Effect of glucose on induction of delta-aminolevulinic acid synthase, ferrochelatase and cytochrome P-450 hemoproteins in isolated rat hepatocytes by phenobarbital and lead

In the present work we have been able to demonstrate that phenobarbital and lead exert an inducing effect on the biosynthesis of delta-aminolevulinic acid synthase, ferrochelatase and cytochrome P-450 hemoproteins in isolated rat hepatocytes of normal adult rats. Dibutyryl cyclic AMP enhances the induction effect produced by phenobarbital in this in vitro system. Glucose inhibits the induction of delta-aminolevulinic acid synthase and ferrochelatase. This repression effect can be reversed with increasing concentrations of dibutyryl cyclic AMP. No glucose effect was observed on the phenobarbital- and lead-mediated inductions of cytochrome P-450. he present results add more experimental evidence to support the concept that the last enzyme of the heme pathway is inducible, and as such may have a significant role in regulatory mechanisms of porphyrin and heme biosynthesis.     

Effect of glucose on induction of δ-aminolevulinic acid synthase,ferrochelatase and cytochrome P-450 hemoproteins in isolated rat hepatocytes by phenobarbital and lead

In the present work we have been able to demonstrate the phenobarbital and lead exert an inducing effect on the biosynthesis of δ-aminovulenic acid synthase, ferrochelatase and cytochrome P-450 hemoproteins in isolated rat hepatocytes of normal adult rats. Dibutyryl cyclic AMP enhances the induction effect produced by phenobarbital in this in vitro system. Glucose inhibits the induction of δ-aminolevulinic acid synthase and ferrochelatase. This repression effect can be reversed with increasing concentrations of dibutyryl cyclic AMP. No glucose effect was observed on the phenobarbital- and lead-mediated inductions of cytochrome P-450. The present results add more experimental evidence to support the concept that the last enzyme of the heme pathway is inducible, and as such may have a significant role in regulatory mechanisms of porphyrin and heme biosynthesis.     

Role of heme in phenobarbital induction of cytochromes P450 and 5-aminolevulinate synthase in cultured rat hepatocytes maintained on an extracellular matrix

When hepatocytes are cultured on matrigel, a reconstituted basement membrane matrix, mRNAs for cytochrome P450 class IIB1/2 and class III genes can be induced by treatment with phenobarbital. We took advantage of this new system to critically evaluate the role of heme as a regulator of these cytochromes P450 and of 5-aminolevulinate synthase (ALA-S), the rate-limiting enzyme in heme biosynthesis. Phenobarbital treatment of rat cultures increased the total amount of cytochrome P450, activities catalyzed by IIB1/2 (benzyloxy- and pentoxyresorufin O-dealkylases) and ALA-S activity, and ALA-S mRNA. Treatments with phenobarbital combined with succinyl acetone, an inhibitor of heme biosynthesis at the step of 5-aminolevulinate dehydrase, blocked the induction of the proteins for cytochrome P450IIB1/2 and cytochrome P450IIIAI, as indicated by spectral, immunological, and enzymatic assays. However, at the same time, succinyl acetone cotreatment failed to inhibit the induction of the mRNAs for cytochrome P450IIB1/2 and cytochrome P450IIIA. Lack of effect on the cytochrome P450 mRNAs was selective inasmuch as treatment with phenobarbital combined with succinyl acetone synergistically increased both ALA-S activity and ALA-S mRNA, presumably by blocking formation of heme, the feedback repressor of ALA-S. Indeed, the increase in ALA-S mRNA caused by the combined treatment was abolished by adding heme itself to the cultures. In contrast to earlier concepts, we conclude that in the intact hepatocyte, phenobarbital-induced cytochrome P450 induction is independent of changes in heme synthesis.     

