Alterations in hepatic heme and cytochrome P-450 metabolism in murphy-sturm lymphosarcoma-bearing rats implications for drug metabolism

Previous studies have shown that tumor-bearing rats have significantly decreased hepatic microsomal cytochrome P-450 content and NADPH-cytochrome c reductase activity with, consequently, significantly decreased capacity for microsomal oxidative drug metabolism. Subsequent investigations have revealed that the rates of hepatic cytochrome P-450 apo-protein synthesis and degradation are decreased significantly and hepatic microsomal heme oxygenase activity is increased significantly in rats bearing an extra-hepatic tumor. Further studies have been done to attempt to clarify the pathogenesis and significance of these observations. Hepatic delta-aminolevulinic acid (ALA) synthetase activity in male Wistar rats declined to a nadir of 162 ± 34 (S.E.) pmoles ALA per mg protein per 30 min 6 days following i.m. transplantation of Murphy-Sturm lymphosarcoma (vs control = 218 ± 36 pmoles per mg per 30 min). Turnover of ³H-labeled heme in microsomal CO-binding particles (i.e. cytochrome P-450 heme) was increased significantly 8 days following i.m. transplantation of Murphy-Sturm lymphosarcoma with a T_{$\text{1}\text{2}$} of 5.5 hr for the fast phase of hepatic cytochrome P-450 heme disappearance in tumor-bearing rats as compared with a T_{$\text{1}\text{2}$} of 7 hr in control rats. Hepatic cytochrome P-450 apo-protein concentration was slightly, but not significantly, increased in Murphy-Sturm lymphosarcoma-bearing rats as compared with control rats up to 10 days following tumor transplantation. These results suggest that, in Murphy-Sturm lymphosarcoma-bearing rats, decreased microsomal cytochrome P-450 concentration is the result of both decreased cytochrome P-450 apo-protein synthesis and increased cytochrome P-450 heme turnover. Apo-cytochrome P-450 concentration was not appreciably altered because increased cytochrome P-450 heme turnover and decreased cytochrome P-450 apo-protein degradation were balanced by decreased cytochrome P-450 apo-protein synthesis. Because of their effects on cytochrome P-450 concentration and action, these alterations in heme and hemoprotein metabolism may be of importance in regulating oxidative drug metabolism in the tumor-bearing state.     

Differential effects of chronic ethanol feeding on cytochrome P-448- and P-450-mediated drug metabolism in the rat

The effects of chronic ethanol feeding on cytochrome P-448- and P-450-mediated drug metabolism have been studied both in vivo and in vitro in the rat, using caffeine, phenacetin, antipyrine and aminopyrine as test substrates. N-Demethylation of aminopyrine (P-450 mediated) was increased both in vivo and in vitro in rats after chronic ethanol feeding (P < 0.05) whereas in vivoN-demethylation of caffeine and O-dealkylation of phenacetin (P-448 mediated) were unchanged in the same animals. N-rmDemethylation of antipyrine was increased by both phenobarbital and 3-methylcholanthrene pretreatment and by chronic ethanol feeding (P < 0.05), possibly due to cytochrome P-450 induction. Furthermore, the Michaelis affinity constants. K_m, for hepatic microsomal aminopyrine N-demethylase and antipyrine N-demethylase were lower in chronic ethanol-fed animals (P < 0.05), suggesting a qualitative change in the enzymes resulting in greater substrate affinity. These findings suggest a differential effect of chronic ethanol feeding on the induction of cytochrome P-450- and cytochrome P-448-mediated drug metabolism, with a greater effect on the former microsomal system.     

Role of hepatic and renal cytochrome P-450 IVA1 in the metabolism of lipid substrates

The role of clofibrate-inducible cytochrome P-450 IVA1 in the metabolism of endogenous lipids in both rat liver and kidney microsomal fractions has been investigated. 20(omega)-hydroxyarachidonic acid has been identified as a major metabolite after incubation with both tissue fractions and the structure confirmed by mass spectrometry. The arachidonic acid 20-hydroxylase activity is inducible by clofibrate in both liver and kidney, indicating that cytochrome P-450 IVA1 is probably the enzyme responsible for this activity. In addition, the kidney exhibited higher rates of arachidonate 20-hydroxylase activity than the liver (in both control and induced states). Although leukotriene B4 was also hydroxylated in the 20-position in both liver and kidney, clofibrate induction resulted in a decrease (approximately 50%) in hydroxylase activity. In addition, the absolute level of leukotriene B4 20-hydroxylase activity in both tissue homogenates and by purified cytochrome P-450 IVA1 in a reconstituted system, was 2-3 orders of magnitude lower than the corresponding activity for lauric acid and arachidonic acid as substrates, indicating that the leukotriene was not the preferred substrate for this enzyme. Computer modelling of the conformational geometries of the above three potential cytochrome P-450 IVA1 substrates have shown that both lauric and arachidonic acids adopt a compact, 'hairpin' structure that are almost superimposed on each other, thereby rationalizing why they are relatively good substrates for this isoenzyme. By contrast, leukotriene B4 adopts a more bulky geometry than the two fatty acids, thereby providing a coherent structural reason why it is a poorer substrate for the cytochrome P-450 IVA1 isoenzyme.     

