Control of delta-aminolaevulinate synthase and haem oxygenase in chronic-iron-overloaded rats.

The hepatic porphyrias are inborn errors of porphyrin and haem biosynthesis characterized biochemically by excessive excretion of delta-aminolaevulinate (ALA), porphobilinogen and other intermediates in haem synthesis. Clinical evidence has implicated iron in the pathogenesis of several types of genetically transmitted diseases. We investigated the role of iron in haem metabolism as well as its relationship to drug-mediated induction of ALA synthase and haem oxygenase in acute and chronic iron overload. Acute iron overload in rats resulted in a marked increase in hepatic haem oxygenase that was associated with a decrease in cytochrome P-450 and an increase in ALA synthase activity. Aminopyrine N-demethylase and aniline hydroxylase activities, which are dependent on the concentration of cytochrome P-450, were also decreased. In contrast, in chronic-iron-overloaded rats, there was an adaptive increase in haem oxygenase activity and an increase in ALA synthase that was associated with normal concentrations of microsomal haem and cytochrome P-450. The induction of ALA synthase in chronic iron overload was enhanced by phenobarbital and allylisopropylacetamide, in spite of the fact that these agents did not increase haem oxygenase activity. Small doses of Co2+ were potent inducers of the haem oxygenase in chronic-iron-overloaded, but not in control, animals. We conclude that increased hepatic cellular iron may predispose certain enzymes of haem synthesis to induction by exogenous agents and thereby affect drug-metabolizing enzyme activities.     

The effect of fluroxene [(2,2,2-trifluoroethoxy)ethane] on haem biosynthesis and degradation

Acute fluroxene treatment of male Wistar rats decreases the amounts of hepatic microsomal cytochrome P-450 and haem, increases the activities of hepatic delta-aminolaevulinate synthase and haem oxygenase, and increases the amounts of haem precursors (delta-aminolaevulinate and porphobilinogen) in the urine. All of the above effects of fluroxene are enhanced by pretreatment of the experimental animals with 3-methylcholanthrene and phenobarbital. The amounts of porphyrins in the urine and faeces were generally unaffected by acute fluroxene treatment of uninduced or 3-methylcholanthrene- or phenobarbital-induced Wistar rats. 2,2,2-Trifluoroethyl ethyl ether, the saturated analogue of fluroxene, did not affect the amounts of hepatic cytochrome P-450 and haem, the amounts of any of the haem precursors in the urine or faeces, or the activity of hepatic haem oxygenase in phenobarbital-induced male Wistar rats. The amounts of hepatic cytochrome P-450 and haem and of the haem precursors in urine and faeces, and the activity of delta-aminolaevulinate synthase, were generally not altered by acute fluroxene treatment of uninduced male Long-Evans rats. Chronic treatment of Wistar rats with fluroxene resulted in small increases in the amounts of delta-aminolaevulinate and porphyrins in urine. The amounts of porphobilinogen in urine were elevated up to 2000%, whereas the amounts of the porphyrins in faeces were generally unaffected. After chronic fluroxene treatment, the activity of delta-aminolaevulinate synthase was increased, whereas the activity of uroporphyrinogen synthase was decreased. It is concluded that acute fluroxene treatment may affect haem biosynthesis and degradation by a mechanism similar to allylisopropylacetamide, namely by stimulating an atypical cytochrome P-450-dependent pathway for haem degradation. The effects of chronic fluroxene treatment on haem biosynthesis may be a consequence of this mechanism or a result of the inhibition by fluroxene of uroporphyrinogen synthase. Chronic fluroxene treatment of male rats affects the haem biosynthetic pathway in a manner similar to that seen in human genetic acute intermittent porphyria.     

Effect of endotoxin on tryptophan pyrrolase and delta-aminolaevulinate synthase: evidence for an endogenous regulatory haem fraction in rat liver.

