Induction of 5-aminolaevulinate synthase by two- to five-carbon alcohols in cultured chick-embryo hepatocytes. Relationship to induction of cytochrome P-450.

The induction of 5-aminolaevulinate synthase and of cytochrome P-450 by short-chain aliphatic alcohols was compared in primary cultures of chicken-embryo hepatocytes. Isopropyl alcohol, isobutanol, pentan-1-ol and isopentanol alone caused up to a 4-fold increase in 5-aminolaevulinate synthase, whereas ethanol and propan-1-ol did not. Induction of the synthase by isopentanol was maximal at 8 h, and reached a plateau thereafter, whereas the activity induced by 2-propyl-2-isopropylacetamide continued to increase for 20 h. In the presence of 3,4,3',4'-tetrachlorobiphenyl, an inhibitor of haem synthesis at the uroporphyrinogen decarboxylase step, synergistic induction of 5-aminolaevulinate synthase was observed with all the alcohols except ethanol. Ethanol, but not isopentanol, decreased the extent of induction of 5-aminolaevulinate synthase by 2-propyl-2-isopropylacetamide and 3,4,3',4'-tetrachlorobiphenyl (50% decrease at 112 mM-ethanol). Total protein synthesis was not inhibited by ethanol in these cells. The composition of porphyrins was determined after treatment of cells with ethanol, isopentanol or 2-propyl-2-isopropylacetamide. Untreated cells, when incubated with 5-aminolaevulinate for 6 h, accumulated mainly protoporphyrin. However, when cells were pretreated with ethanol, isopentanol or 2-propyl-2-isopropylacetamide for 20 h, and 5-aminolaevulinate was added, 8- and 7-carboxyporphyrins increased, whereas protoporphyrin decreased. The dose responses for induction of either 5-aminolaevulinate synthase or cytochrome P-450 after a 20 h exposure to 3- to 5-carbon alcohols were identical. The results indicate that: simple alcohols can induce both enzymes; hydrophobicity increases their effectiveness; and induction of both enzymes are probably mediated by a common mechanism.     

The effect of allyl compounds on hepatic microsomal mixed function oxidation and porphyrogenesis.

The activities of 5-aminolaevulinate (5-ALA) synthetase and of various microsomat drug-metabolising enzymes have been determined in the livers of rats pretreated with different drugs and chemicals containing the allyl group. Safrole, isosafrole and secobarbital gave rise to slight increases in 5-ALA synthetase, whereas alclophenac and triallyl cyanurate almost doubled the enzyme activity and the known porphyrogenic agents, allylisopropylacetamide (AIA) and allobarbital caused increases of 1.5- and 2.5-fold, respectively. Allobarbital induced the microsomal drug-metabolising enzymes while secobarbital had only a weak effect and alclophenac and triallyl cyanurate had no effect at all. From these results it is suggested that induction of the synthesis of cytochrome P-450 is not rate dependent on the synthesis haem and induction of porphyrin biosynthesis.     

Stimulation of liver 5-aminolaevulinate synthetase by drugs and its relevance to drug-induced accumulation of cytochrome P-450. Studies with phenylbutazone and 3,5-diethoxycarbonyl-1,4-dihydrocollidine

The relevance of the stimulation of 5-aminolaevulinate synthetase to the accumulation of cytochrome P-450 after administration of drugs was examined in rats treated with phenylbutazone and with 3,5-diethoxycarbonyl-1,4-dihydrocollidine. 3,5-Diethoxycarbonyl-1,4-dihydrocollidine alone stimulated 5-aminolaevulinate synthetase without increasing the concentration of cytochrome P-450, whereas phenylbutazone alone increased the microsomal cytochrome P-450 without significantly affecting the activity of the enzyme. When the two drugs were given together both effects were found. It is concluded that if an increased amount of 5-aminolaevulinate and haem must be made to provide for the accumulation of cytochrome P-450, it need only be a small amount. It is also concluded from these findings that stimulation of the drug-metabolizing system on the one hand and marked enhancement of 5-aminolaevulinate synthetase activity and porphyria on the other are likely to result from different actions of the drugs. Evidence is presented suggesting that porphyrogenic drugs stimulate markedly the activity of 5-aminolaevulinate synthetase by lowering the concentration of haem in the liver, thereby decreasing the normal feedback control. With 3,5-diethoxycarbonyl-1,4-dihydrocollidine a rapid inhibition of mitochondrial ferrochelatase and of liver haem synthesis may be the primary mechanism involved.     

