Effect of liposomes on substrate uptake by isolated guinea-pig liver mitochondrial and microsomal membranes

The effect of adding small unilamellar lecithin liposomes, prepared in the presence of cytidine-diphosphoryl-1,2-diglycerides (CDP-diglycerides) or cytochrome c, on microsomal biosynthesis of phosphatidylinositol and NADPH-cytochrome c reduction and on mitochondrial biosynthesis of polyglycerophosphatides and succinate-cytochrome c reduction was studied in isolated guinea-pig liver subcellular membranes. Both microsomal biosynthesis of phosphatidylinositol and mitochondrial biosynthesis of phosphatidylglycerol were significantly reduced when CDP-diglycerides associated with liposomes were used, suggesting that some CDP-diglycerides were entrapped by liposomal membranes and were not available to subcellular membranes as substrates. The degree of decrease in phospholipid biosynthesis depended on the membrane and the nature of fatty acids in CDP-diglycerides. The composition of mitochondrial polyglycerophosphatides synthesized in the presence of CDP-diglycerides-liposomes was also affected in respect to the amount of phosphatidylglycerol formed. The reduction of cytochrome c in both microsomal and mitochondrial membranes was also decreased when liposomes were present in the assay system, but to a lesser degree than the phospholipid biosynthesis. These results indicate that the cytochrome c liposome association did not provide efficient protection of this substrate from the subcellular reduction. When chlorpromazine was also present with liposomes in the assay system, the NADPH-cytochrome c reduction in microsomes was scarcely affected, while the succinate-cytochrome c reduction in mitochondria was dependent on the concentration of chlorpromazine and could be completely abolished. These results were interpreted in terms of liposomal interaction with substrates in competition with subcellular membranes for the same substrates.     

Effect of chlorpromazine associated with liposomes on the biosynthesis of acidic lipids in subcellular membranes

The study examined the effect of an association of chlorpromazine, phosphatidic acid and cytidine-diphosphoryl-1,2-diglycerides (CDP-diglycerides) with small unilamellar lecithin liposomes on the formation, hydrolysis and transfer of lipids and cytidine liponucleotides in microsomal and mitochondrial membranes isolated from guinea-pig liver. Association with liposomes undermined the effect of chlorpromazine on these processes, but the type of effect, i.e. inhibitory or stimulatory, was retained. Association of CDP-diglycerides with small unilamellar lecithin liposomes tended to protect this substrate from subcellular uptake, thereby inhibiting phosphatidylinositol and polyglycerophosphatide formation. Phosphatidic acid in the form of liposomes stimulated CDP-diglyceride formation. The nature of fatty acids influenced the magnitude of these effects in polyglycerophosphatide biosynthesis. Transfer of CDP-diglycerides from microsomal to mitochondrial membranes was inhibited by both chlorpromazine associated with liposomes and liposomes alone.     

Monooxygenase Activities of Human Liver,Lung, and Kidney Microsomes – A Study of 42 post mortem Cases

Abstract: The cytochrome P-450-dependent monooxygenase system was examined in microsomal fractions prepared from 42 post mortem human livers and 9 lungs and kidneys. Electron microscopy studies indicated that the human liver samples were relatively free of mitochondrial and plasma membrane contamination, but samples of kidney and lung were less pure. The microsomal fractions from all organs were judged to be relatively free of haemoglobin and methaemoglobin. The specific enzyme activities for several drug substrates for the monooxygenase, NADPH-cytochrome c reductase activity and the content of the microsomal cytochromes were measured. The values of the biochemical parameters studied were found to be quite variable and the values for the human liver were appreciably lower than those obtained with liver microsomes from laboratory rodents. The enzyme activities of the human kidney and lung microsomal fractions were 1–10% of those seen for human liver samples, except for NADPH-cytochrome c (P-450) reductase activity. In order to evaluate any post mortem changes in human liver, correlations between drug metabolism activities and either cytochrome P-450 or NADPH-cytochrome c (P-450) reductase content were examined. Strong correlations (r>0.91) were seen only between aminopyrine or ethylmorphine demethylase activity and cytochrome P-450 content in samples obtained within 4 hours of death. Longer post mortem times gave poorer correlation between activity and cytochrome content. These studies document several conditions required in order to obtain human microsomal fractions representative of the activities in fresh, viable tissue.     

