Sodium cholate extraction of rat liver nuclear xenobiotic-metabolizing enzymes

DNA is the purported target of several carcinogenic and mutagenic agents. Nuclear enzymes which could generate or detoxify reactive metabolites are of major concern. Several such enzymes have been identified within nuclei, but obtaining samples with enriched content or activity is difficult, time-consuming, and uses harsh isolation techniques. Extraction of rat liver nuclear suspensions with cholate-containing buffer results in solubilization of 25-30% of the protein. Linear extraction was obtained for total protein and cytochromes P-450 and b5, NADPH-cytochrome P-450 reductase, NADH-cytochrome b5 reductase, DT-diaphorase, and microsomal-like epoxide hydrolase with specific activities comparable to values reported for isolated nuclear membrane, while the yield was five to ten times greater. Detergent extracts of rat liver nuclei were employed to study the comparative response of microsomal and nuclear enzymes to chemical treatment. While the responses to acute inductive (phenobarbital and 3-methylcholanthrene) and toxic (carbon tetrachloride and dibromochloropropane) treatments were qualitatively similar, an initiation-promotion protocol (diethylnitrosamine with phenobarbital promotion) resulted in divergent responses between the enzymes in the two subcellular fractions. Detergent extracts of nuclei offer an efficient means of recovering xenobiotic-metabolizing enzymes from rat liver nuclei, and have been utilized to demonstrate a differential response of nuclear enzymes during preneoplastic development.     

Oxidation of thiobenzamide by the FAD-containing and cytochrome P-450-dependent monooxygenases of liver and lung microsomes

Two distinct microsomal pathways involved in the metabolism of thiobenzamide to thiobenzamide S-oxide have been identified and quantitated in the liver and lungs of mice and rats, using a highly inhibitory antibody against NADPH-cytochrome P-450 reductase. Approximately 50 and 65% of the oxidation in mouse and rat liver microsomes, respectively, was due to the FAD-containing monooxygenase, the remainder being catalyzed by cytochrome P-450. In the mouse lung, S-oxidation was predominantly via the FAD-containing monooxygenase while that in the rat lung was about 60% via the FAD-containing enzyme and 40% via cytochrome P-450. Cytochrome P-450-dependent S-oxidation of thiobenzamide was induced in the liver by treatment of mice with phenobarbital and slightly increased by treatment with 3-methylcholanthrene, while in rat liver either of these treatments caused only a small increase in metabolism due to cytochrome P-450. Thermal inactivation of the FAD-containing monooxygenase left the cytochrome P-450 component essentially unchanged. Thermally treated microsomes had a pH activity profile characteristic of cytochrome P-450 and were less inhibited by methimazole and thiourea when compared to untreated microsomes. Female mouse liver microsomes had a much higher, and female rat liver microsomes a lower, ability to S-oxidize thiobenzamide when compared to the males.     

DT-diaphorase-like quinone reductase in rat plasma.

The present study provides the evidence that DT-diaphorase-like quinone reductase exists in rat plasma. The quinone reductase activity toward menadione was found in rat plasma in the presence of NADH or NADPH. The enzyme activity was induced by pretreatment with 3-methylcholanthrene, but was not affected by phenobarbital. The 3-methylcholanthrene-induced quinone reductase activity was separated into three fractions (F1, F2, and F3) by gel filtration, which showed NAD(P)H-linked, NADH-linked, and NAD(P)H-linked activities, respectively. F1, which was induced by 3-methylcholanthrene, was inhibited by dicumarol, and cross-reacted with rat liver DT-diaphorase antibody.     

Effect of drugs on progesterone metabolism in the female rat

Various drugs brought about a reduction of serum progesterone level irrespective of whether or not a potent inducer (phenobarbital, 4-methyl-coumarin) or a hepatotoxin (carbon tetrachloride, α-naphthylisothiocyanate, coumarin) has been administered. The decrease by hepatotoxins was highly significant during the estrus phase of the cycle. These treatments affected the hepatic level of progesterone and altered the uptake of [4-¹⁴C]progesterone in vivo. The serum level of progesterone was significantly decreased by phenobarbital and carbon tetrachloride; however, the incorporation into the liver was enhanced by phenobarbital and reduced by carbon tetrachloride. This opposing hepatic action showed selectivity; phenobarbital increased the oxidative pathway of progesterone metabolism (formation of 6β-, 16α-, 20α-hydroxyprogesterone) but the reductive pathway remained unaltered (formation of pregnanediol, pregnanolone). Conversely, carbon tetrachloride diminished oxidation and raised reduction of progesterone. These results have been confirmed by measurements of progesterone metabolism in vitro using isolated microsomes. Phenobarbital brought about an induction of progesterone 16α-, 6β- and 20α-hydroxylase, did not affect progesterone Δ⁴-5α-dehydrogenase, whereas carbon tetrachloride inhibited hydroxylase and raised dehydrogenase activities. The action of these test compounds on serum and liver levels of progesterone and on the variation of progesterone metabolism seemed to be related to changes manifest in the function of the hepatic endoplasmic reticulum. Similar changes might be associated with the development of mild hepatic lesions induced by various steroids.     