Iron and the liver. Acute and long-term effects of iron-loading on hepatic haem metabolism.

We have determined the dose-response curves (100-900 mg of Fe/kg body wt.) and the time course over 84 days for the effects of a single injection of iron-dextran on rat hepatic 5-aminolaevulinate synthetase, cytochrome P-450, iron content, and GSH (reduced glutathione). Porphyrins in liver and urine have also been measured. (1) At 2 days after treatment, a dose of 500 mg of Fe/kg produced a 20-fold increase in iron concentration, which was maintained for 14 days. Total hepatic iron remained constant over 63 days, falling slightly by 84 days. (2) The activity of 5-aminolaevulinate synthetase was maximally increased (6-fold) 12-24 h after iron treatment. By 48 h the activity fell to less than twice the control value and thereafter remained slightly above the control value (1.1-1.5-fold) until 84 days after iron treatment. Liver GSH concentrations were unaffected by iron. Porphyrins in liver and urine were either unchanged or decreased. (3) Hepatic cytochrome P-450 decreased after iron treatment to a minimum (63% of control) at 48 h after iron administration and gradually returned to the control value by 28 days. (4) Iron-dextran potentiated 2 allyl-2-isopropyl-acetamide-induced synthesis of hepatic 5-aminolaevulinate. Potentiation occurred if the drug was given at the same time or 36 h after iron administration, but did not occur if the drug was given 14 or 64 days after iron administration. (5) The results are discussed in relation to proposed mechanisms for the effects of iron on hepatic haem metabolism.     

Decreased liver activity of porphyrin–metal chelatase in hepatic porphyria caused by 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Studies in rats and mice

1. A difference has been found between rats and mice in their sensitivity to the porphyrogenic effect of drugs. Mice are more sensitive than rats to 3,5-diethoxycarbonyl-1,4-dihydrocollidine, but less sensitive than rats to 2-allyl-2-isopropylacetamide. 2. Use has been made of this difference in sensitivity to ascertain the importance of the decrease of liver porphyrin-metal chelatase activity in porphyria caused by 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Mice, which are more sensitive than rats to the stimulation of 5-aminolaevulinate caused by this drug, are also more sensitive with respect to the decrease of chelatase activity. 3. In both species, after treatment with 3,5-diethoxycarbonyl-1,4-dihydrocollidine, the ratio between chelatase activity and 5-aminolaevulinate activity is linear with respect to the reciprocal of the liver porphyrin concentration. This suggests that under these conditions the degree of porphyrin accumulation depends on the balance between rate of porphyrin formation and rate of porphyrin utilization. 4. Compound SKF 525-A (2-diethylaminoethyl 3,3-diphenylpropylacetate) when given before 3,5-diethoxycarbonyl-1,4-dihydrocollidine prevents the appearance of porphyria in the rat and also largely prevents the decrease of chelatase activity. In the mouse it is much less effective in preventing porphyria and it is almost completely inactive in protecting the chelatase from a decrease in activity. 5. Cycloheximide, when given before 3,5-diethoxycarbonyl-1,4-dihydrocollidine also inhibits the induction of 5-aminolaevulinate synthetase and the appearance of porphyria in the rat, but does not prevent the decrease of chelatase activity. These results suggest that two successive stages can be distinguished in the induction process: a first stage leading to inhibition of haem synthesis and a second stage requiring synthesis of protein in the liver and leading to stimulation of 5-aminolaevulinate synthetase.     