Mixed-function oxidases and the alveolar macrophage

Biphenyl 4-hydroxylase, benzpyrene hydroxylase and D-(+)-benzphetamine N-demethylase activities in subcellular fractions of the alveolar macrophage were investigated and found to be extremely low. Cytochrome P-450 was absent from the microsomal fraction but both NADPH-cytochrome c reductase and NADH-cytochrome c reductase activities were present. A b-type cytochrome, which was probably cytochrome b₅, was detected in the microsomal fraction. Glutathione S-aryltransferase and UDP-glucuronyl transferase activities were absent from the soluble and microsomal fractions respectively. These results indicate that alveolar macrophages may not play a significant role in the detoxication of foreign compounds by the lung.     

Perinatal development of hepatic microsomal mixed function oxidase activity in swine

Hepatic microsomal cytochrome P-450, cytochrome b₅, NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase levels were measured in fetal (107-days gestation), newborn and 1-, 2-, 3-, 4- and 6-week-old swine. Cytochrome P-450 levels and NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase activities increased in near parallel with ethylmorphine demethylase (V_max) activity between the first and the sixth postnatal week. The activities or levels of all parameters measured appeared to plateau between the fourth and sixth week post-partum. The only qualitative change observed after 1 week of age was a slight increase in the K_m for ethylmorphine demethylation. NADPH-cytochrome c reductase activity of fetal liver was relatively high, being approximately 40 per cent of the values attained at 6 weeks of age. This was in contrast to very low levels of NADPH-cytochrome P-450 reductase activity and cytochrome P-450 content of fetal liver. Clearly the activity of the flavoprotein NADPH-cytochrome c reductase does not limit the rate of reduction of cytochrome P-450 in the microsomal fraction of fetal liver. The possibility that cytochrome P-450 exists in a different form, or ratio of forms, in fetal liver could not be ascertained from carbon monoxide (CO) or ethylisocyanide (EtCN) difference spectra of fetal microsomal preparations. However, the dithionite difference CO spectra of cytochrome P-450 did not change with age.     

Effect of washing the hepatic microsomal fraction in sucrose solutions and in sucrose solution containing edta upon the metabolism of foreign compounds

Washing the hepatic microsomal fraction of the rat in 0·25 M sucrose containing 0·05 M Tris buffer (pH 7·4) resulted in the metabolism and binding to cytochrome P-450 of Type I substrates, although not of a Type II substrate. The levels of microsomal cytochrome b₅, cytochrome P-450 and NADPH-cytochrome c reductase was unchanged, whilst the activity of NADPH-cytochrome P-450 reductase was reduced. The inhibitors of lipid peroxidation EDTA and Mn²⁺ added to the washing medium prevented the decrease in the metabolism of Type I substrates. It is suggested that the effects of washing may be related to the increase in the level of peroxidised microsomal lipid which could lead to a selective destruction of the Type I binding site. EDTA added to the washing medium also produced an increase above control values, in the metabolism and binding of both Type I and Type II substrates, which may be related to the apparent increase in the amount of microsomal cytochrome P-450. Washing the microsomal fraction almost completely abolished the ability of acetone to enhance aniline hydroxylation. It is concluded that the effects of acetone are not due to the displacement of endogenous substrates bound to the microsomal fraction.     

