The stimulation of microsomal azoreduction by flavins

The reduction of the azo dye, amaranth, by rat liver microsomes is inhibited about 90% by carbon monoxide, suggesting that the reaction largely depends on cytochrome P-450. Reducing equivalents for this reaction are supplied by NADPH. This reaction is stimulated by riboflavin, FMN and FAD, as well as by methylviologen. A large fraction of the stimulated reaction is not blocked by CO, indicating that there is a pathway of electron transfer which is dependent of cytochrome P-450. Rat liver microsomes can reduce FAD, with reducing equivalents supplied by NADPH. The FADH₂ thus produced is quickly oxidized by amaranth so that two FADH₂ are oxidized for every amaranth reduced. The same stoichiometry is observed with photochemically prepared FADH₂, formed in the absence of microsomes.     

The induction of liver microsomal proteins by 3-methylcholanthrene in rats of different age,sex and strain

The treatment of male and female adult and male and female immature rats with 3-methylcholanthrene results in increases in the intensity of two liver microsomal protein bands separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The induced protein species have estimated molecular weights of about 55 000 and 52 500. The time course of the induction of these species has been followed in a semi-quantitative manner. In immature rats an additional band, corresponding to a protein species with a molecular weight of about 43 000, is also increased by 3-methylcholanthrene treatment. In male adults rats exclusively, treatment with 3-methylcholanthrene also results in a decrease in the intensity of a band corresponding to a protein species of about 51 000. The appearance of this band in the liver microsomal fraction of adult male rats occurs following sexual maturation and its presence represents a significant difference in the microsomal proteins between male and female rats, this protein is not detected in younger male rats or in female rats at any age. These findings are discussed in terms of their possible relevance to the cytochrome P-450 content of the endoplasmic reticulum of rat hepatocytes.     

Topology of glutathione-insulin transhydrogenase in rat liver microsomes

Glutathione-insulin transhydrogenase (EC 1.8.4.2) catalyzes the inactivation of insulin through scission of the disulfide bonds to form insulin A and B chains. In the liver, the transhydrogenase occurs primarily in the microsomal fraction where most of the enzyme is present in a latent (‘inactive’) state. We have isolated rat hepatic microsomes with latent transhydrogenase activity being an integral part of the vesicles. We have used these vesicles to study the topological location of glutathione-insulin transhydrogenase by investigating the effects of detergents (Triton X-100 and sodium deoxycholate), phospholipase A₂ and proteinases (trypsin and thermolysin) on the latent enzyme activity. Treatment of intact vesicles with variable concentrations of detergents and phospholipase A₂ resulted in the unmasking of latent transhydrogenase activity. The extent of unmasking of transhydrogenase activity is dependent upon the concentration of detergent or phospholipase used and is accompanied by a parallel release of the enzyme into the soluble fraction. Activation of the transhydrogenase by phospholipase A₂ is partially inhibited by bovine serum albumin and the extent of inhibition is inversely proportional to the phospholipase concentration. In intact vesicles, latent transhydrogenase activity is resistant to proteolytic inactivation by both trypsin and thermolysin, while in semipermeable and permeable vesicles these proteases inactivate 60 and 25% of the total transhydrogenase activity, respectively. Together these results indicate that in microsomes transhydrogenase is probably weakly bound to membrane phospholipid components and that most of the enzyme is present on the cisternal surface (i.e., the luminal surface of endoplasmic reticulum) of microsomes. Each detergent and phospholipase apparently unmasks glutathione-insulin transhydrogenase activity through disruption of the phospholipid-enzyme interaction followed by translocation of the enzyme to the soluble (cytoplasmic) fraction and not through increases in substrate availability.     

