 2 hr in culture and 80% had disappeared in 2 days. The activity of ALAS increased 3- to 4-fold when measured 2 hr after plating, and it reached the maximum level in 19–24 hr when its activity was about eight times the original activity. In 2–4 days in culture, the activity of ALAS was four to five times above the original level. When the amount of δ-aminolevulinic acid (ALA) in the medium was increased from 1 to 100 μM, a decrease in ALAS was obtained, but no significant increase in cytochrome P-450 level was observed. Addition of heme to the medium gave a dose-dependent decrease in the activity of ALAS. Our data indicate that during the first 24 hr in culture the increase of ALAS activity was prevented by exogenous heme. This effect may be due to inhibition of the catalytic activity, suppression of the synthesis of the enzyme, or accelerated breakdown of the enzyme by heme.

Inhibition of uroporphyrinogen I synthase activity and depression of microsomal heme and cytochrome P-450 in rat liver by bilirubin

Purified rat hepatic uroporphyrinogen (UROgen) I synthase (URO-S) was inhibited by bilirubin or the ditaurine derivative. Inhibition was reversible and non-competitive to the substrate porphobilinogen (PBG). The inhibition constants (Ki values) for bilirubin and the conjugate were 1.5 μM and 0.26 μM respectively. Rats afflicted with hyperbilirubinemia caused by biliary obstruction had decreased levels of hepatic microsomal heme (58% of control) and cytochrome P-450 (60% of control) at day 3. Hepatic δ-aminolevulinic acid synthetase (ALAS) activity was increased (39% of control) at day 3.

Studies on the porphyrinogenic action of 1,2,4-trichlorobenzene in birds

The porphyrinogenic action of 1,2,4-trichlorobenzene (TCB) was examined in 17-day-old embryos, day-old chicks, 18-day-old chickens and adult Japanese quail. The quail was found to be the most sensitive species towards TCB induced porphyria whereas the chick embryo was totally non-responsive. The liver porphyrins of Japanese quail were increased in a dose-dependent manner 1 day after TCB. Elevation in porphyrin levels in quail was associated with comparable increases in δ-aminolevulinic acid synthetase (ALA-S) activity 1 day after TCB treatment. In contrast, ferrochelatase activity was found to be unchanged 1 day after TCB. Multiple administration of TCB produced only a slight increase in liver porphyrin levels and ALA-S activity in quail. However, there was a marked induction in ferrochelatase activity suggesting increased porphyrin turnover. Liver glutathione and glutathione S-transferase activity were also significantly increased following repeated administration of TCB in quail, which could indicate an enhancement of detoxication of reactive metabolites of TCB. Thus, it is suggested that the inability of low multiple doses of TCB to cause porphyria in Japanese quail may be related to the low responsiveness of ALA-S but high inducibility of ferrochelatase liver GSH and glutathione S- transferase.

Mechanisms of diethyldithiocarbamate-induced loss of cytochrome P-450 from rat liver

A single, intraperitoneal injection of diethyldithiocarbamate (DDTC) to adult, male Sprague-Dawley rats decreased hepatic cytochrome P-450 (P-450) concentrations. This effect was dose-dependent over a range of 250 to 750 mg/kg and most prominent 24–36 hr after dosing. Depletion of hepatic glutathione (GSH) by diethylmaleate (DEM) administration significantly decreased P-450 8 hr after concurrent treatment with DDTC at a dose which given alone had little effect on P-450 concentrations. When hepatic microsomes were incubated with DDTC in the presence of NADPH, P-450 was converted to cytochrome P-420 (P-420). Similar incubations employing [ 35S]DDTC demonstrated strict NADPH-dependent binding of labeled sulfur to microsomal membranes, suggesting that diminished P-450 concentrations are related to the metabolic activation of DDTC. Addition of reduced GSH to incubation mixtures blocked the binding of 35S to microsomal membranes, as well as conversion of P-450 to P-420. DDTC inhibited NADPH-ADP 3+ mediated peroxidation of microsomal lipids in vitro, suggesting that the effect of DDTC on P-450 does not result from stimulation of lipid peroxidation, but may be influenced by the levels of hepatic GSH. DDTC treatment 1 hr after P-450 was pulsed labeled by an intravenous injection of [ 3H]δ-aminolevulinic acid resulted in a 2-fold increase in the rate of loss of radioactivity associated with membrane-bound P-450 heme during the next 20 hr. Within this time interval, hepatic heme oxygenase (HO) activity increased and at 8 hr after dosing was 7-fold greater than control values in the livers, but was unchanged in the kidneys and spleens of DDTC-treated animals. An elevation of hepatic δ-aminolevulinic acid synthetase (δ-ALAS) activity occurred at 8 and 24 hr after DDTC treatment. Since this enzyme is rate limiting in the biosynthesis of heme, its increased activity may represent a compensatory response to offset the DDTC-mediated loss of P-450 heme.