Induction of xenobiotic-metabolizing enzymes and peroxisome proliferation in rat liver caused by dietary exposure to di(2-ethylhexyl)phosphate

Abstract

1. Exposure of rats to 1% or 3% (w/w) di(2-ethylhexyl)phosphate in the diet for five days results in two- to three-fold inductions of liver cytosolic epoxide hydrolase activity and microsomal cytochrome P-450 content. Cytochromes P-450b + e were induced 20- to 35-fold, but no increase was observed in cytochrome P-450c.

2. Considerably smaller effects were obtained on NADPH-cytochrome c reductase, microsomal epoxide hydrolase and microsomal cytochrome b₅ content, and there was no effect on cytosolic glutathione transferase activity, under the same conditions.

3. A dramatic increase in cyanide-insensitive palmitoyl-CoA oxidation and total mitochondrial protein, together with smaller increases in total catalase and cytochrome oxidase activities, were observed after treatment with di(2-ethylhexyl)phosphate, indicating that this compound causes proliferation of both peroxisomes and mitochondria.

4. It is suggested that the induction of cytosolic epoxide hydrolase and the proliferation of peroxisomes may be related processes.     

Sites of action of cadmium in vitro on hepatic,adrenal, and pulmonary microsomal monooxygenases in guinea pigs

Preincubation of hepatic, adrenal, or pulmonary microsomal preparations with cadmium produced time-dependent decreases in monooxygenase (benzphetamine demethylase, benzo(a)pyrene hydroxylase) activities. Addition of cadmium after the preincubation period had little or no effect on microsomal metabolism. As a result of preincubation with cadmium, hepatic cytochrome P-450 levels declined and the magnitude of the benzphetamine-induced type I spectral change in hepatic microsomes decreased. Cadmium also decreased hepatic NADPH-cytochrome c and NADPH-cytochrome P-450 reductase activities but had no effect on NADH-cytochrome c reductase activity. Cadmium similarly decreased cytochrome P-450 concentrations and NADPH-cytochrome c reductase activity in lung microsomes without affecting NADH-cytochrome c reductase activity. Preincubation of adrenal microsomes with cadmium had no effects on cytochrome P-450 levels, on the benzphetamine-induced type I spectrum, or on NADH-cytochrome c reductase activity. However, decreases in adrenal NADPH-cytochrome c and NADPH-cytochrome P-450 reductase activities resulted which closely paralleled the decline in adrenal monooxygenase activities. EDTA extraction of hepatic, adrenal, or pulmonary microsomes after the preincubation exposure removed about 95% of the cadmium but did not diminish the effects of the metal on microsomal monooxygenases. The results indicate that cadmium has somewhat varying sites of action on hepatic, adrenal, and pulmonary monooxygenases, but in all three tissues electron transfer to cytochrome P-450 is compromised. In addition, the effects of cadmium on microsomal metabolism persist fully even after removal of approximately 95% of the metal.     

Cationic amphiphilic drugs as a potential tool for modifying phospholipids of tumor cells. An in vitro study of chlorpromazine effects on Krebs II ascites cells

[³²P]orthophosphate and [U-¹⁴C]glycerol incorporation into Krebs ascites cell lipids was studied in vitro in the presence of chlorpromazine (CPZ)¹. At concentrations not exceeding 0.1 mM, the drug produced no cell damage within 3 hr incubation. Under these conditions, CPZ inhibited the incorporation of [³²P]orthophosphate into phosphatidylcholine and phosphatidylethanolamine and of [U-¹⁴C]glycerol into phosphatidylcholine and triglycerides, in a dose-dependent manner. On the other hand, synthesis of phosphatidic acid and phosphatidylglycerol was greatly enhanced, whereas phosphatidylinositol showed no major change. These results are compatible with an inhibition of phosphatidate phosphohydrolase, redirecting phospholipid biosynthesis towards the anionic classes. In vitro treatment of cells for 3 hr induced profound changes of phospholipid composition, which displayed a relative increase of phosphatidylglycerol and phosphatidic acid at the expense of phosphatidylcholine and phosphatidylethanolamine. The use of amphipathic cationic drugs can thus offer an interesting model for studying the relationship between cell proliferation and membrane phospholipid biosynthesis.     