Studies on regulatory mechanisms of heme biosynthesis in hepatocytes from experimental-diabetic rats

Isolated hepatocytes from rats with experimental diabetes exhibit increased content of cytochrome P-450 and cyclic AMP and normal activities of the regulatory enzymes delta-aminolevulinic acid synthase and ferrochelatase. The inducing effect exerted by phenobarbital on cytochrome P-450, delta-aminolevulinic acid synthase and ferrochelatase biosynthesis and cyclic AMP content in diabetic hepatic cells is markedly greater than that observed in normal hepatocytes. This stimulatory response is neither enhanced by added dibutyryl cyclic AMP nor repressed by glucose. The present results suggest that the heme pathway of diabetic hepatocytes is more susceptible to porphyrinogenic factors.     

Biochemical characterization of porphobilinogen deaminase-deficient mice during phenobarbital induction of heme synthesis and the effect of enzyme replacement

Acute intermittent porphyria (AIP) is a genetic disorder caused by a deficiency of porphobilinogen deaminase (PBGD), the 3rd enzyme in heme synthesis. It is clinically characterized by acute attacks of neuropsychiatric symptoms and biochemically by increased urinary excretion of the porphyrin precursors porphobilinogen (PBG) and 5-aminolevulinic acid (ALA). A mouse model that is partially deficient in PBGD and biochemically mimics AIP after induction of the hepatic ALA synthase by phenobarbital was used in this study to identify the site of formation of the presumably toxic porphyrin precursors and study the effect of enzyme-replacement therapy by using recombinant human PBGD (rhPBGD). After 4 d of phenobarbital administration, high levels of PBG and ALA were found in liver, kidney, plasma, and urine of the PBGD-deficient mice. The administration of rhPBGD intravenously or subcutaneously after a 4-d phenobarbital induction was shown to lower the PBG level in plasma in a dose-dependent manner with maximal effect seen after 30 min and 2 h, respectively. Injection of rhPBGD subcutaneously twice daily during a 4-d phenobarbital induction reduced urinary PBG excretion to 25% of the levels found in PBGD-deficient mice administered with only phenobarbital. This study points to the liver as the main producer of PBG and ALA in the phenobarbital-induced PBGD-deficient mice and demonstrates efficient removal of accumulated PBG in plasma and urine by enzyme-replacement therapy.     

Inhibition of the biosynthesis of uroporphyrinogen and heme in rat liver during obstructive jaundice produced by bile duct ligation

Altered hepatic microsomal drug metabolism has been reported to occur in afflicted with hyperbilirubinemia. Similarities of the chemical structures of hydroxymethylbilane, an intermediate in the biosynthesis of uroporphyrinogen, to bilirubin prompted investigations of the effect of bilirubin on the activity of uroporphyrinogen I synthase (porphobilinogen deaminase, EC 4.3.1.8) and the biosynthesis of heme. Bilirubin was found to be a reversible, noncompetitive inhibitor of uroporphyrinogen I synthase. The inhibition constant (Ki) for bilirubin was 1.5 microM. Bile acids had no effect on rat hepatic uroporphyrinogen I synthase activity. Hyperbilirubinemia was achieved in rats by biliary ligation in order to investigate whether elevated levels of bilirubin impair the biosynthesis of hepatic heme in vivo. The relative rate of heme biosynthesis, as measured by the rate of incorporation of delta-[4-14C]aminolevulinic acid into heme, was decreased 59% 24 h after biliary obstruction. The levels of hepatic microsomal heme and cytochrome P-450 were decreased by 43 and 40%, respectively, 72 h after biliary obstruction. The activities of hepatic delta-aminolevulinic acid synthase and uroporphyrinogen I synthase were increased by 39 and 46%, respectively, 72 h after biliary obstruction. During the 48- to 72-h period following biliary obstruction, the urinary excretion of porphobilinogen and uroporphyrin was increased 3.0- and 3.5-fold, respectively, whereas, the urinary excretion of delta-aminolevulinic acid was not altered. During this 48-to 72-h time interval following biliary obstruction, 100% of the uroporphyrin was excreted as isomer I. These results indicate that bilirubin is capable of depressing the biosynthesis of rat hepatic heme and thus cytochrome P-450-mediated drug metabolism by inhibition of the formation of uroporphyrinogen. These findings are a plausible mechanism for reports of impaired clearance of various drugs in patients afflicted with hyperbilirubinemic disease states.     