Effect of allylisopropylacetamide on glutathione metabolism in the rat liver. The possible role of glutathione in the induction of 5-aminolaevulinate synthase

Administration of allylisopropylacetamide to rats caused a marked decline in the concentrations of reduced and oxidized glutathione in the liver. However, this decrease occurred in the presence of uninhibited activities of gamma-glutamylcysteine synthase and glutathione reductase, and unaltered activities of glutathione transferases A, B and C. The administration of cysteine, the rate-limiting precursor of glutathione formation, to rats treated with allylisopropylacetamide potentiated the inductive effects of the agent on 5-aminolaevulinate synthase, and markedly decreased the extent of decrease in glutathione concentrations by the agent. Conversely, the administration of diethyl maleate, which depletes the hepatic glutathione concentrations, to allylisopropylacetamide-pretreated rats (1h) diminished the extent of 5-aminolaevulinate synthase induction and the production of porphyrins by nearly 50%, when measured at 16h. This treatment did not alter the extent of non-enzymic degradation of liver haem by allylisopropylacetamide. When diethyl maleate was administered to the animals possessing high 5-aminolaevulinate synthase activity (at 3, 7 and 15h after allylisopropylacetamide), in 1h the enzyme activity was markedly decreased. Diethyl maleate had no effect on induction of 5-aminolaevulinate synthase by 3,5-diethoxycarbonyl-1,4-dihydrocollidine, also a potent porphyrinogenic agent. Diethyl maleate alone neither inhibited 5-aminolaevulinate synthase activity nor decreased the cellular content of porphyrins and haem. The data suggest that the decreases observed in the glutathione concentrations after allylisopropylacetamide administration are not the result of decreased production of the tripeptide. Rather, they most likely reflect the increased utilization of glutathione. The findings further suggest that the inhibition by diethyl maleate of allylisopropylacetamide-stimulated 5-aminolaevulinate synthase involves the inhibition of induction processes.     

Haem synthesis during cytochrome P-450 induction in higher plants. 5-Aminolaevulinic acid synthesis through a five-carbon pathway in Helianthus tuberosus tuber tissues aged in the dark.

Chlorophyll and haem synthesis in illuminated Jerusalem artichoke tuber tissues were very efficiently inhibited by gabaculine (3-amino-2,3-dihydrobenzoic acid). This inhibition seems to be due specifically to a blockade of the pathway for 5-aminolaevulinate biosynthesis which used glutamate as a substrate (the so-called C5 pathway) since we could not detect any inhibition of protein synthesis in the treated tissues and there was no effect of gabaculine on the glycine-dependent yeast 5-aminolaevulinate synthase used as a model. In dark-aged artichoke tissues, gabaculine also effectively blocked cytochrome P-450 induction, peroxidase activity and 5-aminolaevulinic acid synthesis, thus suggesting the involvement of a C5 pathway in cytoplasmic and microsomal haemoprotein synthesis in this higher plant. Allylglycine and (2-amino-ethyloxyvinyl)glycine, two olefinic glycine analogues which are potential suicide inhibitors of pyridoxal phosphate enzymes, were also demonstrated to be effective blockers of chlorophyll synthesis in artichoke tuber and Euglena cells exposed to light.     

Reversibility of the antiproliferative effect of interferon

The reversibility of the antiproliferative effect of interferon (IFN) and its correlations to the induction of (2',5') oligoadenylate synthetase (2-5A synthetase) activity was studied on NIH/3T3 cells transformed by Moloney murine sarcoma virus. The cells were treated with various doses of mouse beta-IFN. At 72 h after treatment, the cultures were subdivided. While half received fresh doses of IFN, the second half received no IFN. Reversibility of the IFN effect was then followed. Three different parameters as indicators for cell proliferation were used: cell growth, protein synthesis and cloning efficiency. In parallel, the IFN-induced activity of 2-5A synthetase was determined. The data obtained led to the following conclusions. (1) The antiproliferative effect of IFN increases with increased IFN concentration (90-1,800 IU/ml) and with time of treatment, up to 72 h after treatment. (2) The induced activity of 2-5A synthetase increases with a much faster rate, reaching maximum activity at 24 h after treatment with 450 IU/ml. This means that the induction of the enzyme precedes the antiproliferative effects of IFN. (3) There is almost no recovery of the IFN antiproliferative effect following treatment for 72 h with high doses of IFN (1,200-1,800 IU/ml). However, at lower doses, recovery is evident. (4) Removal of IFN after treatment for 3 days with 450 IU/ml resulted in a gradual decrease of 2-5A synthetase activity, reaching the basal level at 72 h after removal. However, there is no reduction of enzyme activity following treatment for 72 h with 1,800 IU/ml of IFN.     