Freeze‐dried liposomes as potential carriers for ocular administration of cytochrome c against selenite cataract formation

Objectives In this study, the preparation, stability and anti‐cataract effect of cationic freeze‐dried liposomes containing cytochrome c, along with nicotinamide and adenosine, are described. Methods Cytochrome c‐loaded cationic liposomes (CC‐L) were prepared by the thin‐layer evaporation technique and lyophilized to obtain freeze‐dried cytochrome c liposomes (CC‐F). The influence of the preparation components on the liposomal encapsulation efficiency and the stability were studied. The anti‐cataract effect of the CC‐F was demonstrated through attenuating lens opacity development with slit lamp examination in rats with selenite‐induced cataract. Key findings Our study indicates that: (1) the liposomal encapsulation efficiency increased with increasing phosphatidylcholine content and reduced in the presence of stearylamine. Moreover, optimal encapsulation efficiency was obtained at an appropriate ratio of phosphatidylcholine to cholesterol; (2) CC‐F was stable for at least 12 months at 4°C; (3) satisfactory improvements in lens opacity were shown in the cytochrome c‐treated groups, especially for the CC‐F‐treated group with the decreased percentage of lens opacity at about 28% at the final examination. Conclusions CC‐F were shown to be stable superior ophthalmic carriers and were able to markedly retard the onset of cataract development.     

Liposomes as cyclosporin A carriers: positively charged lecithin-cholesterol liposomes associated with cyclosporin A do not inhibit biosynthesis of mitochondrial polyglycerophosphatides and microsomal phosphatidylinositol

Phosphatidylcholine (from egg yolk)-cholesterol-stearylamine (7:2:2.25, molar ratio) liposomes when associated with cyclosporin A (phosphatidylcholine:cyclosporin A = 2:0.07, molar ratio) do not inhibit significantly the biosynthesis of [3H]polyglycerophosphatides and [3H]phosphatidylinositol in mitochondrial and microsomal membranes, respectively, isolated from rat liver. These results are explained by possible protection of the liposomes by cyclosporin A against phospholipase A2 hydrolysis.     

Effects of chlorpromazine and imipramine on rat heart subcellular membranes

The effects of chlorpromazine and imipramine at concentrations ranging from 25 to 120 (μm on ATPase activities, as well as calcium binding and uptake abilities of the rat heart subcellular membranes, were studied in vitro. Chlorpromazine significantly decreased calcium binding. Mg²⁺ ATPase and Na⁺?K⁺ ATPase activities of the sarcolemmal fraction, whereas imipramine decreased calcium binding, Ca²⁺ ATPase and Mg²⁺ ATPase activities. Chlorpromazine also produced significant inhibition of the calcium binding and uptake abilities of the microsomal and mitochondrial fractions, while imipramine depressed the mitochondrial calcium uptake activity only at concentrations of 80 μm or higher. The mitochondrial respiratory and oxidative phosphorylation activities were depressed at high concentrations of these drugs. Since different membrane systems have been considered to be involved in the regulation of heart function and metabolism, the observed decreases in ATPase and calcium-accumulating activities of the heart subcellular membranes may represent one of the molecular mechanisms for the cardiodepressant actions of chlorpromazine and imipramine.     