Regulation of rat hepatic δ-aminolevulinic acid synthetase and heme oxygenase activities: Evidence for control by heme and against mediation by prosthetic iron

1. 1. The effects of in vivo administration of 6 compounds on the activity of δ-aminolevulinic acid (ALA) synthetase and heme oxygenase were determined. 2. 2. The order of decreasing potency in reducing ALA synthetase activity was heme. bilirubin. protoporphyrin IX, bilirubin dimethyl ester, CoCl 2 and FeCl 3. 3. 3. The chelating agents EDTA and deferoxamine did not prevent heme's repression of ALA synthetase or induction of heme oxygenase activity. 4. 4. The dose response, time course, enzyme subcellular distribution and chelation antagonism studies all suggest that heme itself, and not iron, regulates the rate limiting enzymatic steps of rat hepatic heme synthesis and degradation.

Effect of endotoxin and its degradation products on hepatic mixed-function oxidase and heme enzyme systems in mice

Effect of Salmonella minnesota R595 endotoxin (R595 GL) and its chemical degradation products on hepatic cytochrome P-450, δ-aminolevulinic acid (ALA) synthetase and heme oxygenase activity in mice was examined. The levels of cytochrome P-450 and ALA synthetase activity were significantly decreased following the administration of R595 GL, whereas heme oxygenase activity was markedly increased. The effects of endotoxin were dose-dependent for the range of about 1 to 20 μg per mouse. The result of a comparative study using R595 GL and its degradation products suggested that intact lipid A moiety plays an important role in the activity of endotoxin. In addition, it was found that the depressant effect of endotoxin on the hepatic mixed-function oxidase system in mice was abrogated by treatment with cortisone.

Assay, properties, and regulation of rat ovarian δ-aminolevulinic acid synthetase activity

δ-Aminolevulinic acid synthetase (EC 2.3.1.37) has been detected in homogenates of rat ovaries. Optimal substrate and coenzyme concentrations, and parameters for assay of ovarian δ-aminolevulinic acid synthetase have been determined. Subcellular fractionation studies have shown that enzyme activity is predominantly localized in the mitochondrial fraction. Fasting, which is known to increase enzyme activity in the adrenal and to have no effect on activity in the testis, had no effect on enzyme activity in the ovary. Administration of the hepatic inducer allylisopropylacetamide or the hormone progesterone failed to alter activity of the ovarian enzyme. The activity of the enzyme was significantly increased during the diestrus-1 phase of the estrus cycle, during pregnancy, and by human chorionic gonadotropin at 24 and 48 h, suggesting that ovarian δ-aminolevulinic acid synthetase and the synthesis of heme may be under hormonal control.