Induction of (2'-5')oligoadenylate synthetase in serum-starved HeLa S3 cells by growth factors and its role in growth regulation

When serum-starved HeLa S3 cells were stimulated to proliferate by addition of fetal calf serum (FCS), (2'-5')oligoadenylate synthetase (2-5A synthetase) activity was induced. Although no interferon (IFN) activity was detectable in the HeLa S3 cell-conditioned culture medium after growth stimulation, addition of anti-IFN-beta monoclonal antibody inhibited both the expression of the 2-5A synthetase gene and the production of the enzyme, suggesting that endogenous IFN-beta was involved in 2-5A synthetase induction. Purified preparations of three growth factors, epidermal growth factor, platelet-derived growth factor, and insulin, also induced 2-5A synthetase through IFN-beta. When serum-starved HeLa S3 cells were treated with FCS, DNA synthesis was initiated synchronously, with peaks after 12 and 32 h, although the level of 2-5A synthetase reached a maximum after the first peak of DNA synthesis. Inhibition of 2-5A synthetase induction by anti-IFN-beta antibody enhanced the second, but not the first cycle of DNA synthesis. These results suggested that in HeLa S3 cells, after stimulation with growth factors the IFN/2-5A synthetase system played a role in cell growth negative regulatory mechanisms.     

Iron loading of cultured hepatocytes. Effect of iron on 5-aminolaevulinate synthase is independent of lipid peroxidation.

Cultured chick embryo hepatocytes were iron-loaded with ferric nitrilotriacetate. Iron-loading was confirmed by both quantitative cellular iron determinations and ultrastructural studies. With iron-loading, lipid peroxidation, as detected by malonaldehyde released into the medium, occurred at a linear rate for 12h, after which time the rate of malonaldehyde production decreased. No cell toxicity, as detected by lactate dehydrogenase release, was noted. The amount of malonaldehyde recovered in the medium after 18h of exposure to iron represented 24-33% of the total malonaldehyde that could be produced by incubating lysed cells with iron and ascorbate. Cellular glutathione was not affected by iron-stimulated lipid peroxidation, but was increased by allylisopropylacetamide. Although iron-loading by itself had no effect on activity of 5-aminolaevulinate synthase, the first and rate-limiting step in haem synthesis, iron-loading in the presence of the porphyrogenic drug allylisopropylacetamide increased levels of 5-aminolaevulinate synthase 6-fold over levels induced by the drug alone. The antioxidant, butylated hydroxytoluene, totally inhibited iron-stimulated lipid peroxidation, but did not interfere with the effect of iron-loading to potentiate an increase in 5-aminolaevulinate synthase. After 18h of exposure to iron, followed by a change to fresh medium, the iron remaining within the cells did not stimulate further lipid peroxidation over the following 18h, but did potentiate an increase in 5-aminolaevulinate synthase on exposure to allylisopropylacetamide. It therefore appears that lipid peroxidation is not the mechanism by which iron potentiates induction of hepatic 5-aminolaevulinate synthase.     

Mechanism and stereochemistry of the 5-aminolaevulinate synthetase reaction

1. Two mechanisms for the biosynthesis of 5-aminolaevulinate from glycine and succinyl-CoA (3-carboxypropionyl-CoA) are considered. One of the mechanisms involves the retention of both the C-2 H atoms of glycine during the synthesis of 5-aminolaevulinate, whereas the other predicts the retention of only one of the C-2 H atoms of glycine. 2. Highly purified 5-aminolaevulinate synthetase from Rhodopseudomonas spheroides was used to show that the C-2 H atom of glycine with R configuration is specifically removed during the biosynthesis of 5-aminolaevulinate. 3. The mechanism of the condensation therefore differs from the analogous reaction of the biosynthesis of sphinganine from palmitoyl-CoA and serine, in which the C-2 H of serine is retained (Wiess, 1963).     