Growth hormone modulation of liver drug metabolic enzyme activity in the rat—I: Effect of the hormone on the content and rate of reduction of microsomal cytochrome P-450

The liver metabolism of hexobarbital and aniline was decreased 48 hr after the first injection of growth hormone (GH) in adult male rats. The content and rate of reduction of hepatic microsomal cytochrome P-450 were lowered in these rats as compared with control animals. Liver NADPH-cytochrome c reductase showed a similar decrease in activity after GH treatment. The decrease in hexobarbital metabolism paralleled the change in cytochrome P-450 reductase activity as measured with or without addition of this drug substrate to a suspension of liver microsomes from GH-treated rats. The change in aniline metabolism approximated the extent and rate of cytochrome P-450 reduction after GH treatment only when cytochrome P-450 reductase activity was measured without addition of aniline. Injection of GH produced a parallel decrease in the metabolism of both drugs as compared with cytochrome c reductase activity. Differences in optimal requirements for drug substrates (hexobarbital or aniline) or NADPH for cytochrome P-450 reductase were not detected. Preincubation studies showed no differences in microsomal drug metabolic enzyme system stability in rats injected with GH. Inhibitors of this system in vitro were not demonstrated in liver from GH-treated rats. GH is presumed to affect the level of liver drug metabolism through mechanisms in vivo operative at the first stage transfer of reducing equivalents to cytochrome P-450. An additional effect of this hormone on the level or catalytic properties of the hemoprotein cytochrome P-450 may contribute to the decrease in aniline metabolism.     

Mechanism of 2-Naphthylamine Oxidation Catalysed by Pig Liver Microsomes

1. In pig liver microsomes 2-naphthylamine-dependent NADPH oxidation, oxygen reduction, and hydroxylamine formation are linear with time for several minutes. A sharp increase in NADPH oxidation and oxygen uptake then coincides with an abrupt loss of hydroxylamine from the medium.

2. The initial rate of 2-naphthylamine N-oxidation correlates with the micro-somal concentration of mixed-function amine oxidase and the extent of linear accumulation of hydroxylamine is dependent on microsomal NADPH-cyto-chrome c reductase activity and concentration of lipid (microsomes).

3. Antisera to NADPH-cytochrome c reductase markedly decreased hydroxylamine accumulation during incubation but had no effect on the rate of 2-naphthylamine N-oxidation.

4. A system duplicating all of the kinetic properties of the microsomal 2-naphthylamine oxidase was constructed with two purified flavoproteins, (mixed-function amine oxidase and NADPH-cytochrome c reductase) and a lipid phase (erythrocyte ghosts or synthetic lecithin liposomes).

5. By independently varying the concentrations of each component in the reconstituted system, the contribution of each to the observed kinetics was defined.

6. In addition to the initial JV-oxidation of 2-naphthylamine, at least six other reactions contribute to the kinetic patterns of 2-naphthylamine oxidation catalysed by the reconstituted system.     

Changes in phospholipid biosynthetic enzymes in Type II cells and alveolar macrophages isolated from rat lungs after NO2 exposure

The activities of phospholipid biosynthesizing enzymes and subcellular marker enzymes were measured in type II cells and alveolar macrophages isolated from rat lungs 48 hr after exposure to air or 40 ppm NO₂. DNA and protein increased in cell fractions from NO₂-exposed lungs, but NO₂ exposure had no effect on the percentage of type II cells in isolated cell fractions. A general increase in cell content of biosynthetic enzyme activities (units/mg DNA) was observed in type II cells from NO₂-exposed rats, but no change was detected in the activity of the microsomal marker enzyme, NADPH cytochrome c reductase. Glycerolphosphate acyltransferase and choline phosphotransferase increased 171 and 168%, respectively, and phosphatidate phosphohydrolase increased 69%. Glycerolphosphate phosphatidyltranferase increased 143% and succinate cytochrome c reductase, the mitochondrial marker enzyme, increased 111%. Type II cells from control lungs contained a greater than threefold higher activity of the phosphatidylglycerol synthesizing enzyme, glycerolphosphate phosphatidyltransferase, compared to alveolar macrophages, and comparable activities of the enzymes of phosphatidylcholine synthesis and of the microsomal and mitochondrial reductases. Exposure to NO₂ resulted in a significant increase in NADPH cytochrome c reductase activity (46%) in macrophages, but no change in any biosynthetic enzymes. The increases in protein content and activity of phospholipid biosynthetic enzymes in type II cells are consistent with a general hypertrophy of type II cells which includes stimulation of surfactant phospholipid biosynthesis 2 days after exposure to NO₂, when type II cell proliferation is occurring.     