Effects of lipid peroxidation on adrenal microsomal monooxygenases

Incubation of guinea pig adrenal microsomes with 10^?6 M ferrous (Fe²⁺) ion and adrenal cytosol initiated high levels of lipid peroxidation as measured by the production of malonaldehyde. Cytosol or Fe²⁺ alone had little effect on microsomal malonaldehyde formation. When microsomes were incubated in the presence of Fe²⁺ and cytosol, malonaldehyde levels continued to increase for at least 60 min. Accompanying the lipid peroxidation was a decline in adrenal microsomal monooxygenase activities. The rates of metabolism of xenobiotics (benzphetamine demethylase, benzo[α]pyrene hydroxylase) as well as steroids (21-hydroxylation) decreased as malonaldehyde levels increased. In addition, cytochrome P-450 levels, NADPH- and NADH-cytochrome c reductase activities, and substrate interactions with cytochrome(s) P-450 decreased as lipid peroxidation progressed. Inhibition of lipid peroxidation by increasing microsomal protein concentrations during the incubation period prevented the changes in microsomal metabolism. Malonaldehyde had no direct effects on adrenal microsomal enzyme activities. The results indicate that lipid peroxidation may have significant effects on adrenocortical function, diminishing the capacity for both xenobiotic and steroid metabolism.     

Time-course of effects of toluene on microsomal enzymes in rat liver, kidney and lung during and after inhalation exposure

Inhalation of toluene vapour of 2000 ppm increased the activities of aniline hydroxylase, aminopyrine N-demethylase, aryl hydrocarbon hydroxylase and NADPH-cytochrome c reductase and the concentrations of cytochromes P-450 and b5 in liver microsomes of adult male rats after an exposure period of 1 day or less. Repeated treatments, 8 h daily for 1-16 days, had only a slight further effect. In lung microsomes, the activities of monooxygenases and the concentration of cytochrome P-450 decreased after 6-24 h toluene exposure, but those of cytochrome b5 and NADPH-cytochrome c reductase did not change. In kidney microsomes the changes were mostly insignificant. After discontinuation of exposure the activities of enzymes and the concentrations of cytochromes returned to the control level in 1-4 days. The results obtained resemble the time-courses for the induction of monooxygenases by other inducers. The tissue differences suggest the unequal distribution of various cytochrome P-450 forms and their individual responsiveness to induction in liver, kidneys and lungs.     

Effects of iron-EDTA on uroporphyrinogen oxidation by liver microsomes

Uroporphyrinogen oxidation by hepatic microsomes from chick embryos or mice pretreated with methylcholanthrene was increased by addition of iron-EDTA. This increase was partially prevented by catalase, mannitol, ketoconazole and piperonyl butoxide, whereas only ketoconazole and piperonyl butoxide inhibited the oxidation in the presence and absence of iron-EDTA. These data suggest that the oxidations of uroporphyrinogen in the presence and absence of added iron occur by different mechanisms.     

Topography of phosphatidylcholine,phosphatidylethanolamine and triacylglycerol biosynthetic enzymes in rat liver microsomes

The topography of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol biosynthetic enzymes within the transverse plane of rat liver microsomes was investigated using two impermeant inhibitors, mercury-dextran and dextran-maleimide. Between 70 and 98% of the activities of fatty acid : CoA ligase (EC 6.2.1.3), $\text{sn-}\text{glycerol}\text{-3-}\text{phosphate}$ acyltransferase (EC 2.3.1.15), phosphatidic acid phosphatase (EC 3.1.3.4), diacylglycerol acyltransferase (EC 2.3.1.20), diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) were inactivated by mercury-dextran. Dextran-maleimide caused 52% inactivation of the $\text{sn-}\text{glycerol}\text{-3-}\text{phosphate}$ acyltransferase. Inactivation of each of these activities except fatty acid : CoA ligase occurred in microsomal vesicles which remained intact as evidenced by the maintenance of highly latent mannose-6-phosphatase activity (EC 3.1.3.9). These glycerolipid biosynthetic activities were not latent, indicating that substrates have free access to the active sites. Moreover, ATP, CDP-choline and CMP appeared unable to penetrate the microsome membrane. These data indicate that the active sites of these enzymes are located on the external surface of microsomal vesicles.It is concluded that the biosynthesis of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum.     

