Laser photo-CIDNP NMR of pyridoxal phosphate and pyridoxyllysine residues: an extrinsic probe for non-aromatic amino acid residues in proteins

The interferon inducing agents, polyriboinosinic: polyribocytidylic acid and tilorone, and Freund's complete adjuvant cause a marked depression of several components of the hepatic mixed-function oxidase system. Separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantitation by fluorescence gel scanning of different molecular weight species of cytochrome P-450 indicate that the depressant effect of these agents on the apoprotein moieties of cytochrome P-450 is of a specific nature.     

Induction of xanthine oxidase and depression of cytochrome P-450 by interferon inducers: genetic difference in the responses of mice

Interferon and interferon inducing agents depress hepatic cytochrome P-450 systems. They also induce hepatic xanthine oxidase activity. It has been suggested that free radicals produced by xanthine oxidase may cause the loss of P-450. High titers of serum interferon are induced by poly IC (poly riboinosinic acid.polyribocytidylic acid) in both C57Bl/6J and C3H/HeJ mice; Newcastle disease virus (NDV) induces a high titer of interferon in C57Bl/6J mice but not in C3H/HeJ mice. The induction of xanthine oxidase activity by NDV in C3H/HeJ mice was less than half that seen in C57Bl/6J mice, thus demonstrating a relationship between the induction of xanthine oxidase, the depression of P-450 and a genetically determined difference in responsiveness of mice to interferon inducers.     

Depression of cytochrome P-450 and alterations of protein metabolism in mice treated with the interferon inducer polyriboinosinic acid X polyribocytidylic acid

Treatment of mice with the interferon inducer polyriboinosinic acid X polyribocytidylic acid [poly(IC)] results in the depression of several hepatic proteins. In this study we examined synthesis and degradation of the proteins of liver cell organelles in mice treated with poly(IC). Effects on synthesis were determined by using [14C]- and L-[3H]leucine incorporation into control and poly(IC)-treated mice, respectively. At selected times after poly(IC) treatment the 3H/14C ratio was established for preparations of nuclei, mitochondria, lysosomes, smooth endoplasmic reticulum, rough endoplasmic reticulum, and 105,000g supernatant (cytosol). Time-dependent alterations in de novo protein synthesis were greatest in lysosomal and rough endoplasmic reticular fractions; both were depressed 9 h after treatment. The effects of poly(IC) on protein degradation were determined with [14C]bicarbonate. Poly(IC) treatment decreased the time required for disappearance of 50% of 14C-labeled protein (t1/2) of smooth and rough endoplasmic reticula. Examination of endoplasmic reticulum marker enzymes showed depression of cytochromes P-450 and b5 from 9 h onward after poly(IC) administration. Tyrosine aminotransferase activity was elevated 6 h after treatment with poly(IC), and then depressed after 9 h. The other organelle marker enzymes were not affected significantly. We conclude that poly(IC) decreases the content of proteins of the hepatic endoplasmic reticulum, including certain cytochrome P-450 isozymes, by decreasing rates of protein synthesis and increasing rates of protein degradation.     

Treatment with poly I.C. enhances lipid peroxidation and the activity of xanthine oxidase, and decreases hepatic P-450 content and activities in mice and rats

Treatment of mice and rats with polyriboinosinic acid-polyribocytidylic acid (poly I.C., 5 mg/kg i.p.), a potent interferon inducer, decreased hepatic cytochrome P-450 system content and activities without influencing P-450-independent xenobiotic metabolizing enzymes. Treatment with poly I.C. decreased the content of P-450 by 28% in mice (P less than 0.05) and 30% in rats (P less than 0.05) but did not alter the activity of cytochrome c reductase. With treatment of poly I.C., the activity of XO increased 87% in mice (P less than 0.01) and 30% in rats (P less than 0.01). Lipid peroxidation was enhanced by 82% in mice (P less than 0.01) and 95% in rats (P less than 0.05). These results raise the possibility that a part of the depression of P-450 system content and activities by poly I.C. might be caused by enhanced lipid peroxidation associated with increased activity of XO.     