Possible role of the acetone-inducible cytochrome P-450IIE1 in the metabolism and hepatotoxicity of thiobenzamide

The effect of acetone pretreatment (5% in drinking water for 10 days on rat liver metabolism and toxicity of thiobenzamide (TB) was investigated. Hepatic microsomes from acetone-pretreated rats showed a significant increase of TB-S-oxidation rate which, on the basis of selective thermal inactivation of FAD-containing monooxygenase (FADM), appeared dependent only on cytochrome P-450. Furthermore, TB was able to competitively inhibit acetone hydroxylase (AcH), an enzymatic reaction highly specific for the P-450IIE1 isozyme. Acetone pretreatment of rats also produced an exacerbation of liver damage induced by acute administration of TB (150 mg/kg), as judged by the extent of liver necrosis and serum alanine-amino transferase (ALAT) activities. Coadministration of acetone with TB reduced on the other hand the extent of liver damage. The findings suggest that P-450 species induced by acetone, and in particular the P-450IIE1 isozyme, could be involved in the biotransformation of TB.     

Alterations in hepatic microsomal drug metabolism and cytochrome P-450 proteins in spontaneously hypertensive rats

Experiments were conducted to determine if substrate-specific changes in microsomal metabolism and liver proteins occurred in young (12-13 weeks) spontaneously hypertensive rats (SHR) fed ad libitum compared to age-matched normotensive Wistar Kyoto (WKY) control rats. The hepatic microsomal protein content in SHR rats was significantly increased compared to WKY rats while cytosolic and total liver protein levels did not differ between the two groups. Liver microsomal ethylmorphine-N-demethylase activity was substantially enhanced in SHR rats with only slight increases in cytochrome P-450 content and aniline hydroxylase activity compared to WKY rats. The substrate-specific increases in the microsomal drug metabolism in SHR rats were accompanied by an increase in the prominence of a protein with molecular weight 55,000 in the cytochrome P-450 region. These preliminary observations may be clinically relevant in that alterations in hepatic drug metabolism may be associated with endogenous biochemical processes underlying the hypertensive state.     

Tetrachloroethylene metabolism by the hepatic microsomal cytochrome P-450 system

The interaction of tetrachloroethylene with hepatic microsomal cytochromes P-450 has been investigated using male Long-Evans rats. The spectral binding of tetrachloroethylene to cytochromes P-450 in hepatic microsomes from uninduced rats was characterized by a K_s of 0.4 mM. The K_s was not affected by phenobarbital induction, but was increased following pregnenolone-16α-carbonitrile induction. The K_M of 1.1 mM, calculated for the conversion of tetrachloroethylene to total chlorinated metabolites by the hepatic microsomal cytochrome P-450 system, was decreased by phenobarbital induction and increased by pregnenolone-16α-carbonitrile induction. The maximum extents of binding (ΔA_max) and metabolism (V_max) of tetrachloroethylene were increased by both phenobarbital and pregnenolone-16α-carbonitrile induction. Induction with β-naphthoflavone was without effect on any of the above parameters. The effects of the inducing agents on tetrachloroethylene-stimulated CO-inhibitable hepatic microsomal NADPH oxidation followed the same trend as their effects on V_max for the metabolism of tetrachloroethylene, although in all cases the extent of NADPH oxidation was 5- to 25-fold greater than the extent of metabolite production. The inhibitors of cytochromes P-450, viz. metyrapone, SKF 525-A, and CO, inhibited the hepatic microsomal binding and metabolism of tetrachloroethylene. Free trichloroacetic acid was found to be the major metabolite of tetrachloroethylene from the hepatic microsomal cytochrome P-450 system. Neither 2.2,2-trichloroethanol nor chloral hydrate was produced in measurable amounts from tetrachloroethylene. A minor but significant metabolite of tetrachloroethylene by cytochrome P-450 was the trichloroacetyl moiety covalently bound to components of the hepatic microsomes. Incubation of tetrachloroethylene. an NADPH-generating system. EDTA and hepatic microsomes was without effect on the levels of microsomal cytochromes P-450, cytochrome b₅, beme, and NADPH-cytochrome c reductase. It is concluded that hepatic microsomal cytochromes P-450 bind and metabolize tetrachloroethylene. The major product of this interaction is trichloroacetic acid, which is also the major urinary metabolite of tetrachloroethylene in vivo. The forms of cytochrome P-450 that bind and metabolize tetrachloroethylene include those induced by pregnenolone-16α-carbonitrile and by phenobarbital. Cytochrome P-448. which was induced in rat liver by β-naphthoflavone, does not appear to spectrally bind or metabolize tetrachloroethylene. The metabolism and toxicity of tetrachloroethylene are considered in relation to other chlorinated ethylenes.     