Glutathione-dependent protection by rat liver microsomal protein against lipid peroxidation

GSH is an important cellular defense against oxidant injury. Its effect in the rat liver microsomal lipid peroxidation system has been examined. Incubation of fresh rat liver microsomes with ascorbic acid and ADP-chelated iron leads to the peroxidation of microsomal lipids (production of thiobarbituric acid-reactive substances and destruction of polyunsaturated fatty acids) following a 2 to 5 min lag. Addition of 0.1 mM GSH to the system lengthened the lag period by 5 to 15 min without affecting the rate or the extent of lipid peroxidation. GSH could not be replaced in prolonging the lag by cysteine, mercaptoethanol, dithiothreitol, propylthiouracil, or GSSG. The GSH effect on the lag was abolished by heating or trypsin digestion of the microsomes, indicating that microsomal protein is required for its expression. Progressively longer lags were observed as the GSH concentration was increased from 0.1 to 5 mM, but there was no evidence of GSH oxidation as a consequence of the protection against lipid peroxidation. GSH protected against heat inactivation of the microsomal protein responsible for the GSH effect. Experiments with an oxygen electrode revealed that the GSH protection did not alter the ratio of O₂ consumed to thiobarbituric acid-reactive substances produced. This implicated free radical scavenging as the mechanism of protection. These results indicate the existence of a GSH-dependent rat liver microsomal protein which scavenges free radical. This protein may be an important defense against free radical injury to the microsomal membrane.     

Purification of rat liver microsomal cytochrome P-450b without the use of nonionic detergent

Sodium cholate, Emulgen 911, and (3-[(-cholamidopropyl)-dimethyl- ammonio]-1-propanesulfonate) (CHAPS) were selected to examine the effects of ionic, nonionic, and zwitterionic detergents on testosterone hydroxylation catalyzed by four purified isozymes of rat liver microsomal cytochrome P-450, namely P-450a, P-450b, P-450c, and P-450h, in reconstituted systems containing optimal amounts of dilauroylphosphatidylcholine and saturating amounts of NADPH- cytochrome P-450 reductase (reductase). The major phenobarbital-inducible form of rat liver microsomal cytochrome P-450, designated P-450b, was extremely sensitive to the inhibitory effects of Emulgen 911, which is used in several procedures to purify this and other forms of cytochrome P-450. In contrast, sodium cholate and CHAPS had little effect on the catalytic activity of cytochrome P-450b, even at ten times the concentration of Emulgen 911 effecting 50% inhibition (IC-50). By substituting the zwitterionic detergent CHAPS for Emulgen 911, we purified cytochrome P-450b without the use of nonionic detergent. The protein is designated cytochrome P-450b^* to distinguish it from cytochrome P-450b purified with the use of Emulgen 911. NADPH-cytochrome P-450 reductase was also purified both with and without the use of nonionic detergent. The absolute spectra of cytochrome P-450b and P-450b^* were indistinguishable, as were the carbon monoxide (CO)- and metyrapone-difference spectra of the dithionite-reduced hemoproteins. When reconstituted with NADPH-cytochrome P-450 reductase and dilauroylphosphatidylcholine, cytochromes P-450b and P-450b^* catalyzed the N-demethylation of benzphetamine and aminopyrine, the 4-hydroxylation of aniline, the O-dealkylation of 7-ethoxycoumarin, the 3-hydroxylation of hexobarbital, and the 6-hydroxylation of zoxazolamine. Both hemo-proteins catalyzed the 16α- and 16β-hydroxylation of testosterone, as well as the 17-oxidation of testosterone to androstenedione. Both hemoproteins were poor catalysts of erythromycin demethylation and benzo[a]pyrene 3-/9-hydroxylation. The rate of biotransformation catalyzed by cytochrome P-450b^* was up to 50% greater than the rate catalyzed by cytochrome P-450b when reconstituted with either reductase or reductase^*. The activity of cytochrome P-450b and P-450b^* increased up to 50% when reconstituted with reductase^* instead of reductase. In addition to establishing the feasibility of purifying an isozyme of rat liver microsomal cytochrome P-450 without the use of nonionic detergent, these results indicate that the catalytic activity of cytochrome P-450 is not unduly compromised by residual contamination with the nonionic detergent Emulgen 911.     