Homogeneous interferon from E. coli depresses hepatic cytochrome P-450 and drug biotransformation

A highly purified homogeneous human interferon produced from cloned genes depressed the levels of hepatic cytochrome P-450 and related xenobiotic metabolism. Using another cloned human interferon and several impure preparations of human and mouse interferon, it appears that only interferons with antiviral activity in the mouse depress cytochrome P-450 in that species. This is the first direct evidence that interferon decreases hepatic drug biotransformation and likely explains the depression of drug elimination which occurs during viral infections or following the administration of interferon inducers.     

Suppression of the inductive effects of phenobarbital and 3-methylcholanthrene on ascorbic acid synthesis and hepatic cytochrome P-450-linked monooxygenase systems by the interferon inducers, poly rI.rC and tilorone.

The interferon inducing agents, poly rI·rC and tilorone, cause a marked depression of hepatic cytochrome P-450-linked monooxygenase systems. Ascorbate synthesis and hepatic monnoxygenase systems are induced by phenobarbital and 3-methylcholanthrene. Poly rI·rC and tilorone suppressed the induction of ascorbate synthesis, P-450 and monooxygenase activity (ethylmorphine N-demethylase and benzo[a]pyrene hydroxylase) by phenobarbital. 3-Methylcholanthrene-induced ascorbate synthesis was suppressed by poly rI·rC, but equivocal results were obtained with tilorone. Induction of P-450 by 3-methylcholanthrene was suppressed completely by poly rI·rC or tilorone, but that of benzo[a]pyrene hydroxylase was lowered by only 40%, thus demonstrating the selective depressive action of interferon inducing agents on different species of P-450.     

Fatty acid hydroxylation in rat kidney cortex microsomes

Rat kidney microsomes have been found to catalyze the hydroxylation of medium-chained fatty acids to the omega- and (omego-1)-hydroxy derivatives. This reaction, which requires NADPH and molecular oxygen, is a function of monooxygenase system present in the kidney microsomes, containing NADPH-cytochrome c reductase and cytochrome P-450K. NADH is about half as effective as an electron donor as NADPH and there is an additive effect in the presence of both nucleotides. Cytochrome P-450K absorbs light maximally at 452-3 nm, when it is reduced and bound to carbon monoxide. The extinction coefficient of this complex is 91 mM(-1) cm(-1). Electrons from NADPH are transferred to cytochrome P-450K via the NADPH-cytochrome c reductase. The reduction rate of cytochrome P-450K is stimulated by added fatty acids and the reduction kinetics reveal the presence of endogenous substrates bound to cytochrome P-450K. Both cytochrome P-450K concentration and fatty acid hydroxylation activity in kidney microsomes are increased by starvation. On the other hand, phenobarbital treatment of the rats has no effect on either the hemoprotein or the overall hydroxylation reaction and 3,4-benzpyrene administration induces a new species of cytochrome P-450K not involved in fatty acid hydroxylation. Cytochrome P-450K shows, in contrast to liver P-450, high substrate specificity. The only substances forming enzyme-substrate complexes with cytochrome P-450K are the medium-chained fatty acids and certain derivatives of these acids. The chemical requirements for substrate binding include a carbon chain of medium length and at the end of the chain a carbonyl group and a free electron pair on a neighbouring atom. The distance between the binding site for the carbonyl group and the active oxygen is suggested to be in the order of 16 A. This distance fixes the ratio of omega- and (omega-1)-hydroxylated products formed from a certain fatty acid by the single species of cytochrome P-450K involved. The membrane microenvironment seems also to be of importance for the substrate specificity of cytochrome P-450K, since removal of the cytochrome from the membrane lowers its binding specificity to some extent. A comparison between the liver and kidney cytochrome P-450 systems suggests that the kidney cytochrome P-450K system is specialized for fatty acid hydroxylation.     

Biophysical and enzymological studies upon the interaction of trans-cinnamic acid with higher plant microsomal cytochromes P-450.