Endotoxin was administered to rats at a dose shown previously to stimulate hepatic haem oxygenase activity and to block induction of delta-aminolaevulinate synthase, apparently by causing redistribution of haem from cytochrome P-450 to a regulatory haem pool in the liver. Within 5h of the administration of endotoxin (at a time when the effect of the compound on cytochrome P-450 is maximal) the relative saturation of tryptophan pyrrolase with intrinsic haem rose, from a basal value of 50% to 90%, indicating that 'free' haem had become available. Concurrently, the activity of delta-aminolaevulinate synthase was decreased to 25% of its basal value. Haem oxygenase reached peak activity 13h after endotoxin administration. These findings provide new evidence for the existence of an 'unassigned' hepatic haem fraction, which exchanges with cytochrome P-450 haem and regulates these three enzyme functions.     

Metal ion interactions in the control of haem oxygenase induction in liver and kidney.

Mn2+ and Zn2+ exhibit a striking ability to block the induction by Sn2+ and Ni2+ of haem oxygenase (EC 1.14.99.3) in kidney. The blocking effects of Mn2+ and Zn2+ were found to be greatest on simultaneous administration, time-dependent when administered up to 8 h before the inducing metal ions, and ineffective when administered as little as 10 min after the inducing metal ions. The decreases in cytochrome P-450 and haem contents and the sequential changes in delta-aminolaevulinate synthase (EC 2.3.1.37) activity that occur concomitant with haem oxygenase induction were largely eliminated with simultaneous or prior treatment with Mn2+ or Zn2+, but not when Mn2+ or Zn2+ was administered after Sn2+ or Ni2+. Mn2+ and Zn2+ did not increase the catabolism of the enzyme in vivo. Zn2+ on simultaneous administration was also able substantially to block the induction of haem oxygenase by Co2+, Cd2+ and Ni2+ in liver. The Zn2+ blockade of Cd2+ induction was examined in detail, and prior or simultaneous administration of Zn2+ was found to be effective in blocking the induction of haem oxygenase and the concomitant decreases in cytochrome P-450 and haem contents, ethylmorphine demethylase activity and the sequential changes in delta-aminolaevulinate synthase activity. Zn2+ administration 10 min or more after Cd2+ was ineffective in preventing the occurrence of these perturbations in haem metabolism. These findings describe a new and striking biological property of Mn2+ and Zn2+, and indicate the existence of significant metal ion interactions in the control of haem metabolism.     

Accelerated hepatic haem catabolism in the selenium-deficient rat.

1. Hepatic microsomal cytochrome P-450 concentrations are lower in selenium-deficient rats treated with phenobarbital for 4 days than in similarly treated control rats. 2. No defect in haem synthesis was found on the basis of measurements of delta-aminolaevulinate synthase (EC 2.3.1.37), delta-aminolaevulinate dehydratase (EC 4.2.1.24) and ferrochelatase (EC 4.99.1.1) activities, and urinary excretion of delta-aminolaevulinate, porphobilinogen, uroporphyrin and coproporphyrin. 3. No defect in apo-(cytochrome P-450) separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. An increase in haem catabolism was found. An 8-fold increase in hepatic microsomal haem oxygenase (EC 1.14.99.3) activity occurred in selenium-deficient rats after phenobarbital treatment, compared with a less than 2-fold increase in control rats. Also excretion of 14CO in the breath after administration of delta-amino[5-14C]laevulinate was greater by phenobarbital-treated selenium-deficient rats than by similarly treated controls. 5. These studies demonstrate that the defective induction of cytochrome P-450 by phenobarbital in selenium-deficient rats is accompanied by increased haem catabolism. This could be due to increased breakdown of cytochrome P-450 or to catabolism of haem before it attaches to the apo-cytochrome. The role of selenium in stabilizing cytochrome P-450 and/or in protecting haem from breakdown remains to be determined.     

Evidence that in chick embryos destruction of hepatic microsomal cytochrome P-450 haem is a general mechanism of induction of delta-aminolaevulinate synthase by porphyria-causing drugs.