Activity of key enzymes of heme synthesis and degradation and contents of cytochromes b5 and P-450 in rat liver

The relation between the delta-aminolevulinate-synthase and heme-oxygenase activities and the contents of cytochromes b5 and P-450 in rat liver after phenobarbital and CoCl2 injections was studied. Two hours after a single injection of phenobarbital the delta-aminolevulinate-synthase activity is increased, showing a further rise after 24 hrs. The content of cytochrome b5 is not changed, while that of cytochrome P-450 is increased 24 hrs after the injection. The heme-oxygenase activity remains unaffected thereby. The increase in the enzyme activity and cytochrome P-450 content induced by phenobarbital is eliminated by a preliminary administration of actinomycin D. The administration of CoCl2 is accompanied by a decrease in the delta-aminolevulinate-synthase activity after 2 hrs and its further increase after 24 hrs. The heme-oxygenase activity shows a sharp rise 24 hrs after the injection. The rise in the delta-aminolevulinate-synthase activity induced by CoCl2 is removed by actinomycin D. CoCl2 decreases the content of cytochromes b5 and P-450 24 hrs after the injection. It is assumed that the correlation between the delta-aminolevulinate-synthase activity and cytochrome P-450 content is observed only in the case when the heme-oxygenase activity is not increased. The cytochrome b5 content is independent of the changes in the activity of the key enzyme of heme synthesis and depends to a certain extent on the rate of heme degradation by heme-oxygenase.     

Induction of delta-aminolevulinate synthase and cytochrome P-450 hemoproteins in hepatocyte culture. Effect of glucose and hormones

Addition of glucose to cultured chick embryo hepatocytes caused a concentration-dependent impairment of phenobarbital-mediated induction of delta-aminolevulinate (ALA) synthase resembling the "glucose effect" observed in rodents in vivo. This glucose effect occurred in the complete absence of extrahepatic factors such as serum and hormones. Fructose, glycerol, and lactate mimicked the inhibitory glucose effect on ALA synthase induction, whereas 2-deoxyglucose and 3-O-methylglucose augmented the induction evoked by phenobarbital. 2-Deoxyglucose reversed the effect of glucose, glycerol, and lactate on ALA synthase induction suggesting that the glucose effect is mediated by free glucose or glucose 6-phosphate or a nonglycolytic metabolite of glucose 6-phosphate. The phenobarbital-mediated induction of cytochrome P-450 hemoprotein(s) and its monooxygenase function were concomitantly diminished by glucose. However, this inhibitory effect or glucose was reversible by the addition of exogenous heme or ALA suggesting that the primary target of the glucose effect is ALA synthase induction and not synthesis of apocytochrome P-450. Glucagon and dibutyryl cAMP enhanced the induction of ALA synthase and cytochrome P-450 by phenobarbital and partially counteracted the glucose effect on both enzymes suggesting that the glucose effect may be mediated by changes in cAMP levels. Although insulin did not alter induction of ALA synthase, it impaired induction of cytochrome P-450 even in the presence of glucagon and cAMP. These data may be relevant for the treatment with glucose and heme of patients with "inducible" hepatic porphyria.     

The role of heme in the regulation of tryptophan-2,3-dioxygenase activity and content of cytochrome P-450 in rat liver

The effects of exogenous heme on the activity of delta-aminolevulinate synthase, heme oxygenase, tryptophan-2.3-dioxygenase and microsomal cytochrome content in rat liver were studied. It was shown that hemin chloride diminishes the delta-aminolevulinate synthase activity and provokes heme oxygenase induction. This is paralleled with the induction of the tryptophan 2.3-dioxygenase apoenzyme and an increase in the saturation of the enzyme with heme. The cytochrome b5 content does not change thereby, whereas that of cytochrome P-450 shows a decrease. Upon combined administration of actinomycin D and hemin the cytochrome P-450 level is markedly increased. Actinomycin D by itself has no effect on the hemoprotein concentration. It is concluded that the increase in the cytochrome P-450 level results from the activation of heme-induced mRNA translation.     