VIP induces in HT-29 cells 2'5'oligoadenylate synthetase and antiviral state via interferon beta/alpha synthesis.

Vasoactive intestinal peptide (VIP), composed of 28 amino acids, is a multifunctional neurotransmitter. We have demonstrated here that its action on human transformed colonic epithelial (HT-29) cells is mediated through the induction of interferon (IFN) synthesis. We have found that these cells have a functional receptor for IFN alpha 2; binding was specific to either IFN alpha 2 or IFN beta but not to IFN gamma. VIP induced the 2'5'oligoadenylate synthetase (2'5'A synthetase) and the antiviral state with the same efficiency as poly (I).poly (C). The induction of 2'5'A synthetase activity required cellular RNA and protein synthesis, and the maximum induction occurred with 10(-7) M VIP at 24 h. VIP, like some IFN inducers, induced the synthesis of the 70 hsp which, however, preceded the expression of 2'5'A synthetase. VIP treatment caused the induction and secretion of IFN, having a titer value of 32 international units/ml. This IFN has been identified as type beta/alpha, because both 2'5'A synthetase and the antiviral activities were abolished by anti-human IFN beta/alpha antibodies, but not by anti-IFN gamma antibodies. Thus the pathway of VIP action on HT-29 cells may be outlined as 1) binding of VIP, 2) synthesis of 70 hsp, 3) induction of IFN synthesis and its secretion, 4) binding of the secreted IFN to cell surface receptors and 5) turning on the induction of 2'5'A synthetase and antiviral activities.     

Role of haem in the induction of cytochrome P-450 by phenobarbitone. Studies in chick embryos in ovo and in cultured chick embryo hepatocytes.

The role of haem synthesis during induction of hepatic cytochrome P-450 haemoproteins was studied in chick embryo in ovo and in chick embryos hepatocytes cultured under chemically defined conditions. 1. Phenobarbitone caused a prompt increase in the activity of 5-aminolaevulinate synthase, the rate-limiting enzyme of haem biosynthesis, and in the concentration of cytochrome P-450. This induction response occurred without measurable initial destruction of the haem moiety of cytochrome P-450. 2. When intracellular haem availability was enhanced by exogenous haem or 5-aminolaevulinate, phenobarbitone-medicated induction of cytochrome P-450 was not affected in spite of the well known repression of 5-aminolaevulinate synthase by haem. These data are consistent with the concept that haem does not regulate the synthesis of cytochrome P-450 haemoproteins. 3. Acetate inhibited haem biosynthesis at the level of 5-aminolaevulinate formation. When intracellular haem availability was diminished by treatment with acetate, phenobarbitone-medicated induction was decreased. 4. This inhibitory effect of acetate on cytochrome P-450 induction was reversed by exogenous haem or its precursor 5-aminolaevulinate. These data suggest that inhibition of haem biosynthesis does not decrease synthesis of apo-cytochrome P-450. Moreover, they indicate that exogenous haem can be incorporated into newly formed aop-cytochrome P-450.     

The effects of chemical porphyrogens and drugs on the activity of rat liver tryptophan pyrrolase

1. Drugs such as phenobarbitone and phenylbutazone, which increase the concentration of microsomal haem and cytochrome P-450, also increase the saturation of rat liver apo-(tryptophan pyrrolase) with its haem activator, as does the haem precursor 5-aminolaevulinate. 2. At 4h after the administration of the porphyrogens 2-allyl-2-isopropylacetamide, 3,5-diethoxycarbonyl-1,4-dihydrocollidine and griseofulvin, the total pyrrolase activity is increased whereas the haem saturation of the apoenzyme is decreased. This decreased saturation is prevented by pretreatment of the animals with the inhibitor of drug-metabolizing enzymes, SKF 525-A. 3. Pretreatment of rats with the above porphyrogens inhibits the rise in holo-(tryptophan pyrrolase) activity produced by subsequent administration of cortisol, tryptophan and 5-aminolaevulinate with two single exceptions, the possible reasons for which are discussed. 4. At 24h after the administration, in starved rats, of a single daily injection of the above porphyrogens for 1 or 2 days, the holoenzyme activity is significantly increased. 5. It is suggested that the saturation of rat liver apo-(tryptophan pyrrolase) with its haem activator can be modified by treatment known to cause destruction, inhibition of synthesis, increased utilization and enhanced synthesis of liver haem. The possible involvement of the latter phenomenon in the aetiology of mental disorders in some patients with porphyria is discussed.     

