Drug metabolizing enzyme system and heme pathway in hepatocarcinogenesis

Chemically induced and spontaneous liver tumors share some metabolic alterations. The decline in hemoprotein levels during hepatocarcinogenesis may result from a diminution of the intracellular heme pool. To elucidate if the onset of the pre-initiation stage alters the natural regulation mechanism of heme pathway, animals were fed with p-dimethylaminoazobenzene (DAB) and treated or not with 2-allylisopropylacetamide (AIA). The induction of 6-Aminolevulinic acid synthase (ALA-S) activity and the diminution in microsomal heme oxygenase (MHO) did not change when DAB fed animals were treated with AIA. Cytochrome P-450 (P-450) levels and glutathione S-transferase activity were increased in all the groups tested. Tryptophan pyrrolase, sulphatase and beta-glucuronidase activities were altered in DAB fed animals but AIA treatment did not produce any effect. Changes in drug metabolizing enzymes in livers of DAB fed animals could be the result of a primary deregulation of heme metabolism. These results give additional support to our hypothesis about a mechanism for the onset of hepatocarcinogenesis.     

Heme regulation in mouse mammary carcinoma and liver of tumor bearing mice--I. Effect of allyl-isopropylacetamide and veronal on delta-aminolevulinate synthetase, cytochrome P-450 and cytochrome oxidase

1. Basal levels and allyl-isopropylacetamide (AIA) or veronal induced levels of delta-aminolevulinate synthetase (ALA-S), cytochrome P-450 (cyt P-450) and cytochrome oxidase were determined in tumor (T) and liver of both normal mice (NM) and T bearing mice (TBM). 2. Basal levels of ALA-S were nearly the same in either source. The amount of cyt P-450 was lower in TBM liver than in NM liver, and no detectable in T. While the basal activity of cytochrome oxidase in TBM liver and T were higher than those of NM liver. 3. In AIA intoxicated animals there was a lower induction of ALA-S in liver of TBM than in NM liver. There was no induction in T ALA-S. The loss of cyt P-450 was less in TBM liver when compared with NM liver. 4. The induction level of cyt P-450 after veronal administration was nearly the same in liver of both TBM and NM. 5. We conclude that lower induction of liver ALA-S activity in TBM liver is due to correspondingly lower drug metabolism ability of TBM liver. Otherwise our results suggest that the control mechanism operating in T and probably in its original tissue are different from those described for normal liver.     

Spectroscopic evidence of interaction between 2-allyl-2-isopropylacetamide and cytochrome P-450 of rat liver microsomes

2-allyl-2-isopropylacetamide (AIA) causes marked induction of heme synthesis in rats and other species, degrades cytochrome P-450 in the presence of NADPH and causes experimental porphyria. Using difference spectroscopy we sought evidence of an interaction between AIA and P-450 in microsomes prepared from rat liver. AIA alone caused small and variable changes in the spectral properties of liver microsomes but markedly inhibited the Type I spectral change due to hexobarbitone. Phenobarbitone exhibited behaviour qualitatively similar to AIA. It is concluded that AIA binds to cytochrome P-450 without much altering its spectral properties but in such a way as to prevent the change induced by the Type I substrate hexobarbitone.     

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.     

Amplifying effect of dietary taurine on the induction of cytochrome P-450 and on the urinary excretion of ascorbic acid in rats fed on phenobarbital-containing diets

Dietary taurine amplified the induction of cytochrome P-450 and the urinary excretion of ascorbic acid in rats fed on phenobarbital (PB)-containing diets. These facts suggest that taurine could influence the hepatic metabolism of xenobiotics via the induction of drug-metabolizing enzymes (DME) and the ascorbic acid metabolism. Taurine might improve the function of DME exposed by some xenobiotics.     

Effect of ascorbic acid on heme metabolism in hepatic microsomes

Hepatic microsonal cytochrome P-450 levels are significantly decreased (46–68%) in ascorbic acid-deficient guinea pigs. Studies attempting to elucidate the mechanism responsible for decreased cytochrome P-450 demonstrated that ascorbic acid status did not influence the turnover $\text{(}\text{t}\text{1}\text{2}\text{)}$ or the degradation of hepatic cytochrome P-450 heme. Urinary excretion of Δ-aminolevulinic acid (ALA) and coproporphyrin was significantly decreased (30 and 69% respectively) in ascorbic acid-deficient guinea pigs. Injections (ip) of ALA into ascorbic acid-deficient guinea pigs were not effective in returning cytochrome P-450 levels to values found in ascorbic acid-supplemented guinea pigs. In addition, plasma and hepatic iron and blood heme were related directly, while hepatic copper and plasma copper or ceruloplasmin were related inversely, to the ascorbic-acid status of the guinea pig. These data, along with past investigations on heme synthesis in the ascorbic acid-deficient guinea pig, are consistent with mechanisms proposing that ascorbic acid may influence: 1) apocytochrome P-450 synthesis, 2) binding of heme and apo-cytochrome P-450 to form active cytochrome P-450, and/or 3) incorporation of Fe++ into the heme moiety of cytochrome P-450, perhaps via changes in copper metabolism.     