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.     

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.     

Coordinate elevations of liver delta-aminolevulinate synthase and cytochrome P-450 RNA by phenobarbital in chicken embryos: the effects of heme

1. The role of heme in the coordinate elevations of liver delta-aminolevulinate (ALA) synthase activity and microsomal cytochrome P-450 concentration induced by phenobarbital (PB) was investigated in the chicken embryo. 2. Eighteen day old chicken embryos were given PB, and the changes in liver content of PB-inducible cytochrome P-450 RNA and of ALA synthase RNA were determined at different times after exposure to the drug. 3. The concentrations of both types of RNA increased rapidly after PB administration, and by 9 hr the level of ALA synthase RNA was 55-fold higher than control and that of cytochrome P-450 RNA was 7-fold higher than normal. 4. While the rate of increase in ALA synthase activity paralleled closely that of the enzyme's RNA concentration, the rate of increase of spectrally active cytochrome P-450 concentration in microsomes lagged behind that of the apoprotein's RNA by several hours. 5. To test whether heme depletion was responsible for the coordinate inductions of the two enzymes, embryos were loaded with ALA 2 hr before exposure to PB. 6. The protocol led to a drop in the PB-inducible ALA synthase RNA concentration and to an increase in that of cytochrome P-450 RNA, measured 6 hr after drug administration. 7. In primary cultures of hepatocytes, hemin in the culture medium caused a modest drop in ALA synthase RNA concentration but had a variable effect on that of cytochrome P-450 RNA in cells incubated with PB for 9 hr.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of phenobarbital-induced cytochrome P-450 in rat hepatic microsomes using an enzyme-linked immunosorbent assay

The major phenobarbital-inducible form of cytochrome P-450 (cytochrome P-450 PB) was purified to homogeneity from rat liver microsomes and rabbit antibodies prepared against the purified enzyme. Using these antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of cytochrome P-450 PB in microsomes which was sensitive at the nanogram level. The content of cytochrome P-450 PB was determined in hepatic microsomes from rats treated with various xenobiotics. Phenobarbital and Aroclor 1254 pretreatments resulted in several-fold increases in immunoreactive cytochrome P-450 PB over control levels. ELISA measurements of cytochrome P-450 PB were also carried out over a 48-h time course of phenobarbital induction in liver microsomes. Significant increases over control levels were seen at 16 h and beyond. Measurements of ELISA-detectable cytochrome P-450 PB were made in microsomes following the administration of CCl4 to phenobarbital-pretreated rats. Immunoreactive cytochrome P-450 PB was observed to decrease less rapidly than the spectrally detectable enzyme in the microsomal membranes. Inhibition of heme synthesis was carried out by the administration of 3-amino-1,2,4-triazole (AT) to rats. Concomitant pretreatment with phenobarbital and AT resulted in levels of ELISA-detectable cytochrome P-450 PB which were significantly increased over control levels, while spectrally detectable levels of total holoenzyme remained unchanged. These results support the idea that this cytochrome P-450 may exist, at least partly, in the microsomal membrane in an inactive or apoprotein form.     

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.     