Haem synthesis from exogenous 5-aminolaevulinate in cultured chick-embryo hepatocytes. Effects of inducers of cytochromes P-450.

The effects of inducers of cytochrome P-450 on haem biosynthesis from 5-aminolaevulinate were examined by using cultured chick-embryo hepatocytes. Cultures treated with either 2-propyl-2-isopropylacetamide or 3-methylcholanthrene contained increased amounts of cytochrome P-450 and haem. After treatment for 3 h with 5-amino[4-14C]laevulinate, the relative amounts of radioactivity accumulating as haem corresponded to the relative amounts of total cellular haem, but not to increases in the amounts of cytochrome P-450. Treatment with 5-aminolaevulinate did not alter cellular haem or cytochrome P-450 concentrations in either control or drug-treated cultures. The mechanism of the enhanced accumulation of radioactivity in haem was investigated. Although 2-propyl-2-isopropylacetamide enhanced the uptake of 5-aminolaevulinate and increased the cellular concentration of porphobilinogen 1.5-fold, these changes did not account for the increases in haem radioactivity. The inducing drugs had no effect on the rates of degradation of radioactive haem, but appeared to enhance conversion of protoporphyrin into haem. This latter effect was shown by: (1) a decreased accumulation of protoporphyrin from 5-aminolaevulinate in cells treated with inducers, and (2) complete prevention of this decrease if the iron chelator desferrioxamine was present. We conclude that inducers of cytochrome P-450 may increase haem synthesis not only by increasing activity of 5-aminolaevulinate synthase, but also by increasing conversion of protoporphyrin into haem.     

Tryptophan pyrrolase in haem regulation. The mechanisms of enhancement of rat liver 5-aminolaevulinate synthase activity by starvation and of the glucose effect on induction of the enzyme by 2-allyl-2-isopropylacetamide

1. Rat liver tryptophan pyrrolase activity is enhanced by a hormonal-type mechanism during the first 2 days of starvation and by a substrate-type mechanism during the subsequent 2 days. 5-Aminolaevulinate synthase activity is also enhanced during the first 2 days of starvation, but returns thereafter to values resembling those observed in the fed rat. Treatments that prevent or reversé the enhancement of tryptophan pyrrolase activity in 24–48h-starved rats also abolish that of 5-aminolaevulinate synthase activity. Starvation of guinea pigs, which does not enhance the pyrrolase activity, also fails to alter that of the synthase. It is suggested that the decrease in 5-aminolaevulinate synthase activity in 72–96h-starved rats represents negative-feedback repression of synthesis, possibly involving tryptophan participation, whereas the enhancement observed in 24–48h-starved animals is caused by positive-feedback induction secondarily to increased utilization of the regulatory-haem pool by the newly synthesized apo-(tryptophan pyrrolase). 2. Glucose, fructose and sucrose abolish the 24h-starvation-induced increases in rat liver tryptophan pyrrolase and 5-aminolaevulinate synthase activities. Cortisol reverses the glucose effect on 5-aminolaevulinate synthase activity, presumably by enabling pyrrolase to re-utilize the regulatory-haem pool after induction of synthesis of this latter enzyme. 3. The impaired ability of 2-allyl-2-isopropylacetamide to enhance markedly 5-aminolaevulinate synthase activity in 24h-starved rats treated with glucose is associated with a failure of the porphyrogen to cause loss of tryptophan pyrrolase haem. Cortisol restores the ability of the porphyrogen to destroy tryptophan pyrrolase haem and to enhance markedly 5-aminolaevulinate synthase activity, presumably by enhancing tryptophan pyrrolase synthesis and, thereby, its re-utilization of the regulatory-haem pool. It is tentatively suggested that 2-allyl-2-isopropylacetamide destroys the above pool only after it has become bound to (or utilized by) apo-(tryptophan pyrrolase).     