Cholestasis as an in vivo model for analysis of the induction of liver microsomal monooxygenases by sodium phenobarbital and 3-methylcholanthrene.

Cholestasis was used as a model of sharp decrease in the amounts of substrate-binding and catalytic centres of cytochrome P-450 for sodium phenobarbital and 3-methylcholanthrene. Based on this model, the induction effects of sodium phenobarbital and 3-methylcholanthrene on rat liver microsomal monooxygenases were analyzed. Under conditions excluding the primary binding and metabolism of the inducer by monooxygenases, sodium phenobarbital retains its capacity for induction. By contrast, 3-methylcholanthrene exerted no inducing effects under the same conditions as confirmed by the lack of increase of cytochrome P-448 content. From the data obtained it is suggested that in mechanism of sodium phenobarbital induction of liver microsomal monooxygenases the activation of protein synthesis is affected by the inducer itself. As for 3-methylcholanthrene, it is assumed that the synthesis of specific protein (cytochrome P-448) could be initiated by the microsomal metabolites of this inducer.     

Metabolism of dihalomethanes to carbon monoxide: IV. Studies in isolated rat hepatocytes

The effects of SKF 525-A, phenobarbital, and 3-methylcholanthrene on dihalomethane metabolism were studied in intact animals as well as in isolated rat hepatocytes (IRH). Treatment of rats with phenobarbital or 3-methylcholanthrene resulted in increased metabolism of dibromomethane to carbon monoxide in vivo. Treatment of rats with SKF 525-A or diethyl maleate prior to dibromomethane administration resulted in decreased blood carbon monoxide levels in vivo. The biotransformation of dibromomethane to carbon monoxide was also studied in IRH and was characterized with respect to time, number of cells, substrate concentration and effects of inhibitors and inducers of drug metabolism. Carbon monoxide production was linear for about 30 min and increased with increasing cell concentration. The apparent K_m and V_max for dibromomethane were 15.9 ± 2.0 mM and 1.14 ± 0.25 nmoles carbon monoxide/1 × 10⁶ cells/min, respectively, in IRH from control rats, while the values in IRH from phenobarbital treated animals were 18.5 ± 9.6 mM and 1.77 ± 0.10 nmoles carbon monoxide/1 × 10⁶ cells/min, respectively. SKF 525-A, diethyl maleate and ethanol inhibited the biotransformation of dibromomethane to carbon monoxide in IRH. The rate of metabolism of various dihalomethanes in IRH was found to be diiodomethane > dibromomethane > bromochloromethane = dichloromethane suggesting a relationship between decreased electronegativity of the halogen and increased metabolism. These studies demonstrate that dibromomethane metabolism is increased in vivo by treatment with phenobarbital and 3-methylcholanthrene as well as in IRH from phenobarbital treated animals. Dibromomethane metabolism can be decreased by a variety of inhibitors in both systems.     

In vitro metabolism of penicillium roqueforti toxin (PRT) and a structurally related compound, eremofortin A, by rat liver.

The metabolism of two closely related compounds (PRT and Eremofortin A) isolated from a culture of Penicillium roqueforti was studied in vitro using the rat liver system. Enzymatic activity was enhanced by treatment of rats with phenobarbital (PB) or 3-methylcholanthrene (MC). Metabolites were isolated by silica gel column chromatography and their structures elucidated by spectral analysis and confirmed by chemical synthesis.Three types of enzymatic reactions were observed: hydroxylation, reduction and deacetylation; the metabolic pathways of the two compounds give the same final product. The authors suggest that the pathway described here is a route of detoxication.     