Biochemical basis for impaired drug metabolism in tumor-bearing rats: Evidence for altered regulation of hepatic microsomal hemeprotein synthesis

The pharmacologie effects of many drugs are enhanced in animals bearing tumors. This apparently stems from a decrease in the microsomal metabolism of these compounds in liver cells, owing to a decrease in either the activities of certain enzymes or in the content of cytochrome P-450 in microsomes, or both. Since impaired drug metabolism may have a direct bearing on the outcome of chemotherapy, a study of the biochemical basis for this alteration was begun. In female Sprague-Dawley rats bearing Walker 256 solid tumors i.m., pentobarbital metabolism was impaired as judged from prolonged sleeping-times. This effect was accompanied by a decrease in the microsomal content of cytochrome P-450. Analysis of hepatic microsomes by sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed that a microsomal protein of ~53,000 daltons was diminished in livers of tumor-bearing animals, when compared to normal controls, as determined by staining with Coomassie Blue. When rats were injected with [ 3H]leucine, although the dpm/100 μg of microsomal protein were the same for both normal and tumor-bearing rats, ~40% less 3H was associated with the microsomal proteins in the cytochrome P-450 region (~43,000 to ~58,000 daltons) of tumor-bearing animals than with those of normal controls. This was attributed to a decrease in the rate of synthesis of these, but not of other, microsomal proteins. Either staining of microsomal proteins with 3,3',5,5'-tetramethyl-benzidine, or labelling of them with δ-[ 14C]aminolevulinic acid, revealed that the overall content of microsomal hemeproteins of tumor-bearing rats was reduced considerably, when compared to normal controls (~70% as judged by the incorporation of 14C). These observations prompted investigations of the apparent rate-limiting synthetic and degradative enzymes of heme. We found that in the livers of 7-day tumor-bearing rats, the activity of δ-aminolevulinic acid synthetase was only 16% of control activity; conversely, the activity of hepatic microsomal heme oxygenase in the tumor-bearing rats was nearly eight-times greater than that of the normal animals. Together, these data indicate that perturbations in heme- and hemeprotein-synthesis cause the reduced content of cytochrome P-450 seen in tumor-bearing rats, and they provide a partial explanation for diminished drug metabolism by hepatic microsomes in the presence of a growing, transplantable, non-hepatic tumor.

Detection and regulation of δ-aminolevulinic acid synthetase activity in rat skeletal muscle

The presence of δ-aminolevulinic acid synthetase (ALAS) in mitochondria obtained from rat skeletal muscles has been observed. Optimal conditions for the meausurement of this activity are described. The activity of skeletal muscle ALAS was investigated under conditions known to affect the activity of this enzyme in other tissues. ALAS activity in skeletal muscle mitochondria was decreased 55% by a 48-h fast. Treatment with dexamethasone did not reverse the effect of starvation on ALAS activity and did not change the activity in the fed controls. ALAS activity was decreased 56% in skeletal muscle mitochondria obtained from rats in which diabetes mellitus had been induced by streptozotocin. Administration of insulin to the diabetic animals partially reversed the effect of diabetes on skeletal muscle ALAS; however, administration of insulin to control animals caused a 21% decrease in skeletal muscle ALAS activity. By contrast, treatment with inducers of hepatic ALAS such as allylisopropylacetamide or 3,5-dicarbethoxy-1,4-dihydrocollidine had no effect on skeletal muscle ALAS. These results confirm our previous suggestion that ALAS activity is regulated in a tissue-specific manner.

Heme biosynthesis in the heart

The rates of biosynthesis of heme a and heme b in hearts of fed and fasted rats were measured using an isolated heart perfusion system. δ-Aminolevulinic acid synthetase activity was decreased in hearts of fasted rats to about 30% of values in hearts obtained from fed rats. [ 14C]Glycine incorporation into hemes a and b of cardiac tissue obtained from fasted rats was also decreased to about 30% of values obtained in hearts from fed rats. Cobalt addition to the perfusion fluid led to a decrease in cardiac δ-aminolevulinic acid synthetase activity just as cobalt administration to rats does in vivo. These studies strongly suggest that δ-aminolevulinic acid synthetase activity regulates the rate of synthesis of hemes a and b in the heart.