Serum cyclosporin levels, hepatic drug metabolism and renal tubulotoxicity

The present study was designed to examine inter-relationships between serum cyclosporin (CsA) levels, hepatic drug metabolising enzyme activity and CsA induced nephrotoxicity. CsA (25 mg/kg p.o.) was administered daily to male Sprague-Dawley rats: groups of animals were killed on days 0, 4, 7, 10 and 14 and thereafter at weekly intervals over the 7-week course of the experiment. Nephrotoxicity was evaluated by measuring tubular enzymuria and by light microscopy and serum CsA levels (parent drug plus certain metabolites) were determined by radioimmunoassay. The hepatic microsomal mono-oxygenase enzyme system was monitored by measurement of cytochrome P-450, aminopyrine N-demethylase and NADPH-cytochrome c reductase. Nephrotoxicity appeared within 4 days of starting treatment and continued for 4 weeks. Between weeks 4 and 6 there was a period of complete remission followed by the return of renal damage. Aminopyrine N-demethylase activity fell during the first 4 weeks. During the period of remission, however, N-demethylase activity rose to a point significantly higher than pretreatment values and serum CsA levels fell to their lowest concentration. With relapse, hepatic N-demethylase activity again fell below normal and serum drug levels rose to their pre-remission values. From the third week onward, changes in NADPH-cytochrome c reductase activity paralleled those in N-demethylase activity. The hepatic microsomal concentration of cytochrome P-450 did not, however, change significantly during the 7-week period of CsA treatment. Our results suggest that the spontaneous remission of CsA-induced nephrotoxicity is due to a reduction in circulating drug levels caused by increased hepatic CsA metabolism.     

Morphine-mediated effects on rat hepatic heme and cytochrome P-450 in vivo: Antagonism by naloxone in the liver

Chronic morphine administration to adult male rats has long been known to lower hepatic cytochrome P-450 content and its dependent mixed function oxidase activity. More recently, we found that acute treatment of mature male rats with a dose of morphine higher than that used chronically also reduces their hepatic cytochrome P-450. In the present study, we demonstrate that this acute reduction of cytochrome P-450 in the rat liver is a result of morphine-mediated accelerated turnover (degradation) of its heme moiety and apparently is associated with hepatotoxicity of the drug. These morphine-mediated effects are largely prevented by concomitant administration of naloxone, a morphine antagonist.     

Differential effects of 3 dipyridyl isomers on hepatic microsomal cytochrome P450 and heme oxygenase in rats

We compared the effects of 3 dipyridyl isomers, 2,2′-dipyridyl, 2,4′-dipyridyl and 4,4′-dipyridyl, on hepatic microsomal heme oxygenase and drug-metabolizing enzyme activities in male rats. 2,2′-Dipyridyl increased cytochrome P450 (P450) content at lower doses, but decreased with increasing dose levels. Immunoblot analysis revealed that 2,2′-dipyridyl did not induce both P450 1A1/2 and P450 2B1/2, in contrast to 2,4′- and 4,4′-dipyridyls, both of which were inducers of either P450 1A1/2 and/or P450 2B1/2. Some drug-metabolizing enzyme activities gradually declined with the increasing dose level of 2,2′-dipyridyl. 2,2′-Dipyridyl was able to induce hepatic microsomal heme oxygenase in a dose-dependent manner, but 2,4′- and 4,4′-dipyridyls did not, even at the highest dose (0.80 mmol/kg) examined. Treatment of rats with 2,2′-dipyridyl resulted in the increase of glutathione (GSH) content in a dose-dependent manner, but not 4-substituted isomers. A time course study with 2,2′-dipyridyl revealed that it produced a significant decrease in hepatic GSH content at early time periods (2–6 h) after its administration with an inverse increase in heme oxygenase activity. The present investigation has revealed that in contrast to the induction of P450 by 4-substituted dipyridyl compounds, 2,2′-dipyridyl is a novel inducer of hepatic microsomal heme oxygenase, together with the change in hepatic GSH content. This study would provide information on the differential effects of simple dipyridyl isomers on hepatic enzymes involved in heme and drug metabolism.     