One-electron reduction of mitomycin c by rat liver: Role of cytochrome P-450 and NADPH-cytochrome P-450 reductase

1. The role of cytochrome P-450 in the one-electron reduction of mitomycin c was studied in rat hepatic microsomal systems and in reconstituted systems of purified cytochrome P-450. Formation of H₂O₂ from redox cycling of the reduced mitomycin c in the presence of O₂ and the alkylation of ρ-nitrobenzylpyridine (NBP) in the absence of O₂ were taken as parameters.

2. With liver microsomes from both 3-methylcholanthrene (MC)- and phenobarbital (PB)-pretreated rats, reverse type I difference spectra were observed, indicative of a weak interaction between mitomycin c and the substrate binding site of cytochrome P-450. Mitomycin c inhibited the oxidative dealkylation of aminopyrine and ethoxyresorufin in both microsomal systems.

3. Under aerobic conditions the H₂O₂ production in the microsomal systems was dependent on NADPH, O₂ and mitomycin c, and was inhibited by the cytochrome P-450 inhibitors, metyrapone and SKF-525A.

4. Although purified NADPH-cytochrome P-450 reductase was also effective in reduction of mitomycin c and the concomitant reduction of O₂, complete microsomal systems and fully reconstituted systems of cytochrome P-450b or P-450c and the reductase were much more efficient.

5. Under anaerobic conditions in the microsomal systems both reduction of mitomycin c (measured as the rate of substrate disappearance) and the reductive alkylation of NBP were dependent on cytochrome P-450.

6. The relative rate of reduction of mitomycin c by purified NADPH-cytochrome P-450 reductase was lower than that by a complete microsomal system containing both cytochrome P-450 and a similar amount of NADPH-cytochrome P-450 reductase.

7. It is concluded that although NADPH-cytochrome P-450 reductase is active in the one-electron reduction of mitomycin c, the actual metabolic locus for the reduction of this compound in liver microsomes under a relatively low O₂ tension is more likely the haem site of cytochrome P-450.     

The effects of adriamycin in vitro and in vivo on hepatic microsomal drug-metabolizing enzymes: role of microsomal lipid peroxidation

The quinone-containing anticancer drug adriamycin augments the reduction of dioxygen to reactive oxygen species and thereby stimulates (sixfold) NADPH-dependent microsomal lipid peroxidation. In vitro the extensive adriamycin-promoted peroxidation depleted (85%) rat liver microsomal cytochrome P-450, severely inhibited cytochrome P-450-dependent monooxygenation (70%), and glucose-6-phosphatase activity (80%), and activated (450%) UDP-glucuronyltransferase activity. When lipid peroxidation was blocked by EDTA, adriamycin selectively decreased cytochrome P-450 and aminopyrine N-demethylase activity; NADPH-cytochrome c reductase, UDP-glucuronyltransferase, and glucose-6-phosphatase activities were unchanged. Washing and resedimenting peroxidized microsomes to remove adriamycin and soluble lipid peroxidation products failed to restore enzyme activities to control values. Adriamycin administered subacutely (5 mg/kg × three doses) to rats significantly descreased hepatic microsomal cytochrome P-450 content and reduced aminopyrine N-demethylase and NADPH-cytochrome c reductase activities compared to saline-treated controls. Microsomal lipid peroxidation was increased following the above adriamycin treatment. Thus, these data suggested that adriamycin was capable of impairing hepatic drug metabolism in vitro by stimulating membrane lipid peroxidation in a manner similar to carbon tetrachloride; the mechanism by which adriamycin treatment in vivo decreased the activity of the drug monooxygenase system remains unclear.     