Characteristics of two classes of azo dye reductase activity associated with rat liver microsomal cytochrome P450

Azo dyes are reduced to primary amines by the microsomal enzymes NADPH-cytochrome P450 reductase and cytochrome P450. Amaranth, a highly polar dye, is reduced almost exclusively by rat liver microsomal cytochrome P450 and the reaction is inhibited almost totally by oxygen or CO. Activity is induced by pretreatment with phenobarbital or 3-methylcholanthrene. In contrast, microsomal reduction of the hepatocarcinogen dimethylaminoazobenzene (DAB), a lipid soluble, weakly polar compound, is insensitive to both oxygen and CO. However, reconstitution of activity with purified NADPH-cytochrome P450 reductase and a partially purified cytochrome P450 preparation indicates that activity is catalyzed almost exclusively by cytochrome P450. Activity is induced by clofibrate but not phenobarbital, beta-naphthoflavone, 3-methylcholanthrene, isosafrol, or pregnenolone-16 alpha-carbonitrile. These observations suggest the existence of at least two classes of azoreductase activity catalyzed by cytochrome P450. To investigate this possibility, the reduction of a number of azo dyes was investigated using microsomal and partially purified systems and the characteristics of the reactions were observed. Microsomal reduction of azo dyes structurally related to DAB required a polar electron-donating substituent on one ring. Activity was insensitive to oxygen and CO if the substrates had no additional substituents on either ring or contained only electron-donating substituents. Introduction of an electron-withdrawing group into the prime ring conferred oxygen and CO sensitivity on the reaction. Substrates in the former group are referred to as insensitive and substrates in the latter group as sensitive. Inhibitors of cytochrome P450 activity depressed reduction of both insensitive and sensitive substrates. In a fully reconstituted system containing lipid, highly purified NADPH-cytochrome P450 reductase and a partially purified cytochrome P450 preparation, rates of reduction of various insensitive substrates varied several-fold, whereas rates of reduction of sensitive substrates varied by three orders of magnitude. Using purified enzymes, each of the insensitive substrates was shown to be reduced by reductase alone, but only at a fraction of the rate seen in the fully reconstituted system, implying that reducing electrons were transferred to the dyes mainly from cytochrome P450. Conversely, there was substantial, in some cases almost exclusive, reduction of sensitive substrates by purified reductase alone and almost no inhibition by CO. Their reduction, however, was inhibited by CO in microsomal systems.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of 1-phthalidyl 5-fluorouracil in rat liver and enzyme induction by phenobarbital

1. When 1-phthalidyl 5-fluorouracil (PH-FU) was incubated with isolated rat hepatocytes, 5-fluorouracil, 2-carboxybenzaldehyde (CBA) and alpha-hydroxymethylbenzoic acid (HMB) were detected as the major metabolites. 2. The enzymes involved in the metabolism of PH-FU, PH-FU hydrolase and CBA reductase are cytosolic and were induced by treating the rats with phenobarbital (PB). Treatment of rats with 3-methylcholanthrene (3-MC) did not affect either enzyme activity. 3. The PB-induced PH-FU hydrolase was inhibited by NADH and several aldehydes, while NAD stimulated the hydrolase and protected it from inactivation by SH reagents. 4. Study in vivo revealed that treatment of rats with PB accelerated the metabolism of PH-FU in the liver and markedly decreased the blood PH-FU after its oral administration to rats, which resulted in reduction of the anti-tumour activity of PH-FU. This activity was not affected by treatment of the rats with 3-MC.     

Reductase-mediated metabolism of motexafin gadolinium (Xcytrin) in rat and human liver subcellular fractions and purified enzyme preparations

The biotransformation of motexafin gadolinium (MGd, Xcytrin) was investigated in subcellular rat and human liver fractions. Microsomal MGd metabolism was dependent on NADPH in both species. Cytosolic metabolism in rat and human livers was dependent on NADPH or NADH. Under anaerobic conditions, MGd metabolism increased in liver microsomes and purified enzyme preparations. Cytochrome P450 (CYP450) inhibitors ketoconazole, proadifen, and carbon monoxide increased NADPH-dependent MGd metabolism in microsomes. Treatment of rats with beta-naphthoflavone increased cytosolic metabolism of MGd twofold, but had no effect on microsomal metabolism. Conversely, in liver preparations from phenobarbital treated rats microsomal metabolism of MGd was enhanced twofold, but not in cytosolic preparations. Purified CYP450 reductase from phenobarbital-treated rabbit or untreated human livers metabolized MGd suggesting involvement of CYP450 reductase. Dicumarol, a potent DT-diaphorase inhibitor, inhibited MGd metabolism in both rat and human liver cytosol. These data suggest MGd metabolism in rat liver involves CYP450 reductase and/or DT-diaphorase. In human liver preparations only CYP450 reductase is directly involved in MGd metabolism. A metabolite identified in microsomes and cytosol is a metal-free, reduced form of MGd, indicating that both enzymes generate metabolite 1, which appears to be PCI-0108, a synthetic precursor to MGd.     