A model of cytochrome P-450-centered hepatic dysfunction in drug metabolism induced by cobalt-protoporphyrin administration

Cobalt-protoporphyrin treatment disrupts cytochrome P-450-centered drug metabolism and is known to decrease significantly the cytochrome P-450 content of the liver. This study assesses further the correlations between biochemical and functional changes induced by Co-protoporphyrin. Specifically, it confirmed the fall in cytochrome P-450 levels in liver and demonstrated that both NADPH-cytochrome P-450 reductase and NADH-cytochrome b5 reductase activities decreased in a dose-dependent manner, albeit to a lesser degree, upon Co-protoporphyrin administration. Furthermore, plasma clearance of the marker drug aminopyrine fell off abruptly with a minimal decrease in cytochrome P-450 content, and then monotonically with its further depletion. Both aminopyrine and caffeine demethylation, as measured by the amount of radiolabeled CO2 exhaled, also decreased with diminishing cytochrome P-450 content. With aminopyrine the decrease was abrupt but with caffeine biphasic, consistent with preferential isozyme depletion. The drop in oxidative drug metabolism measured by these two in vivo techniques occurred in the absence of organellar damage to hepatocytes, as observed by electron microscopy. In vitro studies of aminopyrine metabolism in microsomes prepared from rats with and without Co-protoporphyrin injection proved to be consistent with the in vivo studies. Moreover aminopyrine Vmax decreased and Km increased with decreasing cytochrome P-450 content, suggesting preferential isozyme depletion. Furthermore, the changes in aminopyrine intrinsic clearance predicted by the in vitro Vmax and Km values agreed with those measured by in vivo plasma clearance. Taken together, these data suggest that Co-protoporphyrin treatment can be used to produce a model of altered cytochrome P-450-centered drug metabolism, as measured consistently by several techniques. However, this model appears to be more complex than one involving nonspecific depletion of cytochrome P-450 alone, and may be influenced also by concomitant changes in the electron transport chain or other aspects of hepatic metabolism.     

Role of human hepatic cytochrome P-450s in territrem A metabolism

The ability of human liver microsomal preparations (HLM1, 2, 3, and 5), microsomes from human lymphoblasts expressing different cytochrome P-450 (CYP450) isoforms, and CYP3A4 cDNA-transfected V79 Chinese hamster cells to metabolize territrem A (TRA) was studied. The only metabolite generated by any of these preparations was 6beta-hydroxymethyl-6beta-demethylterritrem A (MA(1)). MA(1) formation was observed with all four human liver microsomal samples. Of the eight microsomal preparations from human lymphoblasts expressing different cytochrome P-450 enzymes (1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4) examined, only those expressing CYP2C9, CYP2D6, or CYP3A4 metabolized TRA, with that expressing CYP3A4 being the most active. No TRA metabolites were formed by control V79MZ cells, but MA(1) was formed by CYP3A4 cDNA-transfected V79 Chinese hamster cells. In order to investigate which CYP450 isoforms were involved in MA(1) formation in the human liver microsomal preparations, the effects of six isoform-specific chemical inhibitors (furafylline, sulfaphenazole, omeprazole, quinidine, ketaconazole, and diethyldithiocarbamate) and anti-3A4, anti-2C9, and anti-2D6 antibodies on TRA metabolism by HLM2 and HLM5 were examined. MA(1) formation was markedly inhibited by ketaconazole, with quinidine and sulfaphenazole having less of an effect. Anti-CYP3A4 antibody markedly inhibited MA(1) formation, while antibodies against CYP2C9 or CYP2D6 had little effect. The amount of MA(1) formed using different HLM preparations was related to the 6beta-testosterone hydroxylase activity and CYP3A4 protein content of the preparations. These results suggest that CYP3A4 is the major enzyme involved in TRA metabolism by human liver microsomes, with CYP2C9 and CYP2D6 playing a minor role.     