Effect of Acute Treatment with Dimethylnitrosamine on the Contents of Cytochromes and on the Activities of δ-Aminolevulinic Acid Synthetase, Heme Oxygenase and Drug-Metabolizing Enzymes in the Liver of Rats

The effects of acute treatment with dimethylnitrosamine (DMN) on the contents of cytochromes and on the activities of key enzymes in heme metabolic pathways were investigated in male Wistar rats.Oral administration of DMN at a single dose of 30mg/kg markedly decreased the contents of microsomal cytochrome P-450 and b5, and this depression remained during 7 days after treatment. On the other hand, the activity of heme oxygenase was enhanced by 140% at 1 day, reached a peak (about 335% of the control) at 2 days, and returned to the initial level at 7 days after treatment. However, no significant change was observed in the activity of δ-aminolevulinic acid synthetase throughout these experiments.The activities of aminopyrine demethylase, aniline hydroxylase, bilirubin UDP-glucuronyltransferase, NADPH-cytochrome c reductase and NADH-cytochrome b5 reductase were all markedly reduced at 2 or 3 days after DMN treatment. In contrast, the activities of serum GPT and GOT and the content of serum bilirubin were significantly increased at 2 or 3 days after treatment.In the case of phenobarbital pretreatment (80mg/kg/day for 3 days), no remarkable differences were seen in the activities of bilirubin UDP-glucuronyltransferase and of key enzymes in the heme metabolic pathways between groups given simultaneous treatment with phenobarbital and DMN and DMN treatment alone, although phenobarbital apparently potentiated the activities of serum GPT and GOT induced by DMN treatment.

Does iron or heme control rat hepatic δ-aminolevulinic acid synthetase activity?

Disodium ethylenediamine tetraacetic acid and/or allylisopropylacetamide administration to rat pups did not evoke a premature induction of hepatic δ-aminolevulinic acid synthetase. Administration of iron to adult rats did not alter δ-aminolevulinic acid synthetase activity and had little inductive effect on heme oxygenase activity. Both heme and cobalt/dextran rapidly induced microsomal heme oxygenase by 3–8 fold. Induction of heme oxygenase by heme could be totally blocked by concurrent administration of cycloheximide. These results argue against the hypothesis that iron is the physiological mediator of δ-aminolevulinic acid synthetase activity.

An endotoxin-induced serum factor that depresses hepatic delta-aminolevulinic acid synthetase activity and cytochrome P-450 levels in mice.

An endotoxin-induced serum factor that depresses hepatic delta-aminolevulinic acid synthetase activity and cytochrome P-450 levels in mice.

Biosynthesis of δ-aminolevulinic acid in Rhodopseudomonas sphaeroides

The biosynthesis of δ-aminolevulinic acid was investigated in three strains of Rhodopseudomonas sphaeroides. A wild-type strain (NCIB 8253) possessed both δ-aminolevulinic acid synthetase and γ,δ-dioxovaleric acid transaminase in the cytoplasmic and membrane cell fractions. δ-Aminolevulinic acid synthetase activities were not detected in extracts of mutant strains H5 and H5D. However, γ,δ-dioxovaleric acid transaminase was found in the cytoplasmic and membrane fractions of these latter two strains. Strain H5 required exogenously added δ-aminolevulinic acid for growth and bacteriochlorophyll synthesis. Strain H5D did not require this compound for growth and bacteriochlorophyll synthesis. γ,δ-Dioxovaleric acid added in the growth medium did not support the growth of H5, although it was actively transported into the cells. Addition of γ,δ-dioxovaleric acid to the growth medium did not enhance the growth of either the wild-type or H5D strains. These results indicate that ALA synthetase is not required for growth and bacteriochlorophyll synthesis in H5D and that γ,δ-dioxovaleric acid is probably not an intermediate in the formation of δ-aminolevulinic acid in the strains of Rhodopseudomonas sphaeroides studied. In strain H5D another pathway may function in the formation of δ-aminolevulinic acid other than that catalyzed by δ-aminolevulinic acid synthetase or γ,δ-dioxovaleric acid transaminase.