Correlation between the metabolism of hexobarbital and aminopyrine in vivo in rats

Two model substrates for oxidative hepatic enzyme activity, namely hexobarbital and aminopyrine, were simultaneously orally administered to rats, and blood concentrations of the substrates measured by g.l.c. The apparent intrinsic clearances of hexobarbital (Cl*int.HB) and of aminopyrine (Cl*int,AM) were correlated in untreated rats, and in rats pretreated with phenobarbital, 3-methylcholanthrene, polychlorinated biphenyls or carbon tetrachloride. Cl*int,HB and Cl*int,AM were both increased by phenobarbital and polychlorinated biphenyl pretreatment. Pretreatment with 3-methylcholanthrene had hardly any effect, and carbon tetrachloride caused a strong diminution of Cl*int.HB and Cl*int.AM. When the dose of aminopyrine was decreased, both Cl*int,HB and Cl*int,AM increased. This indicated that the primary metabolite of aminopyrine, monomethylaminopyrine, inhibits cytochrome P-450. The correlation coefficient for all clearance data was 0.92 (N = 36). It was concluded that both hexobarbital and aminopyrine are metabolized in vivo by the same or closely related cytochrome P-450 isozymes, and both may be used as model substrates in vivo for metabolic conversions primarily mediated by the major phenobarbital-inducible cytochrome P-450 subspecies.     

Exacerbation of carbon tetrachloride-induced liver injury in the rat by methamphetamine

The effect of methamphetamine cotreatment on carbon tetrachloride-induced liver toxicity was examined in male Sprague-Dawley rats. Concurrent administration of methamphetamine was found to greatly increase the extent of liver injury resulting from carbon tetrachloride treatment, as indicated both by measurement of serum alanine aminotransferase (ALT) activity and from direct histopathologic examination. Concurrent administration of methamphetamine doses less than 10 mg/kg (i.p.), or administration of methamphetamine either before (−3 h) or after (3–9 h) the carbon tetrachloride dose, did not significantly increase liver injury from carbon tetrachloride. These observations indicate that the potentiation by methamphetamine of carbon tetrachloride hepatoxicity previously observed in the mouse also occurs in the rat, and that the timing of the methamphetamine and carbon tetrachloride doses is critical for the interaction.     

Metabolism of thioridazine by microsomal monooxygenases: relative roles of P450 and flavin-containing monooxygenase

1. The metabolism of thioridazine by the flavin-containing monooxygenase (FMO) of mouse liver and several P450 isozymes was examined using microsomes, purified FMO, and expressed P450 isozymes. Metabolites were identified by hplc.

2. Thermal inactivation and antibodies to NADPH P450 reductase were used to selectively inactivate FMO and P450 respectively. Inactivation of FMO by heat-treatment reduced the formation of thioridazine-N-oxide and northioridazine, whereas inactivation of P450 resulted in decreased amounts of thioridazine-2-sulphoxide, northioridazine, and thioridazine-5-sulphoxide.

3. Liver microsomes from mouse induced with phenobarbital, 3-methylcholanthrene, or acetone were compared with control microsomes. Phenobarbital induction resulted in increased formation of all metabolites except thioridazine-N-oxide, while retaining a general metabolic profile similar to that achieved with control microsomes. Neither 3-methylcholanthrene nor acetone induction had any effect on the in vitro metabolism of thioridazine.

4. FMO purified from mouse liver produced thioridazine-N-oxide as the major metabolite.

5. Preliminary experiments with commercially prepared microsomes made from cells expressing recombinant human liver P450 2D6 and 3A4 suggested that thioridazine is metabolized by 2D6 but not 3A4.     