The interaction of trans-cinnamic acid with the cytochrome P-450 of microsomes derived from washed potato slices has been studied. The washing process increased the specific content of microsomal electron transport components and hence provided a useful material in which to study the interaction. Evidence is presented that the trans-cinnamic acid interacts with the cytochrome P-450, and that this interaction is analogous to "type 1" interactions of other cytochrome P-450 systems. This evidence includes the formation of a "type 1" substrate binding spectrum, an increased rate of reduction of cytochrome P-450 by NADPH in the presence of trans-cinnamic acid, an increased oxygen uptake and NADPH oxidation when trans-cinnamic acid is added to the microsomes in the presence of NADPH, and a close correlation between biophysical parameters of electron transport in the cytochrome P-450 system and enzymological parameters of the trans-cinnamic acid 4-hydroxulation reaction. The investigation has been extended to cytochrome P-450 systems of other tissues and it has been found that the trans-cinnamic acid 4-hydroxylation reaction cannot account for the presence of most of th cytochrome P-450 in several tissues. This suggests that other functions of higher plant cytochrome P-450 chains exist, and that the substrate specificityof the hemoprotein may vary in different plant tissues.     

Specific inactivation of hepatic fatty acid hydroxylases by acetylenic fatty acids

The terminal acetylenic analogue of lauric acid, 11-dodecynoic acid (11-DDYA), specifically inactivates hepatic cytochrome P-450 enzymes that catalyze omega- and omega-1-hydroxylation of lauric acid. The inactivation, as required for a suicidal process, is NADPH- and time-dependent and follows pseudo-first order kinetics. In contrast, 11-DDYA causes no measurable change in the spectroscopically-measured concentration of cytochrome P-450 or in the N-demethylation of benzphetamine or N-methyl p-chloroaniline. 10-Undecynoic acid is as effective a suicide substrate for fatty acid hydroxylases as 11-DDYA but 11-dodecenoic acid is much less effective. 11-DDYA is able to completely inhibit omega-hydroxylation but suppresses no more than 50% of omega-1-hydroxylation despite the fact that both activities are completely inactivated by 1-aminobenzotriazole. At least three hepatic cytochrome P-450 fatty acid hydroxylases, one omega-hydroxylase and two omega-1-hydroxylases, are required by these results. The construction of suicide substrates that specifically inactivate cytochrome P-450 fatty acid hydroxylases provides a new experimental probe of the physiological role of this process.     

Effect of the interferone inducer, polyriboinosinic.polyribocytidylic acid on rat liver regeneration following partial hepatectomy

The administration of the interferone inducer, polyriboinosinic.polyribocytidylic acid, inhibited the rise of activities of thymidylate synthase and thymidine kinase as well as DNA content in 24 h-regenerating rat liver in a dose dependent manner. The immunoblotting assay showed that the decrease of thymidylate synthase activity was due to inhibition of the induction of the enzyme. Co-administration of putrescine did not affect the inhibitory effect of polyriboinosinic.polyribocytidylic acid. Polyriboinosinic acid did not affect DNA synthesis in rat liver regeneration.     

Inhibition of arylhydrocarbon hydroxylase and cytochrome P-450 by 20-methylcholanthrene and phenobarbital in guinea pig on excessive doses of ascorbic acid

Treatment of guinea pigs on adequate ascorbic acid (AA) with 20-methylcholanthrene (MCA) and phenobarbital (PB) significantly increased hepatic arylhydrocarbon hydroxylase (AHH), cytochrome P-450 and cytochrome-b5 activities. In lungs, only MCA treatment significantly enhanced the activities of AHH, cytochrome P-450 and cytochrome b5. In animals on excessive doses of AA, there was inhibition of hepatic AHH, cytochrome P-450 and cytochrome b5 levels by treatment with these xenobiotics. Also, inhibition was observed in pulmonary AHH and cytochrome P-450 levels. The relevance of these observations in excessive AA-fed guinea pigs to carcinogenesis requires further extensive investigations.     

Incorporation of haemoglobin haem into the rat hepatic haemoproteins tryptophan pyrrolase and cytochrome P-450.