827 CONGENITAL SIDEROBLASTIC ANEMIA (CSA): A MICRO-ENVIRONMENT DEFECT

Erythroid colony (EC) formation and heme biosynthesis were studied in marrow cultures of a 2 year old girl with severe CSA and neutropenia. Bone marrow showed micronormoblastic erythroid hyperplasia, ringed sideroblasts and decreased granulopoeisis. The patient had not responded to hematinics. and prednisone. EC formation was determined in plasma clot (pc) and methyl cellulose (mc) culture systems after 7 days incubation by benzidine staining and expressed as CFUE/8×105 marrow cells. In pc cultures, EC(140) were fewer than normal adult controls (1100).However, cultures in me resulted in exuberant EC growth (1400). Good EC formation (900) also occurred without added erythropoeitin (ep), in contrast to no growth in controls. Addition of hemin stimulated EC formation (2000) and reversed pyridoxal PO4 inhibition. δ-aminolevulinic acid (ALA) synthetase activity of marrow itself was substantially decreased and heme oxygenase was increased. However, in me culture ALA synthetase, ALA dehydratase, C- 14ALA incorporation into heme and H-3 leucine incorporation into protein were normal and increased by addition of hemin. This patient's excellent capacity to synthesize hemoglobin and form EC in me culture, even in the absence of added ep, suggests that CSA may result from an unfavorable marrow microenvironment.

Effect of Cobalt on Heme Metabolism and Related Enzymes of Rat Liver

Effect of cobalt on heme metabolism and its related enzymes were investigated and obtained the following results. 1. A single administration of cobaltous chloride dosing 40 mg/kg to rat caused an enlargement of the liver and reduction of the kidney and spleen weights. 2. Activity of heme oxygenase increased and contents of cyt. P-450 and activity of catalase decreased. 3. Activity of δ-aminolevulinic acid synthetase (ALAS) in cytoplasm increased but that of mitochondria decreased. These results suggest that cobalt inhibits the translocation of the newly synthesized ALAS from the cytosol to mitochondria. 4. Metabolic rate of aniline (Type 2) per nmol cyt. P-450 was not changed but that of aminopyrine (Type 1) increased. 5. Peroxidation rate of lipids did not change.

Effects of chlorinated benzenes of the activities of delta-aminolevulinic acid synthetase and heme oxygenase and on the content of hemoprotein in the liver of rats.

Effects of chlorinated benzenes on the activities of delta-aminolevulinic acid (delta-ALA) synthetase and heme oxygenase, the rate-limiting enzyme in heme biosynthesis and degradation respectively, and on the incorporation of 3H-delta-ALA into hemoprotein in the liver of male rats were investigated. Monochlorobenzene (MCB) and 1,2,4-trichlorobenzene(TRCB) stimulated significantly the activities of above both hepatic enzymes. After a single injection of 200 mg/kg, heme oxygenase activity was enhanced rapidly and sustained markedly for at least 48 hr by MCB treatment, while that was also enhanced but restored nearly to control levels at 48 hr by TRCB. delta-ALA synthetase activity was once decreased at 6 hr and restored within 12 hr and then reached peak levels (about 2--3 times to control levels) at 24 hr by MCB or TRCB treatment. However, this activity was sustained for 48 hr by TRCB treatment, whereas returned again to nearly control levels by MCB at 48 hr. Cytochrome P-450 content at 48 hr was significantly decreased by MCB (63% to control), in contrast, increased by TRCB (200% to control). When MCB or TRCB injected once daily throughout the study, biphasic disappearance of radioactivity incorporated into CO-binding particles was shown in control and MCB- or TRCB-treated rats. The half-life of the fast phase was about 8 hr in control and about 6 or 13 hr in MCB- or TRCB-treated rats respectively.