Allylisopropylacetamide Treatment Research Articles

Sn-protoporphyrin suppresses chemically induced experimental hepatic porphyria. Potential clinical implications.

The ability of Sn(tin)-protoporphyrin to inhibit the induction of hepatic delta-aminolevulinate (ALA) synthase by allylisopropyl acetamide (AIA) was examined in the adult rat. Doses of Sn-protoporphyrin of 1, 10, and 50 mumol/kg body wt resulted in decreases in AIA-induced hepatic ALA-synthase activity of 32, 52, and 60%, respectively, compared with rats treated with AIA alone; inhibition of ALA-synthase was not a direct effect of Sn-protoporphyrin. This inhibition of the enzyme activity in liver was reflected in concurrent decreases in urinary excretion of ALA and porphobilinogen (PBG). The increased urinary excretion of ALA and PBG observed following AIA treatment was reduced by the lowest dose of Sn-protoporphyrin (1 mumol/kg body wt) and abolished completely by the higher doses of the metalloporphyrin (10 and 50 mumol/kg body wt). These findings in a rat model of hepatic porphyria suggest that Sn-protoporphyrin may be useful in the treatment of acute exacerbations of "inducible" hepatic porphyrias in man, especially since Sn-protoporphyrin, unlike hematin which is presently used for this purpose, is neither degraded by nor induces the activity of heme oxygenase.

Formation of cytochrome P-450 containing haem or cobalt-protoporphyrin in liver homogenates of rats treated with phenobarbital and allylisopropylacetamide.

The potent porphyrogen allylisopropylacetamide and related compounds decrease hepatic concentrations of cytochrome P-450. This decrease occurs particularly in phenobarbital-induced cytochrome P-450 and is caused by suicidal breakdown of the haem of cytochrome P-450. Quantitative rocket immunoelectrophoresis showed that the protein moiety of the major phenobarbital-inducible form of hepatic cytochrome P-450 was not diminished up to 1 h, but was markedly decreased (to 43% of that of the phenobarbital-treated control) at 20 h after allylisopropylacetamide treatment. In contrast, the concentration of total cytochrome P-450, measured spectrophotometrically, decreased to 30-40% of the control at both 1 and 20 h after allylisopropylacetamide. Cytochrome P-450-dependent demethylations of ethylmorphine and benzphetamine decreased to a similar extent. When liver homogenates from rats treated with allylisopropylacetamide 1 h before being killed were incubated with haem, functional holocytochrome P-450 could be reconstituted from the apoprotein. Incubation with haem increased spectrophotometrically measurable cytochrome P-450 to 69%, ethylmorphine demethylase to 64% and benzphetamine demethylase to 93% of the activities in rats treated with phenobarbital alone. At 20 h after allylisopropylacetamide treatment, however, little or no reconstitution of cytochrome P-450 occurred after incubation with haem. When liver homogenates were incubated with cobalt and protoporphyrin, and microsomal proteins were then subjected to polyacrylamide-gel electrophoresis, cobalt-protoporphyrin was found specifically associated with proteins of Mr 50 000-53 000. When homogenates from rats given allylisopropylacetamide for 1 h or 20 h were compared, it was found that the extent of this association was higher in livers from the rats containing more apocytochrome P-450, suggesting that cobalt-protoporphyrin had associated with the apocytochrome. The data provide insight into the association of haem with the protein moiety of cytochrome P-450 and factors affecting breakdown of this protein.

Aging-related decreases in hepatic mitochondrial and cytosolic δ-aminolevulinic acid synthase during experimental porphyria

The basal- and allylisopropylacetamide-induced activities of the first enzyme of heme biosynthesis, δ-aminolevulinic acid synthase (ALAS) were measured in hepatic mitochondria and cytosol of young, adult, and aged Fisher 344 rats. The total cellular ALAS activity induced by allylisopropylacetamide decreased 67% with age. The specific activity of mitochondrial ALAS in normal and induced animals decreased with aging when assayed in whole or broken mitochondria. The levels of ALAS which accumulated in the cytosol after allylisopropylacetamide administration were proportionally greater in both the young and senescent than in the mature animals. During aging, no evidence for a fragile population of mitochondria in either normal or induced animals was observed suggesting that mitochondrial matrix proteins are not released during homogenization. The hepatic mitochondrial content decreased during aging when calculated using both a membrane-bound marker enzyme cytochrome oxidase and a matrix marker enzyme citrate synthase and was unaffected by allylisopropylacetamide treatment. This reduced mitochondrial content further diminishes the level of functional ALAS available in the liver during senescence. This study confirms the age-dependent decrease in mitochondria ALAS in normal and induced animals and also suggests an age-related change in the process by which cytosolic ALAS is translocated into the mitochondria.