A variety of prophyrinogenic compounds were tested for their effect in ovo on chick-embryo liver microsomal cytochrome P-450 haem concentration and mitochondrial delta-aminolaevulinate synthase activity. With all drugs tested, there was a 30--50% decrease in cytochrome P-450 haem concentration within 1 h of treatment, and this was closely followed by an increase in delta-aminolaevulinate synthase activity. The relationship was independent of the extent of enzyme induction and is consistent with the proposal that drug-mediated destruction of cytochrome P-450 haem is the primary mechanism of induction of delta-aminolaevulinate synthase. After induction, synthesis of delta-aminolaevulinate synthase could be maintained by inhibiting further haem synthesis. These studies suggest that induction of porphyria is a combination of two distinct processes: (a) induction of delta-aminolaevulinate synthase synthesis by destruction of cytochrome P-450 haem and consequent depletion of cellular free haem; (b) maintenance of continued delta-aminolaevulinate synthase synthesis by preventing replenishment of cellular haem either by inhibiting haem synthesis and/or by promoting continuous removal of newly synthesized haem.     

A model for the regulation of δ-aminolaevulinate synthetase induction in rat liver

A reciprocal relationship exists between the cytochrome P-450 content and delta-aminolaevulinate synthetase activity in adult rats. In young rats the basal delta-aminolaevulinate synthetase activity is higher and the cytochrome P-450 content is lower compared with the adult rat liver. Administration of allylisopropylacetamide neither induces the enzyme nor causes degradation of cytochrome P-450 in the young rat liver, unlike adult rat liver. Allylisopropylacetamide fails to induce delta-aminolaevulinate synthetase in adrenalectomized-ovariectomized animals or intact animals pretreated with successive doses of the drug, in the absence of cortisol. The cortisol-mediated induction of the enzyme is sensitive to actinomycin D. Allylisopropylacetamide administration degrades microsomal haem but not nuclear haem. Haem does not counteract the decrease in cytochrome P-450 content caused by allylisopropylacetamide administration, but there is evidence for the formation of drug-resistant protein-bound haem in liver microsomal material under these conditions. Phenobarbital induces delta-aminolaevulinate synthetase under conditions when there is no breakdown of cytochrome P-450. On the basis of these results and those already published, a model is proposed for the regulation of delta-aminolaevulinate synthetase induction in rat liver.     

Induction of liver cell haem oxygenase in iron-overloaded rats.

Rats were chronically iron-overloaded by intraperitonel injections of iron-dextran. Electron microscopy revealed that the excess iron was deposited in ferritin-like particles packed in lysosomes and scattered in hepatic cytoplasm. No mitochondrial iron deposition or damage was seen. Furthermore, mitochondrial preparations from chronically iron-overloaded animals were found to be contaminated with lysosomes, which could explain previously reported increases in mitochondrial iron by chemical analysis. Mitochondrial function, as measured by cytochromes a-a3, b and c concentrations as well as activity of the rate-limiting enzyme of haem synthesis, delta-aminolaevulinate synthetase, was not diminished by chronic iron-overloading. Microsomal haem was decreased by 30% at the time that haem oxygenase, the rate-limiting enzyme of haem degradation, was increased approx. 3-fold. Animals were given a single intraperitoneal injection of iron-dextran and the activities of delta-aminolaevulinate synthetase and haem oxygenase were measured over 24 h. delta-Aminolaevulinate synthetase activity increased approx. 2-fold in these acutely iron-overloaded rat livers, but at a time after the increase in haem oxygenase. These results suggest that an early consequence of excess iron in liver is acceleration of the rate of haem degradation, possible by haem oxygenase.     

The effect of exogenous δ-aminolaevulinate on rat liver haem and cytochromes

The activity of delta-aminolaevulinate synthetase is generally regarded as rate-limiting for hepatic haem biosynthesis. It has been suggested that cytochrome synthesis may also be regulated by changes in delta-aminolaevulinate synthetase activity. This hypothesis was studied by injecting product, delta-aminolaevulinate, into adult rats over a 4-240h period. The concentrations of hepatic mitochondrial cytochromes a, b, c and c(1) were unchanged by treatment with delta-aminolaevulinate, allylisopropylacetamide or phenobarbital. In control animals, total microsomal haem content equalled the sum of cytochromes b(5) plus P-450. After delta-aminolaevulinate administration the total amount of microsomal haem, measured as the pyridine haemochromogen, exceeded these components, indicating the formation of a ;free' haem pool. Haem synthesis does not appear rate-limiting for hepatic cytochrome synthesis in the adult rat.     