Multiple forms of cytochrome P-450. Characteristics of isolated aminopyrine-N-demethylase (cytochrome P-450AP)

A previously unidentified cytochrome P-450AP possessing the highest aminopyrine-N-demethylase activity has been isolated from liver microsomes of 4-isopropylaminoantipyrine-induced rats, using affinity chromatography in combination with ion-exchange chromatography with subsequent separation on hydroxyl apatite. Using radioisotope techniques, it was found that 4-isopropylaminoantipyrine induces cytochrome P-450AP synthesis de novo. The isolated cytochrome P-450AP has the following characteristics: Mr = 49,000 Da. CO-peak maximum at 450.5 mm, rate of aminopyrine demethylation in a reconstituted system-20 nmol HCHO/min/nmol of cytochrome P-450, benzphetamine-15. The hemoprotein synthesis is paralleled with the synthesis of a protein with Mr of 51,000 Da. Immunochemical analysis permitted to identify the latter protein as cytochrome P-450b. It was demonstrated that cytochrome P-450AP does not interact with the antibodies to the major phenobarbital-induced form, i.e., with cytochrome P-450b.     

Drug-induced protoporphyria in the olfactory mucosa of the hamster

Administration of 3,5-diethoxycarbonyl-4-ethyl-1,4-dihydro-2,6-dimethylpyridine (4-ethyl-DDC) to hamsters resulted in a marked loss of cytochrome P-450-dependent reactions (peroxidase, 7-ethoxycoumarin O-deethylase, and 7-ethoxyresorufin O-deethylase) in both liver and olfactory epithelium within 2 hr. This inactivation of cytochrome P-450 was accompanied by inhibition of ferrochelatase (FK), stimulation of 5-aminolevulinate synthase (ALA-S), and accumulation of protoporphyrin both in the liver and to a lesser degree, in the olfactory epithelium. These results suggest that the mechanism of induction of protoporphyria in nasal tissues is similar to that occurring in the liver, namely, suicidal metabolism of 4-ethyl DDC by cytochrome P-450 resulting in formation of N-ethylprotoporphyrin, a potent inhibitor of FK. The consequent depletion of heme leads to stimulation of ALA-S and, thus, porphyrin accumulation. Investigation of the dose-response to 4-ethyl DDC demonstrated that, in liver, maximal inhibition of FK and accumulation of protoporphyrin occurred at a dose of 50 mg/kg while ALA-S activity continued to increase up to a dose of 100 mg/kg. This is compatible with an additional effect of the drug on ALA-S involving induction of cytochrome P-450 and, thus, further depletion of heme. In the olfactory epithelium, stimulation of ALA-S was significantly less marked, suggesting that this secondary effect does not operate in nasal tissue. This is consistent with reports that olfactory cytochrome P-450s are noninducible.     

Ascorbic acid deficiency decreases hepatic cytochrome P-450, especially CYP2B1/2B2, and simultaneously induces heme oxygenase-1 gene expression in scurvy-prone ODS rats

The mechanisms underlying the decrease in hepatic cytochrome P-450 (CYP) content in ascorbic acid deficiency was investigated in scurvy-prone ODS rats. First, male ODS rats were fed a diet containing sufficient ascorbic acid (control) or a diet without ascorbic acid (deficient) for 18?days, with or without the intraperitoneal injection of phenobarbital. Ascorbic acid deficiency decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial cytochrome oxidase (COX) complex IV subunit I protein, and simultaneously increased heme oxygenase-1 protein in microsomes and mitochondria. Next, heme oxygenase-1 inducers, that is lipopolysaccharide and hemin, were administered to phenobaribital-treated ODS rats fed sufficient ascorbic acid. The administration of these inducers decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial COX complex IV subunit I protein. These results suggested that the stimulation of hepatic heme oxygenase-1 expression by ascorbic acid deficiency caused the decrease in CYP content in liver.     

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.     