Structural and functional reconstitution of hepatic cytochrome P-450 in vivo. Reversal of allylisopropylacetamide-mediated destruction of the hemoprotein by exogenous heme.

After the heme moiety of hepatic cytochrome P-450 in rats had been destroyed by allylisopropylacetamide (AIA), intravenously injected heme significantly increased the cytochrome P-450 content and mixed function oxidase activity of liver microsomes. (The term "heme" is used throughout this paper for iron protorphyrin IX, irrespective of its redox state). In the absence of AIA treatment, heme administration failed to elicit these effects. The heme-mediated increase of cytochrome P-450 was most pronounced in phenobarbital-pretreated rats (46%), but was detectable also in 3-methylcholanthrene-pretreated (6%) and in untreated animals (7%). Restoration of mixed function oxidase activity by heme appeared to depend on reconstitution of cytochrome P-450 and was greatest for oxidases whose activity is stimulated by phenobarbital. Under optimal experimental conditions, heme enhanced the residual activity of ethylmorphine N-demethylase 3-fold when 92% of the initial enzyme activity had been destroyed by AIA. Reconstitution of cytochrome P-450 was dependent on the relative amount of cytochrome P-450 destroyed by AIA and on the dose of heme administered. Formation of holocytochrome P-450 appeared to be independent of apoprotein synthesis as it was unaffected by cycloheximide. Thus, reconstitution of cytochrome P-450 following AIA-mediated destruction of its original prosthetic heme reflects direct incorporation of administered heme into residual apoprotein in the endoplasmic reticulum. These findings demonstrate that exogenous heme can partially restore the structure and function of hepatic cytochrome P-450 following its drug-mediated destruction.

Allylisopropylacetamide restricts expression of β minor globin gene in Friend cells

Two structural genes, β major and β minor, are present at the β-globin locus (Hbb) of a variety of mice phenotypically characterised by adult haemoglobin of the diffuse type1. These genes may be the result of duplication of an ancestral gene2, but there is increasing evidence that their expression is independently regulated. For example, in erythrocytes of the adult DBA/2 mouse, the ratio of the two nonallelic variant gene products is 4:1 in favour of the β-major chain whereas in Friend erythroleukaemia cells, clone 745, derived from the same mouse strain, the ratio varies from 3 to 0.6, depending on the inducer of erythroid differentiation used3–6. We reported previously that treatment of clone 745 cells for 6 d with 1×10−4 M haemin, an inducer of haemoglobin synthesis7, induces synthesis of the β minor, but not the β major globin chain5. This observation suggested the possibility that intracellular levels of haem (the reduced form of haemin) might act physiologically to selectively control the expression of one of the two β globin genes. To test this hypothesis, we determined the effect of allylisopropylacetamide (AIA) on the relative amounts of β globin chains present in induced erythroleukaemia cells. AIA is a strong inducer of δ-aminolevulinic acid synthetase in rat hepatocytes8 and in mouse erythroleukaemia cells9. Induction of the enzyme is due to breakdown by the drug of the haem moiety of the haemoproteins10. We report here that AIA treatment of Friend cells induced to differentiate by dimethylsulphoxide (DMSO) causes a reduction in the intracellular haem concentration and at the same time a marked decrease in the amount of the β minor globin chain. The ratio of the two β chains thus becomes very close to the ratio observed in normal erythrocytes of adult DBA/2 mouse.

Decrease in hepatic cytochrome P-450 and catalase following allylisopropylacetamide: The effect of concomitant hemin administration

It is known that administration of allylisopropylacetamide (AIA) to rats increases δ-aminolevulinic acid synthetase (ALA-S), urine ascorbic acid excretion and decreases hepatic hemoproteins cytochrome P-450 and catalase. Hemin has been shown to inhibit induction of ALA-S by AIA. To investigate further the regulatory role of hemin, AIA was administered to rats over a 5 day period with and without hemin. Hemin did not prevent the decrease in P-450 or catalase caused by AIA but partially inhibited induction of ALA-S and the increase in ascorbic acid excretion. It is unlikely that the hemin is replacing endogenous heme in hemoprotein synthesis and we conclude that the role of hemin in suppressing induction phenomena that follow AIA treatment reflects a regulatory function not related to a role in hemoprotein synthesis.

Incorporation of radioactive-δ-aminolevulinic acid into microsomal cytochrome P 450: Selective breakdown of the hemoprotein by allylisopropylacetamide and carbon tetrachloride

Administration of allylisopropylacetamide (AIA) or CCl 4 to rats previously treated with phenobarbital leads to a rapid decrease in cytochrome P 450 within 1 hr. The amount of cytochrome b 5 and NADPH cytochrome c reductase in liver microsomes remains unchanged following AIA treatment. In contrast, CCl 4 administration causes a decrease in total microsomal protein thus leading to a net loss in cytochrome b 5 and NADPH cytochrome c reductase. By using 3H-δ-aminolevulinic acid to label microsomal cytochrome P 450 heme, the effect of AIA and CCl 4 on this cytochrome was shown to be caused by destruction of preexisting CO-binding pigment and not from inhibition of synthesis. In addition, the breakdown products of cytochrome P 450 heme accumulate in the liver after AIA or CCl 4 treatment.