The relationship between δ-aminolaevulinate synthetase induction and the concentrations of cytochrome P-450 and catalase in rat liver

The porphyrinogenic drug 2-allyl-2-isopropylacetamide causes the degradation of microsomal cytochrome P-450 and inhibits the synthesis of catalase in rat liver. The inhibition of catalase synthesis follows the induction of delta-aminolaevulinate synthetase and the consequent overproduction of haem. The allylisopropylacetamide-mediated breakdown of cytochrome P-450 is a rapid event and has a reciprocal relationship to the pattern of delta-aminolaevulinate synthetase induction. Breakdown of cytochrome P-450 appears to be one of the conditions leading to the ;derepression' of delta-aminolaevulinate synthetase.     

Prolonged induction of hepatic haem oxygenase and decreases in cytochrome P-450 content by organotin compounds.

The administration of organotin compounds to rats in single doses causes a significant and prolonged induction of haem oxygenase and a sustained decrease in haemoprotein content in the liver. The extent of induction of hepatic haem oxygenase varied between 3 and 5-fold at 72h after a single injection of water-insoluble organotins of differing structure. The alterations in haem metabolism produced by tricyclohexyltin hydroxide were studied in detail. The effects were dose-dependent, with doses as low as 3.75 mg/kg body wt. resulting in significant induction of haem oxygenase and a decrease in cytochrome P-450 and cytochrome b5 contents at 72h in the liver. The effects with time of a single dose of tricyclohexyltin on various parameters of liver haem metabolism were also examined. The organotin produced a substantial and very prolonged induction of haem oxygenase accompanied by a steady decline in cytochrome P-450 content for periods up to 8 days. The long duration of action of these organotins with respect to induction of haem oxygenase and depletion of cellular haemoprotein content provides a highly sensitive metabolic system with which to define further the toxic potential of organometals as well as to study the adaptive responses in liver to long-term perturbations of haem metabolism by foreign chemicals.     

Induction of hepatic and renal ornithine decarboxylase by cobalt and other metal ions in rats.

We previously showed that Cd2+ is able to induce hepatic and renal ornithine decarboxylase (ODC). In addition to Cd2+, the administration of Co2+ and other metal ions such as Se2+, Zn2+ and Cr2+ produced a significant increase of hepatic and/or renal ODC activity. Of the metal ions used in this study, Co2+ produced the greatest increase of ODC activity. The maximum increases in hepatic and renal ODC activity, to respectively 70 and 14 times the control values in male rats, were observed 6 h after the administration of Co2+. A similar response was seen in the liver, but not in the kidney, of female rats. Thereafter, ODC activity gradually returned to control values in the liver, but it was profoundly decreased to 7% of the control value at 24 h in the kidney. The pretreatment of animals with either actinomycin D or cycloheximide almost completely blocked the Co2+-mediated increase of ODC activity. Co2+ complexed with either cysteine or glutathione (GSH) failed to induce ODC. Depletion of hepatic GSH content by treatment of rats with diethyl maleate greatly enhanced the inducing effect of Co2+ on ODC. The inhibitors of ODC, 1,3-diaminopropane and alpha-difluoromethylornithine, were able to inhibit the induction of the enzyme, without affecting the induction of haem oxygenase by Co2+. Methylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, significantly inhibited the Co2+-mediated induction of both ODC and haem oxygenase. It is suggested that the inducing effects of Co2+ on ODC and haem oxygenase are brought about in a similar manner.     

Regulation of hepatic haem metabolism. Disparate mechanisms of induction of haem oxygenase by drugs and metals.