Studies on Cerebellar Haem Metabolism in the Rat In Vivo

Abstract: Rats were injected intraventricularly with 5-amino[4-¹⁴C]laevulinate and the radioactivity recovered in the total cerebellum homogenate and in its haem and porphyrin fractions was determined in time. Two phases could be distinguished in the decline of haem radioactivity, suggesting labelling of at least two pools of widely different turnover rates. Succinyl acetone, when injected intraventricularly, caused a marked and long-lasting inhibition of cerebellar 5-aminolaevulinate dehydratase activity and a corresponding inhibition of the incorporation of [¹⁴C]5-aminolaevulinate into cerebellar haem in vivo. Inhibition of cerebellar haem biosynthesis by succinylacetone was followed by stimulation of the first enzyme of the pathway, 5-aminolaevulinate synthase, whereas intraventricular injection of haematin led to a significant depression of the activity of the enzyme. This suggested that the cerebellar 5-aminolaevulinate synthetase is regulated by haem through a negative feedback mechanism. Rats given repeated doses of succinylacetone, so as to maintain 80% inhibition of their cerebellar 5-aminolaevulinate dehydratase activity for 5 days, failed to exhibit any obvious symptoms of toxicity but became more sensitive to the neurotoxic effects of large intraventricular doses of 5-aminolaevulinate.     

STUDIES ON HAEM BIOSYNTHESIS IN RAT BRAIN

Abstract— Abnormalities involving haem biosynthesis have been postulated as underlying mechanisms in the aetiology of the neural manifestations of acute porphyria and of lead poisoning. This paper reports a study of the enzymes of the haem biosynthetic pathway and their control in mammalian brain. The activity of rat brain 6-aminolaevulinate synthetase (ALA synthetase), 6-aminolaevulinate dehydratase (ALA dehydratase), uroporphyrinogen I synthetase, uroporphyrinogen decarboxylase and ferrochelatase were found to be between 12.5 and 0.002% of the corresponding values for liver. This accords with the lower concentrations of total haem and cytochrome P₄₅₀ found in brain and with the slower rate of incorporation of [4-¹⁴C]ALA into brain haem in vivo . The subcellular distribution of radioactivity following intraventricular injection of [4-¹⁴C]ALA confirmed that the bulk of brain haemoproteins are intramitochondrial in contrast to liver where the major portion is microsomal. Brain haem biosynthesis was apparently unaffected by factors known to influence this pathway in liver, including starvation and treatment with allylisopropylacetamide or phenobarbitone. These findings suggest that brain haem requirements are considerably less than those of liver and are not subject to significant fluctuations under normal circumstances. Apparent non-inducibility of ALA synthetase suggests that deficient haem and consequently haemoprotein production could result where other enzymes in the pathway become rate-limiting due to genetic defects or inhibition by exogenous agents such as lead.     

Heme biosynthesis pathway regulation in a model of hepatocarcinogenesis pre-initiation.

1. Heme regulation before the appearance of hyperplastic nodules was investigated in mice models of hepatocarcinogenesis. 2. With this aim 5-aminolaevulinate synthetase (ALA-S), microsomal heme-oxygenase (MHO), mitochondrial and cytoplasmic rhodanese activities were examined throughout a period of 35 days in animals exposed to dietary p-dimethylaminoazobenzene (DAB). 3. ALA-S activity was significantly diminished (50%) on day 14, then showing a sharply rising profile from day 28 onwards, and reaching 350% on day 35. 4. A similar profile was observed for mitochondrial rhodanese activity. 5. Changes in MHO and cytoplasmic rhodanese activities were almost the opposite to those observed for ALA-S. 6. The distinctive alteration in mitochondrial and cytoplasmic rhodanese would suggest that it plays a subtle role in ALA-S regulation during carcinogenesis initiation through a mechanism that appears to involve subcellular localization controls perhaps by means of the breakage of cystine trisulphide postulated to act as an ALA-S activator. 7. Taking into account the present results, we suggest a probable mechanism for the onset of hepatocarcinogenesis that includes a primary activating liver status, provoking biochemical aberration leading to the stage of initiation of hepatocarcinogenesis involving the whole organ.