The possible relationship between heme synthesis and mitochondrial biogenesis

Administration of allylisopropylacetamide (AIA) 1 1 Abbreviations used: ALA, δ-aminolevulinic acid; AIA, allylisopropylacetamide. to rats causes a rapid induction of hepatic δ-aminolevulinic acid (ALA) synthetase. Liver mitochondria obtained from animals after AIA treatment had a 50–60% greater rate of amino acid incorporation in vitro as compared to mitochondria from control animals. Similarly, pulselabeling by ( 3H) leucine in vivo was stimulated 50% in submitochondrial fractions containing proteins insoluble in acetic acid and a membranous preparation of cytochrome oxidase. Previous studies had indicated that 15–20% of the proteins in these fractions were labeled in vivo by a cycloheximide-insensitive system, presumably that of the mitochondria ( Fed. Eur. Biol. Soc. Lett. 9, 232 1970). In time-course studies, it was observed that increased incorporation of ( 14C)glycine into heme in vivo followed shortly after ALA synthetase induction. A slight decay occurred before the incorporation of ( 3H)leucine into cytochrome oxidase or total mitochondrial protein was stimulated. At these latter times a 20–30% increase in the cytochrome content was also observed. Administration of aminotriazole, an inhibitor of ALA dehydratase and hence overall heme synthesis, prevented the stimulation of leucine incorporation in vivo and the increase in cytochrome content observed after AIA treatment. These results suggest that mitochondrial protein synthesis and cytochrome formation may be stimulated when heme biosynthesis is increased due to ALA synthetase induction.

Catabolism of heme in vivo: comparison of the simultaneous production of bilirubin and carbon monoxide.

The quantitative relationship between the catabolism of heme and the formation of bilirubin and carbon monoxide (CO) was studied in untreated rats and in animals treated with phenobarbital or the porphyrogenic drug, allylisopropylacetamide (AIA). A novel metabolic chamber permitting continuous collection of the bile and breath was utilized for quantitation of bilirubin-(14)C and (14)CO after the administration of hematin-(14)C or glycine-(14)C. After intravenous infusion of hematin-(14)C, control and phenobarbital-treated rats produced equimolar amounts of labeled bilirubin and CO; a minor fraction of the infused radioactivity appeared in the bile in other metabolites. The equimolar relationship in the formation of bilirubin and CO was also observed after pulse-labeling with glycine-2-(14)C; in phenobarbital-treated rats both metabolites were formed at an increased rate as compared to controls. By contrast, AIA treatment reduced the fractional conversion of hematin-(14)C to bilirubin and CO; a major fraction of the infused radioactivity appeared in the bile in metabolites other than bilirubin. In addition, in AIA-treated animals the molar CO/bilirubin recovery ratio was consistently greater than 1.0. Comparable results were obtained in AIA-treated rats after pulse-labeling with glycine-2-(14)C. These findings suggest that (a) in control and phenobarbital-treated rats infused hematin and heme formed in the liver are converted predominantly to bilirubin and CO, appearing in equimolar amounts; only a minor fraction of the hematin is degraded to other metabolites; (b) treatment with phenobarbital results in a proportional increase in the formation of both bilirubin and CO, reflecting increased heme synthesis and degradation in the liver; and (c) treatment with the porphyrogenic drug AIA shifts the CO/bilirubin ratio in favor of the gas, and enhances the formation of nonbilirubin metabolites.

Tryptophan pyrrolase, tryptophan and tyrosine transaminase changes during allylisopropylacetamide-induced porphyria in the rat

Changes in liver tryptophan pyrrolase (EC 1.11.1.4) and in tyrosine and tryptophan transaminase (EC 2.6.1.5) activities after induction of experimental porphyria with allylisopropylacetamide (AIA) were studied in female rats of the Long-Evans strain. All three enzymic activities increased after a single injection of AIA (400 mg/kg) to intact animals. AIA treatment also increased tryptophan pyrrolase in adrenalectomized but not in hypophysectomized animals. Peak levels of enzyme were reached in about 8 hr after drug administration to intact animals. The magnitude of the change in intact animals was enhanced by a 16-hr fast preceding the AIA treatment. Acute administration of AIA also produced behavioral changes and increased urinary excretion of δ-aminolevulinic acid and porphobilinogen. Behavioral, enzymic and excretory changes produced by the drug progressively diminished in magnitude with continued drug treatment.