We studied drug- and metal-mediated increases in activity of haem oxygenase, the rate-controlling enzyme for haem breakdown, in chick-embryo hepatocytes in ovo and in primary culture. Phenobarbitone and phenobarbitone-like drugs (glutethimide, mephenytoin), which are known to increase concentrations of an isoform of cytochrome P-450 in chick-embryo hepatocytes, were found to increase activities of haem oxygenase as well. In contrast, 20-methylcholanthrene, which increases the concentration of a different isoform of cytochrome P-450, had no effect on activity of haem oxygenase. Inhibitors of haem synthesis, 4,6-dioxoheptanoic acid or desferrioxamine, prevented drug-mediated induction of both cytochrome P-450 and haem oxygenase in embryo hepatocytes in ovo or in culture. Addition of haem restored induction of both enzymes. These results are interpreted to indicate that phenobarbitone and its congeners induce haem oxygenase by increasing hepatic haem formation. In contrast, increases in haem oxygenase activity by metals such as cobalt, cadmium and iron were not dependent on increased haem synthesis and were not inhibited by 4,6-dioxoheptanoic acid. We conclude that (1) induction of hepatic haem oxygenase activity by phenobarbitone-type drugs is due to increased haem formation, and (2) induction of haem oxygenase by drugs and metals occurs by different mechanisms.     

Regional distribution of the enzymes of haem biosynthesis and the inhibition of 5-aminolaevulinate synthase by manganese in the rat brain

The activity of 5-aminolaevulinate synthase, the rate-limiting enzyme of haem biosynthesis, is differentially distributed in various regions of the rat brain. The cerebellum possessed the highest enzyme activity of the eight regions studied. The cerebral cortex and the midbrain also exhibited high 5-aminolaevulinate synthase activity; the septum, hypothalamus, thalamus, amygdala and the hippocampus possessed much lower enzyme activity. However, the total porphyrin and haem contents of the different brain segments did not vary greatly. Mn²⁺, when administered subcutaneously to rats, effectively inhibited the activity of 5-aminolaevulinate synthase in the cerebellum, midbrain and cerebral cortex; however, repeated injections of the metal ion neither decreased the haem and porphyrin contents of the brain nor induced haem oxygenase activity. Mn²⁺ was not an effective inhibitor of 5-aminolaevulinate synthase activity in vitro. On the other hand, studies carried out with the liver in vivo suggested that Mn²⁺ may alter the turnover rate of cellular haem and haemoproteins. In that event, it is likely that the inhibition of 5-aminolaevulinate synthase by Mn²⁺ was in part a result of the inhibition of protein synthesis by the metal ion. It is postulated that the haem and porphyrin contents of the brain are maintained at a steady-state level, due in part to the refractoriness to inducers of the regulatory mechanism for haem catabolic enzymes and in part to the ability of the organ to utilize haem precursors derived from extraneuronal sources.     

Metal induction of haem oxygenase without concurrent degradation of cytochrome P-450. Protective effects of compound SKF 525A on the haem protein.

The induction of hepatic haem oxygenase (EC 1.14.99.3) by a series of metals, organometals and metalloporphyrins was examined in vivo in the presence of compound SKF 525A, which is known to complex with the prosthetic group of cytochrome P-450. Concurrent administration of SKF 525A and an inducing metal did not affect the extent and time course of haem oxygenase induction. The decrease in cytochrome P-450 content normally associated with metal administration was, however, prevented, indicating that haem oxygenase induction by metals can proceed without the significant labilization of the haem moiety of cytochrome P-450. In addition, the integrity of this haem protein can be maintained by chemical means in the presence of sustained high activities of haem oxygenase.     

Mechanism of hexachlorobenzene-induced porphyria in rats. Effect of phenobarbitone pretreatment.