Hepatic mixed-function oxidase activities of wild pigeons

Hepatic mixed-function oxidase activities of wild pigeons were determined and compared with those of rat to assess the apparent differences in avian and mammalian drug metabolism. Aminopyrine N-demethylase, benzphetamine N-demethylase, ethylmorphine N-demethylase, aniline hydroxylase, NADPH-cytochrome c reductase, glutathione S-transferase activities and cytochrome P-450 levels in pigeon liver were 30-80% lower than the corresponding activities in rat liver. p-Nitroanisole O-demethylase activity in pigeon liver was similar to that of rat liver. Wild pigeon-liver benzo[a]pyrene hydroxylase activity was approx. five times higher than that in the rat. Pigeons did not reveal any noticeable sex differences in mixed-function oxidase activities. Administration of 3-methylcholanthrene and phenobarbital to pigeons resulted in the induction of demethylase and benzo[a]pyrene hydroxylase activities and in cytochrome P-450 levels.     

1,3-(2-Chloroethyl)-1-nitrosourea potentiates the toxicity of acetaminophen both in the phenobarbital-induced rat and in hepatocytes cultured from such animals

The toxicity of acetaminophen was studied in hepatocytes cultured from phenobarbital-induced male rats. Such cells were less sensitive to acetaminophen than similar ones cultured from animals induced with 3-methylcholanthrene. In both cases, the toxicity of acetaminophen depended on its metabolism. Inhibition of glutathione reductase with 1,3-(2-chloroethyl)-1-nitrosourea (BCNU) potentiated the toxicity of acetaminophen in the presence or absence of 100 mM acetone, an agent that activates the mixed function oxidation of the toxin. BCNU enhanced the rate and extent of the depletion of GSH in the presence or absence of acetone. Pretreatment of the hepatocytes with the ferric iron chelator deferoxamine or addition to the culture medium of the antioxidant N,N'-diphenyl-p-phenylenediamine prevented the toxicity of acetaminophen in the presence of BCNU whether or not there was acetone in the cultures. BCNU similarly potentiated the hepatotoxicity of acetaminophen in the intact, phenobarbital-induced rat. These data indicate that the mechanism of the killing of hepatocytes induced with phenobarbital is similar to that reported previously with hepatocytes prepared from animals induced with 3-methylcholanthrene. In both cases it would seem that the liver cells are killed by acetaminophen as a result of an oxidative stress that accompanies the metabolism of this hepatotoxin.     

Binding of reactive metabolites of CCl4 to specific microsomal proteins

The covalent binding of [14C]carbon tetrachloride to microsomal proteins in rat liver microsomes under anaerobic conditions was investigated by SDS-polyacrylamide slab gel electrophoresis and fluorography. Most of the labeled proteins were observed in the molecular weight range of 52-61 kDa, indicating that cytochrome P-450 forms (EC 1.14.14.1) were labeled. Protein bands at the position of the NADPH-cytochrome P-450 reductase (78 kDa) (EC 1.6.2.4) and NADH-cytochrome b5 reductase (33 kDa) (EC 1.6.2.2) also showed radioactivity. The fluorographic pattern of the protein labeling was cytochrome P-450-dependent, as was demonstrated by CO and metyrapone inhibition as well as by pretreatment of rats with inducing drugs such as 3-methylcholanthrene, benzo(a)pyrene, phenobarbitone and Aroclor 1254. Immuno-precipitation with a purified anti-P-450 immunoglobulin against cytochrome P-450 PB-B (52 kDa) of rat liver indicated that this protein contained about 10-20% of the total bound radioactivity in an average ratio of 0.8 mol [14C]CCl4-metabolite/mol cytochrome P-450 PB-B.     

Divergent effects of classical inducers on rat plasma and microsomal fraction paraoxonase and arylesterase

The effects of three different enzyme-inducing agents (phenobarbital, 3-methylcholanthrene and rifampicin) on plasma and liver microsomal fraction paraoxonase and arylesterase were studied in rats. Although phenobarbital and 3-methylcholanthrene each increased the esterase activities in microsomal fraction, only 3-methylcholanthrene was capable to increase them in plasma. By contrast, the administration of rifampicin decreased both enzyme activities in liver and plasma. The results indicate that at least there exists two esterase activities in rat liver microsomes which hydrolyse both paraoxon and phenylacetate, but only one of them is released into the blood.     

Metabolism of 1-phthalidyl 5-fluorouracil in rat liver and enzyme induction by phenobarbital

1. When 1-phthalidyl 5-fluorouracil (PH-FU) was incubated with isolated rat hepatocytes, 5-fluorouracil, 2-carboxybenzaldehyde (CBA) and α-hydroxymethylbenzoic acid (HMB) were detected as the major metabolites.

2. The enzymes involved in the metabolism of PH-FU, PH-FU hydrolase and CBA reductase are cytosolic and were induced by treating the rats with phenobarbital (PB). Treatment of rats with 3-methylcholanthrene (3-MC) did not affect either enzyme activity.