After its administration to intact rats, haemoglobin haem was incorporated into hepatic tryptophan pyrrolase as shown by the marked increase in functional constitution of this enzyme. Incorporation of haemoglobin haem into cytochrome P-450 was demonstrated in intact rats and in the isolated rat liver perfused with haemoglobin-free medium. In both systems, haemoglobin haem restored cytochrome P-450 content and its dependent mixed-function-oxidase activity after substrate-induced destruction of the cytochrome P-450 haem moiety. Further confirmation that haemoglobin haem could be incorporated prosthetically into cytochrome P-450 was achieved by administration of [3H]haemoglobin to rats and subsequent isolation and characterization of radiolabelled substrate-alkylated products of cytochrome P-450 haem. Our findings indicate that, although hepatic uptake of parenteral haemoglobin is slower than that of haem, it appears to serve as an effective haem donor to the intrahepatic 'free' haem pool. Thus parenteral haemoglobin may warrant consideration as a therapeutic alternative to haem in the acute hepatic porphyrias.     

Oxygenation mechanism in conversion of aldehyde to carboxylic acid catalyzed by a cytochrome P-450 isozyme.

The oxygenation of an aldehyde, 11-oxo-delta 8-tetrahydrocannabinol to a carboxylic acid, delta 8-tetrahydrocannabinol-11-oic acid was catalyzed by cytochrome P-450 MUT-2 purified from hepatic microsomes of male ddN mice. The oxygenation mechanism was confirmed by the incorporation of oxygen-18 from molecular oxygen into the carboxylic acid formed. An aldehyde form but not a hydrated form of 11-oxo-delta 8-tetrahydrocannabinol may be a substrate for the cytochrome P-450. The oxygenation of aldehyde catalyzed by cytochrome P-450 might be a common metabolic reaction in biological systems, and should be considered as an additional role of cytochrome P-450 in biotransformation of endogenous compounds and xenobiotics.     

Interaction of hexane phosphonic acid diethyl ester with phospholipids in hepatic microsomes and reconstituted liposomes as studied by 31P-NMR

By use of 31P-NMR, quasi-elastic light scattering and freeze-fracture electron microscopy it is shown that hexane phosphonic acid diethyl ester (PAE) is incorporated in hepatic microsomes without any alteration of the bilayer structure at two different sites. These findings proved that PAE can be used as molecular 31P-NMR probe in microsomes to get information about lipid-protein interactions. Extensive studies on reconstituted liposomal systems which contained cytochrome P-450 and cytochrome P-450 reductase showed that both proteins influence the localization of incorporated PAE. The results indicate a specific interaction of phosphatidylethanolamine (PE) with cytochrome P-450 in microsomes.     

Dose dependent suppression of hepatic cytochrome P-450 content by doxorubicin and Mitomycin-C: correlation with antipyrine biotransformation

Doxorubicin (DOX) and Mitomycin-C (MMC) are two anthraquinones which, when administered to rats, result in a decrease in the content of hepatic cytochrome P-450 and mixed function oxidase activities. DOX administration produced a dose-dependent immediate decrease in cytochrome P-450 content at all doses but a parallel dose-dependent decrease in the rate of antipyrine metabolite formation of the two higher doses. The lower dose of DOX produced an increase in metabolite formation and produced a less than 20% reduction in cytochrome P-450 content. MMC administration produced an immediate, modest (less than 10% of control levels) suppression of hepatic cytochrome P-450 content, and had no effect on antipyrine metabolite formation. These findings demonstrates that two drugs of the same class can produce similar suppressions of cytochrome P-450 content and that a threshold suppression of cytochrome P-450 content is needed to produce alterations in in vivo drug biotransformations.     

Metabolization of Porphyrinogenic Agents in Brain: Involvement of the Phase I Drug Metabolizing System. A Comparative Study in Liver and Kidney