Microsomal electron transport and xenobiotic monooxygenase activities during the embryonic period of development in the killifish,Fundulus heteroclitus

The developmental patterns of aryl hydrocarbon hydroxylase (AHH) activity and NADPH-cytochrome c reductase activity were followed during embryonic development in Fundulus. AHH activity was localized in microsomal fractions prepared from whole Fundulus embryos and eleutheroembryos. On the basis of this subcellular localization, the requirements of O₂ and NADPH for activity, and sensitivity to carbon monoxide and cytochrome c inhibition, the AHH activity in Fundulus embryos and eleutheroembryos appeared to be cytochrome P-450 dependent. AHH activity was measurable in stages prior to the appearance of the liver rudiment, and during subsequent embryonic development the extrahepatic tissues were likely to have contributed substantially to the AHH activity measured. At all stages assayed before hatching, microsomal AHH specific activity remained uniformly low, but within 24 hr of hatching, AHH specific activity increased about ninefold. This posthatching increase in AHH activity was not age dependent, nor developmental stage dependent, but rather required hatching, and was not due to the presence of endogenous inhibitors in prehatching stages. The levels of NADPH-cytochrome c reductase activity and AHH activity were not closely correlated in whole embryo and eleutheroembryo microsomes, but the AHH activity in these preparations apparently was not limited by the levels of the NADPH-cytochrome c (P-450) reductase. The presence of AHH activity in Fundulus embryos during the period of active organogenesis, prior to hatching, indicates that this species is likely to be susceptible to a variety of teratogens requiring metabolic activation, and this may be the case for other species of fish as well.     

Modification of carbon tetrachloride hepatotoxicity by chemicals.

Cobaltous chloride (60 mg/kg, sc, daily for 2 days), which was found to effectively decrease the microsomal cytochrome P-450 content of mouse liver to approximately half of its normal value and which impaired the oxidative metabolism or hydroxylation of aminopyrine, ethylmorphine, and hexobarbital, offered no protection against CCl₄-induced liver damage. However, this hemoprotein inhibitor had no effect on the rate of reduction of cytochrome P-450 by NADPH and exerted a slight effect on aniline hydroxylation. SKF-525A (50 mg/kg, ip) also failed to protect against CCl₄ hepatotoxicity though it has been shown to inhibit the hydroxylation of a number of substrates. This inhibitor, a type I compound, was found to enhance cytochrome P-450 reduction by NADPH. Further studies revealed that CCl₄-induced hepatic injury could be prevented by phenazine methosulfate (2 mg/kg, ip, 5 doses at 0.5-hr intervals), which in vitro was found to inhibit NADPH-cytochrome c reductase noncompetitively. All of these findings are not satisfactorily explainable by electron transfer from NADPH-cytochrome c reductase to CCl₄ as the activation reaction for CCl₄ but are compatible with the hypothesis previously proposed by others that cytochrome P-450 is the critical site for CCl₄ activation.     

Effects of Phenobarbital and Ethanol on Rat Liver Aldehyde Dehydrogenases

The aldehyde dehydrogenases and the drug-metabolizing system (the mixed function oxidase) were studied in Wistar rats treated with ethanol and phenobarbital. Phenobarbital treatment had no effect on the microsomal aldehyde dehydrogenase activity, whereas the mitochondrial aldehyde dehydro-genase activity was slightly decreased and the activities of ethylmorphine demethylase, NADPH-cytochrome c reductase and the concentration of cytochrome P-450 were increased two to threefold. Ethanol treatment caused no changes in the activities of the aldehyde dehydrogenases, and the drug metabolizing system was only slightly affected. Very little aldehyde dehydrogenase activity was found in the cytosol from livers of either control, ethanol-treated or phenobarbital-treated rats. The results are discussed with particular reference to the oxidation of acetaldehyde and other aldehydes during ethanol metabolism.