3. The PB-induced PH-FU hydrolase was inhibited by NADH and several aldehydes, while NAD stimulated the hydrolase and protected it from inactivation by SH reagents.

4. Study in vivo revealed that treatment of rats with PB accelerated the metabolism of PH-FU in the liver and markedly decreased the blood PH-FU after its oral administration to rats, which resulted in reduction of the anti-tumour activity of PH-FU. This activity was not affected by treatment of the rats with 3-MC.     

Effect of riboflavin deficiency on phenobarbital and 3-methylcholanthrene induction of microsomal drug-metabolizing enzymes of the rat

The effect of riboflavin deficiency on the induction of hepatic microsomal enzymes by phenobarbital and 3-methylcholanthrene has been investigated. A decrease in microsomal flavin levels of 56 per cent was associated with a decrease in NADPH cytochrome c reductase (52 per cent), azoreductase (71 per cent) and benzpyrene hydroxylase (74 per cent). Microsomal cytochrome P-450 content and aminopyrine demethylase were not significantly affected by flavin deficiency. Phenobarbital or 3-methylcholanthrene pretreatment did not affect hepatic microsomal flavin levels in normal or deficient animals. In flavin-deficient animals, phenobarbital pretreatment significantly increased cytochrome c reductase, cytochrome P-450 content, aminopyrine demethylase and azoreductase. Thus the carbon monoxide-sensitive pathway (cytochrome P-450 mediated) of azoreductase was essentially unaffected by flavin deficiency. In deficient animals, the carbon monoxide-insensitive microsomal azoreductase pathway (non-cytochrome P-450 mediated) normally induced by 3-methylcholanthrene was unaffected. Thus, induction of azoreductase by 3-methylcholanthrene was found to be flavin dependent. However, 3-methylcholanthrene did increase cytochrome P-450 content and benzpyrene hydroxylase in flavin-deficient animals. The induction of benzpyrene hydroxylase by 3-methylcholanthrene increased with increasing microsomal flavin content. Part of the mechanism of azoreductase induction by 3-methylcholanthrene was due to an induced change in the structure or composition of microsomal flavoprotein. This interpretation is supported by the findings that: (1) induction by 3-methylcholanthrene in riboflavin-deficient rats required a minimal flavin level, (2) increased enzyme activity was not compensated by an increase in microsomal flavin and (3) induction by 3-methylcholanthrene augmented FMN-stimulation of microsomal azoreductase in vitro.     

Comparative alterations in extrahepatic drug metabolism by factors known to affect hepatic activity.

Various factors known to alter hepatic drug metabolism were examined for their effects on drug metabolism in certain extrahepatic organs, viz. lung and kidney. The prominent sex-related differences in drug metabolism in rat liver were not seen in either lung or kidney. Pretreatment of rats with phenobarbital produced the expected large increases in hepatic NADPH cytochrome c reductase, cytochrome P-450. aminopyrine demethylase and biphenyl hydroxylase activities without concomitant changes in any of these parameters in lung, and only scattered and smaller changes in kidney. 3-Methylcholanthrene (3-MC) pretreatment significantly increased cytochrome P-450 levels in all three organs. Pretreatment of rats with carbon tetrachloride (CCl₄) produced consistent inhibition of mixedfunction oxidation in hepatic microsomes. but the extrahepatic effects were less predictable and were both organ- and enzyme-specific. An increase in renal UDP-glucuronyltransferase activity was observed after CCl₄ treatment that paralleled a similar but larger increase observed in liver. Extrahepatic NADPH cytochrome c reductase and N-methyl-p-chloroaniline demethylase values were unaffected by CCl₄. Lung and kidney responded in a like manner to liver to the additions in vitro of β-diethylaminoethyl diphenylpropylacetate (SKF-525A). Losses in enzyme activities in lung and kidney microsomes roughly paralleled those of liver when stored as pellets for up to 14 days at ?70°. Two or 4 days of starvation produced substrate-specific changes in enzyme-specific activity in liver and kidney, with lung appearing resistant to the effect. When enzyme activity was expressed on a whole organ basis, however, lung cytochrome P-450 values decreased significantly and parameters from liver and kidney increased or decreased in a substrate-specific manner. It is concluded that some physiological and pharmacological factors that influence hepatic drug metabolism produce similar effects in lung and kidney, while other factors produce organ-specific effects.