Oxidative modification of cytochrome P-450 during its function. II. Study of the mechanism of cytochrome P-450 LM2 inactivation in a soluble reconstructed monooxygenase system

Inactivation of cytochrome P-450 LM2 induced by hydrogen peroxide formed in the active site of the enzyme was studied. Catalase did not protect cytochrome P-450 LM2 from inactivation during its operation in a soluble reconstituted system. The hemoprotein inactivation in this system was found to depend on the ratio of hemo- to flavoproteins. It was demonstrated that cytochrome P-450 LM2 inactivation during catalysis is accompanied by cleavage of the hemoprotein molecule. It is probable that this fact plays a key role in regulation of enzyme decay.     

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.     

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.     

Biosynthesis of rat liver cytochrome P-450 in mitochondria-associated rough endoplasmic reticulum and in rough microsomes in vivo

The hypothesis of a preferential biosynthesis of a major phenobarbital inducible form of hepatic cytochrome P-450 (P-450b) in mitochondria-associated rough endoplasmic reticulum (RERmito) was tested by measuring incorporation rates of [35S]methionine and delta-amino[3H]levulinate into the hemoprotein in adult rats. RERmito, rough microsomes (RM representing RER not associated with mitochondria) and smooth microsomes (SM) were quantitatively isolated from the same homogenate by rate zonal centrifugation and their content of P-450b determined by rocket immunoelectrophoresis. P-450b was isolated by immunoprecipitation from detergent-solubilized membrane fractions. The time course and rate of incorporation of [35S] methionine into immunoprecipitable P-450b of RERmito and of RM were similar at all time points studied (2-15 min) both under conditions of maximal induction (4 injections of phenobarbital in 4 days) and after a single injection of phenobarbital. The incorporation of [35S]methionine into P-450b of SM was slower at early time points (2-8 min) but similar to RERmito and RM after 15 min. In contrast, at short labeling periods (less than 8 min) more delta-amino[3H]levulinate was incorporated into P-450b of RERmito than into P-450b of RM and SM. No significant accumulation of free apocytochrome P-450b was found in either membrane fraction. These data indicate a close coordination of the biosynthesis and assembly of apocytochrome P-450b and its prosthetic heme but do not support the hypothesis of a major functional role of MITO X RER complexes in the synthesis of microsomal cytochrome P-450b.     

Heme maintains catalytically active structure of cytochrome P-450

Treatment of purified cytochrome P-450 LM2 and its liposome-bound form with hydrogen peroxide led to complete destruction of the P-450 heme. The apoenzyme thus produced could be reconstituted to catalytically active cytochrome P-450 by incubation with hemin, the reconstitution efficiency being 50% for the soluble enzyme and 80% for the liposome-bound enzyme. The removal of heme from the soluble hemoprotein resulted in a 3-fold decrease in the efficiency of its incorporation into sonicated liposomes. The contents of 5 secondary structure forms in the native, apoand reconstituted holoenzymes were estimated from their circular dichroism spectra. It was thus found that the helix content increased from 34% to 60% upon removal of the heme from the native enzyme. We suggest that the increase in the helix content leads to a reduction of the incorporation efficiency into liposomal membranes.     

Benzonal--a phenobarbital-type of inducer of the mono-oxygenase system]

It has been found that the Soviet anticonvulsant drug Benzonal is an inducer of the liver cytochrome P-450 of the phenobarbital type. The drug causes formation of the cytochrome P-450 form which is immunologically identical to the phenobarbital inducible form of the hemoprotein with identical molecular mass determined with the SDS-gel electrophoresis method in PAAG. The microsomes, obtained from the rats treated with Benzonal display increased rates of metabolism of 7-pentoxiresorufin and 16 beta-hydroxylation of androstenedione which are specific substrates for the cytochrome P-450b.     

Exogenous heme restores in vivo functional capacity of hepatic cytochrome P-450 destroyed by allylisopropylacetamide.

Administration of allylisopropylacetamide (AIA) produces a dose-related destruction of the heme moiety of the phenobarbital-induced subspecies of hepatic cytochrome P-450. This results in delayed plasma disappearance of the inactivating agent as determined after injection of [¹⁴C]AIA. In phenobarbital-pretreated rats, infusion of heme reversed this AIA-mediated impairment of the plasma disappearance of [¹⁴C]AIA. In the absence of phenobarbital pretreatment, cytochrome P-450 destruction by AIA was minimal and heme infusion failed to enhance plasma disappearance of [¹⁴C]AIA. Since exogenously administered heme is incorporated into hepatic cytochrome P-450 $\text{in vivo}$, these observations suggest that the infused heme restored the functional capacity of the phenobarbital-induced mixed function oxidase system by substituting for the prosthetic heme moiety destroyed by AIA. Heme infusion is a potentially useful therapeutic modality for enhancing drug biotransformation after intoxication with compounds that inactivate cytochrome P-450.     

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.     

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.     

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.