The effect of a pretreatment with phenobarbitone (PB) on the porphyrinogenic action exerted by hexachlorobenzene (HCB) was examined in female rats. Kinetic studies of enzyme function after HCB poisoning showed that porphyrinogen carboxy-lyase was the only enzyme of haem biosynthesis that markedly lowered its activity. Both stages of uroporphyrinogen (UPG) III decarboxylation were decreased. This enzyme, together with UPG I synthase (increased levels) were the first enzymes altered. Subsequently, an increase in delta-aminolaevulinate (AmLev) synthase and ferrochelatase was detected; AmLev dehydratase was the last to increase. On long-term exposure, PB alone did not modify the basal values of haem intermediates; only the content of cytochrome P-450 increased. All the enzyme activities studied showed no significant changes, except ferrochelatase, which increased. With both drugs the metabolic impairment promoted by HCB was accelerated and enhanced by prior PB treatment leading to the onset of an earlier and stronger porphyria. A more noticeable accumulation and excretion of higher carboxylated porphyrins and precursors was more promptly observed as a consequence of the early porphyrinogen carboxy-lyase blockade and the concomitant induction of AmLev synthase. Although the enzymic activities of both AmLev dehydratase and ferrochelatase were enhanced, this response differed in time. For UPG I synthase this pretreatment elicited lower values than those found in the HCB group. Cytochrome P-450 contents were immediately and slightly enhanced by all the drugs, but the values for the combined treatment were the lowest. Of the several hypotheses that could explain the action of HCB on the haem pathway, our results would suggest that the porphyrinogenic action of HCB is mediated by some of its metabolic products.     

The effects of acute-phase inducers and dimethyl sulphoxide on delta-aminolaevulinate synthase activity in human HepG2 hepatoma cells.

The effects of acute-phase inducers and dimethyl sulphoxide (Me2SO) on delta-aminolaevulinate (ALA) synthase in HepG2 cells were examined. Treatment of cells with Me2SO resulted in a significant increase in ALA synthase activity. Interleukin-6 increased ALA synthase activity only slightly, but it substantially potentiated the induction of ALA synthase by Me2SO. These data suggest that ALA synthase activity in liver is altered during acute-phase reactions.     

Level of haem oxygenase does not obligatorily reflect the sensitivity of PC12 cells to an oxidative shock induced by glutathione depletion

In order to investigate the function of haem oxygenase in neuronal cell death or survival, we have determined in PC12 cells whether induction of haem oxygenase mRNA and protein or inhibition of haem oxygenase activity may be able to modulate the cell response to an oxidative stress. Inhibition of glutathione biosynthesis by buthionine sulfoximine (BSO) has indeed been demonstrated, in this cell line, to decrease the intracellular content of glutathione and to trigger a gradual and programmed cell death. Inhibition of haem oxygenase by zinc protoporphyrin IX, a potent inhibitor of this enzyme, or by a recently described peptidic inhibitor, induced a significant decrease in the toxicity of BSO. This protective action was not due to an alteration in the metabolism of glutathione and was still observed when the protecting agent was added several hours after BSO treatment. Induction of haem oxygenase-1 mRNA and protein by either haemin or pyrrolidine dithiocarbamate was associated with no protection or a significant reduction in the toxicity of BSO respectively. Our results indicate that induction of haem oxygenase-1 is not obligatorily associated with an improved resistance towards oxidative stress and suggest that a byproduct of haem degradation may also become detrimental.     

Identification of amino-acid substitutions in the proteolipid subunit of the ATP synthase from dicyclohexylcarbodiimide-resistant mutants of Escherichia coli

Polyadenylated mRNA was isolated from chick embryo liver following induction of hepatic porphyria. The RNA was translated in vitro using a wheat germ cell-free system and delta-aminolaevulinate synthase was identified in the translation products by indirect immunoprecipitation. The enzyme was not apparent in the translation products of polyadenylated RNA from non-induced livers. The molecular weight of delta-aminolaevulinate synthase synthesized in vitro was 70000 and the protein was estimated to represent up to 5% of total products synthesised in vitro. These data demonstrate for the first time that induction of chick embryo liver delta-aminolaevulinate synthase activity in hepatic porphyria correlates with a large increase in the translational capacity of isolated polyadenylated RNA for this enzyme and, together with preliminary cDNA . RNA hybridization studies, indicate that an increase in the level of delta-aminolaevulinic synthase mRNA is responsible.