Relation between hepatic microsomal metabolism of N-nitrosamines and cytochrome P-450 species

Effects of SKF 525A (0.1 mM), metyrapone (0.1 mM), alpha-naphthoflavone (ANF) (0.5 mM) and pyrazole (1.0 mM) on N-nitrosodimethylamine (NDMA), N-nitrosomethylbutylamine (NMBuA) and N-nitrosomethylbenzylamine (NMBeA) metabolism by hepatic microsomes from rats pretreated with inducers were investigated. NDMA demethylation was weakly increased by phenobarbital (PB) treatment. The demethylation was inhibited by SKF 525A and enhanced by metyrapone in non-treated and PB-treated microsomes, and weakly inhibited by ANF in 3-methylcholanthrene(MC)-treated microsomes. NMBuA demethylation was increased by PB treatment and inhibited by SKF 525A in all microsomes. Metyrapone inhibited the demethylation in PB-treated microsomes. NMBuA debutylation was increased by PB and MC treatments, and inhibited by metyrapone in all microsomes. The strongest inhibition by metyrapone was observed in PB-treated microsomes. The debutylation was inhibited by SKF 525A in non-treated and PB-treated microsomes and by ANF in MC-treated microsomes. NMBeA demethylation was decreased by MC treatment and weakly inhibited by SKF 525A in all microsomes. The effects of the inducers and inhibitors on NMBeA debenzylation were almost the same as those on NMBuA debutylation except that the increasing effect of MC was small. Pyrazole was a relatively selective inhibitor of NDMA demethylation. These results suggest the following: NDMA demethylation is catalyzed by PB-induced cytochrome P-450 species (P450-PB) and MC-induced cytochrome P-450 species (P448-MC). But their specific activity is low and the other cytochrome P-450 species demethylate NDMA. NMBuA demethylation is catalyzed by P450-PB. But the specific activity is not high and the other cytochrome P-450 species also demethylate NMBuA. NMBuA debutylation is catalyzed by P450-PB and P448-MC. Almost all of NMBeA demethylation is catalyzed by cytochrome P-450 species other than P450-PB and P448-MC. NMBeA debenzylation is catalyzed by P450-PB and P448-MC, but the specific activity of P448-MC is not high.     

Induction of hepatic cytochrome P-450 and drug metabolism by metyrapone in the rat: relevance to its effects in rat-liver cell culture

The ability of metyrapone to induce hepatic cytochrome P-450 and xenobiotic metabolism in rats and rat hepatocyte culture has been studied. Metyrapone is a phenobarbitone-type inducer in rats. When rat hepatocytes were cultured with beta-naphthoflavone, a polycyclic hydrocarbon-type induction pattern was observed. In contrast, the observed induction pattern of both phenobarbitone and metyrapone in cell culture differed from that obtained with these compounds in vivo. The relationship between the nature of the induction of hepatic xenobiotic metabolism by metyrapone in vivo and its effects in liver-cell culture are discussed.     

Regulation of intestinal cytochrome P-450 and heme by dietary nutrients. Critical role of selenium

The intestinal cytochrome P-450 (I-P-450)-dependent mixed function oxidase (MFO) system is regulated to a remarkable extent by various ingested xenobiotics, including drugs and carcinogens, as well as dietary nutrients. Accordingly, acute dietary iron deprivation is found to result in a marked decrease in I-P-450 content and activity. This decrease is most pronounced in the villous tip cells, the very cells committed to absorption of ingested materials. We investigated the mechanistic basis for such acute reduction and report that iron was not only required as a co-substrate for I-P-450 heme formation, but also as a regulator of two key heme-synthetic enzymes, delta-aminolevulinic acid synthetase and ferrochelatase. In addition, our studies revealed that dietary deprivation of selenium for a single day dramatically reduced I-P-450-dependent MFO activity. This prompt reduction apparently reflects impaired I-P-450 formation resulting from lowered ferrochelatase activity and consequently decreased intestinal heme availability, and was not a consequence of intracellular peroxidation presumably enhanced by concomitant lowering of the seleno-dependent glutathione peroxidase. Thus, we report the novel observation that dietary selenium also appears to be a critical modulator of intestinal cytochrome P-450-dependent metabolism of ingested drugs, carcinogens, and toxins that are absorbed by the intestinal mucosa.