Degradation of rat hepatic cytochrome P-450 heme by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine to irreversibly bound protein adducts

Administration of 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) (a structural analog of the dihydropyridine Ca2+ antagonists) to untreated, phenobarbital-, or dexamethasone-pretreated rats results in time-dependent losses of hepatic cytochrome P-450 content. Functional markers for various cytochrome P-450 isozymes have permitted the identification of P-450h, P-450 PB-1/k, and P-450p as the isozymes inactivated preferentially by the drug. DDEP-mediated cytochrome P-450 destruction may be reproduced in vitro, is most prominent after pretreatment of rats with dexamethasone, pregnenolone 16 alpha-carbonitrile or phenobarbital, and is blocked by triacetyloleandomycin. These findings together with the observation that DDEP markedly inactivates hepatic 2 beta- and 6 beta-testosterone hydroxylase and erythromycin N-demethylase tend to indict the steroid-inducible P-450p isozyme as a key protagonist in this event. The precise mechanism of such DDEP-mediated P-450p heme destruction is unclear, but involves prosthetic heme alkylation of the apocytochrome at its active site in what appears to be a novel mechanism-based "suicide" inactivation. Such inactivation appears to involve fragmentation of the heme to reactive metabolites that irreversibly bind to the protein, but the chemical structure of the heme-protein adducts is yet to be established. Intriguingly, such DDEP-mediated P-450p destruction in vivo also results in accelerated loss of immunochemically detectable apocytochrome P-450p. It remains to be determined whether or not this loss is due to enhanced proteolysis triggered by the structural modification of the apocytochrome.     

Reversible transfer of heme between different molecular species of microsome-bound cytochrome P-450 in rat liver

Incorporation of newly synthesized heme into microsome-bound cytochrome P-450 in rat liver was not affected by cycloheximide administration to the animals, indicating that the heme incorporation into cytochrome P-450 is not tightly coupled with the synthesis of the apo-cytochrome. When the heme of microsomal cytochrome P-450 had been labeled in vivo with delta-[14C]aminolevulinic acid, and then the animals were treated with phenobarbital (PB) or 3-methylcholanthrene (MC), PB-induced or MC-induced form of cytochrome P-450 was found to contain labeled heme derived from preexistent cytochrome P-450. These observations indicated that the heme of microsome-bound cytochrome P-450 is not tightly associated with the protein portion, and exchanges reversibly between different molecular species of cytochrome P-450 in vivo.     

Hepatic cytochrome P-450 in chronically hypoxemic rats

The basal level of hepatic cytochrome P-450 and its inducibility by phenobarbital pretreatment have been found to be enhanced by chronic hypoxemia. Pentobarbital sleeping times were decreased in parallel to changes in levels of cytochrome P-450. The increase in level of hepatic cytochrome P-450 in chronically hypoxemic rats occurred despite the increased levels of hepatic heme oxygenase which previously were associated with the chronic hemoglobinemia of the hypoxemic state. Chronically hypoxemic rats may provide a useful model for study of control of hepatic heme and hemoprotein metabolism.     

Maintenance and induction of cytochrome P-450 in cultured rat hepatocytes

Maintenance of microsomal cytochrome P-450 content by cultured rat hepatocytes has proven an elusive goal. It is reported here that exogenous heme maintains cytochrome P-450 content of cultured rat hepatocytes at high levels during the first 72 h of incubation. The maintenance studies have been expanded to demonstrate the in vitro induction of cytochrome P-450 by phenobarbital treatment. The induction of P-450 in vitro by phenobarbital required the trace element, selenium, in the presence of exogenous heme. The present findings suggest that selenium, and other trace elements, may have an essential role in the formation of holocytochrome P-450 in vitro.     

Control of cytochromes P450 expression in Gunn rat liver: implication of the intracellular heme pool

The absence of changes in the overall hepatic cytochrome P450 content after administration of 3-methylcholanthrene (MC) to congenitally jaundiced Gunn rats is believed to be related to a limited heme availability in this strain of rat. The amount of available heme, estimated by tryptophan pyrrolase activity, shows a substantial decrease in control Gunn versus control Wistar rats. This reduction is moderately enhanced by MC treatment in Gunn rats but is abolished after phenobarbital administration. Heme oxygenase activity is diminished in Gunn rats and consequently is not responsible for the decrease in the hepatic heme availability. These data point out that the depletion of the intracellular heme can lead to a limitation in the synthesis of cytochrome P450 isoenzymes in the MC-induced Gunn rat.