(1) We evaluated the involvement of brain mitochondrial and microsomal cytochrome P-450 in the metabolization of known porphyrinogenic agents, with the aim of improving the knowledge on the mechanism leading to porphyric neuropathy. We also compared the response in brain, liver and kidney. To this end, we determined mitochondrial and microsomal cytochrome P-450 levels and the activity of NADPH cytochrome P-450 reductase. (2) Animals were treated with known porphyrinogenic drugs such as volatile anaesthetics, allylisopropylacetamide, veronal, griseofulvin and ethanol or were starved during 24 h. Cytochrome P-450 levels and NADPH cytochrome P-450 reductase activity were measured in mitochondrial and microsomal fractions from the different tissues. (3) Some of the porphyrinogenic agents studied altered mitochondrial cytochrome P-450 brain but not microsomal cytochrome P-450. Oral griseofulvin induced an increase in mitochondrial cytochrome P-450 levels, while chronic Isoflurane produced a reduction on its levels, without alterations on microsomal cytochrome P-450. Allylisopropylacetamide diminished both mitochondrial and microsomal cytochrome P-450 brain levels; a similar pattern was detected in liver. Mitochondria cytochorme P-450 liver levels were only diminished after chronic Isoflurane administration. In kidney only mitochondrial cytochrome P-450 levels were modified by veronal; while in microsomes, only acute anaesthesia with Enflurane diminished cytochrome P-450 content. (4) Taking into account that δ-aminolevulinic acid would be responsible for porphyric neuropathy, we investigated the effect of acute and chronic δ-aminolevulinic acid administration. Acute δ-aminolevulinic acid administration reduced brain and liver cytochrome P-450 levels in both fractions; chronic δ-aminolevulinic acid administration diminished only liver mitochondrial cytochrome P-450. (5) Brain NADPH cytochrome P-450 reductase activity in animals receiving allylisopropylacetamide, dietary griseofulvin and δ-aminolevulinic acid showed a similar profile as that for total cytochrome P-450 levels. The same response was observed for the hepatic enzyme. (6) Results here reported revealed differential tissue responses against the xenobiotics assayed and give evidence on the participation of extrahepatic tissues in porphyrinogenic drug metabolization. These studies have demonstrated the presence of the integral Phase I drug metabolizing system in the brain, thus, total cytochrome P-450 and associated monooxygenases in brain microsomes and mitochondria would be taken into account when considering the xenobiotic metabolizing capability of this organ. Dedicated to the memory of Dr. Susana Afonso     

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.     

Retinoic acid can enhance the stimulation by thyroid hormone of heme oxygenase activity in the liver of thyroidectomized rats.

The effects of retinoic acid (RA) (50 micrograms/100 g body wt. per day) on hepatic heme oxygenase activity, delta-aminolevulinate synthase (ALAS) activity and on cytochrome P-450 content were determined in thyroidectomized rats treated with T3 (10 micrograms/100 g body wt. per day) or diluent. RA, when administered for 3 days, failed to influence significantly the activity of either heme oxygenase or ALAS, however, the retinoid depleted hepatic cytochrome P-450 content by 17% (P less than 0.01) and microsomal heme content by 47% (P less than 0.001). T3 administration enhanced heme oxygenase activity by 72% (P less than 0.001) and ALAS activity by 251% (P less than 0.001) above levels in diluent treated controls and depleted cytochrome P-450 levels by 55% (P less than 0.001) and heme levels by 75% (P less than 0.001). When RA and T3 were administered together, the retinoid markedly enhanced the T3 stimulation of heme oxygenase activity; 173% above controls (P less than 0.001), and 61% above T3 alone (P less than 0.001). However, RA failed to influence the effect of T3 on ALAS activity or cytochrome P-450 depletion. The results indicate that RA can influence the levels of hepatic cytochrome P-450 and can modulate the stimulation of heme oxygenase activity by thyroid hormone in vivo.     

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.     

Depression of hepatic cytochrome P-450-dependent monooxygenase systems with administered interferon inducing agents.

Cytochrome P-450-dependent monooxygenase activities and cytochrome P-450 levels were depressed in hepatic microsomes from rats treated with 12 interferon inducing agents of various types: small molecules (e.g. tilorone), an RNA virus (Mengo), a fungal mycophage (statolon), liver RNA, a synthetic double-stranded polynucleotide (poly rI · poly rC), a bacterial lipopolysaccharide ($\text{E.}\text{coli}$ endotoxin) and an attenuated bacteria ($\text{B.}\text{pertussis}$ vaccine). The results suggest that the depression of hepatic cytochrome P-450-dependent monooxygenase systems may be a general property of interferon inducing agents.