Differential modulation of hepatic cytochrome P-450 enzymes in rat and syrian hamster by 4′-trifluoromethyl-2,3,4,5-tetrachlorobiphenyl

The effects of a single injection (40 mg/kg) of 4′-trifluoromethyl-2,3,4,5-tetrachlorobiphenyl (CF3) on hepatic cytochrome P-450 monooxygenases were assessed in rat and syrian hamster. The CF3 treatment significantly increased the total amount of cytochrome P-450 in both species. In rats, CF3 treatment caused marked increases in ethoxyresorufin O-deethylase (EROD), arylhydrocarbon hydroxylase (AHH), and testosterone 7α-hydroxylase activities but significantly reduced the activities of benzphetamine N-demethylase (BzND), erythromycin N-demethylase (ErND), testosterone 6β, 16α, and 16β-hydroxylases, and formation of androstenedione. Administration of CF3 to hamsters strongly induced the activities of EROD, AHH, BzND, testosterone 15α, and 16α-hydroxylases, and androstenedione production, whereas ErND, testosterone 6β, and 7α-hydroxylases were decreased. Administration of CF3 to rats induced the CYP1A family proteins and CYP2A1, while CF3 reduced the level of CYP2B1, and, to a lesser extent, of CYP6β2. In hamsters, CF3 treatment significantly induced the CYP1A2, CYP2A1, CYP2A8, and CYP2B1 isozymes, whereas the CYP6β2 level was decreased. The ability of hepatic microsomes to activate aflatoxin B1 and benzo(a)pyrene was elevated by CF3 treatment in hamsters, while activation of aflatoxin B1 was decreased in microsomes from CF3-treated rats. These results showed differences in the CF3-induced pattern of rat and hamster cytochrome P-450 monooxygenases.     

Fructose utilization and altered cytochrome P-450 in cultured hepatocytes from adult rats

Cultured adult rat hepatocytes incubated in media containing fructose exhibit increased levels of cytochrome P-450, relative to cells incubated with equimolar glucose, and the effect of fructose is proportional to its concentration between 2 and 10 mM. For investigating the mechanism of the effect of fructose on cytochrome P-450 in cultured cells, [U-¹⁴C]fructose or [U-¹⁴C]-glucose were added to the incubation medium, and their uptake and utilization were compared. While the uptake kinetics of the two hexoses were similar, the rate of phosphorylation of fructose was more than 10-fold that of glucose. Similarly, the appearance of fructose carbon in metabolic pools, as well as its conversion to CO₂ and cellular glycerolipid, was increased. The latter finding suggested that fructose might alter cytochrome P-450 by stimulating glycerolipid synthesis, since the stability of the cytochrome is lipid-dependent. However, the changes in glycerolipid formation failed to parallel changes in the level of cytochrome P-450 in fructose-treated cells. Moreover, the relative distribution of ¹⁴C into specific lipids was similar for both hexoses, suggesting that an increased carbon flux in cells incubated with fructose did not directly impose a qualitative change in cellular lipid synthesis. We conclude that the fructose-mediated alteration of cytochrome P-450 in cultured rat hepatocytes reflects a process other than increased incorporation of fructose carbon into metabolic pools.     

Alcohol-mediated effects on the level of cytochrome P-450 and heme biosynthesis in cultured rat hepatocytes

Interaction of alcohol and drugs in the liver appears to involve common microsomal oxidative enzymes which utilize cytochrome P-450. Since alcohol augments the toxicity of a variety of drugs, the regulation of the P-450 hemoprotein, a primary component in hepatic drug metabolizing systems, may play a vital role in this phenomenon. We utilize an adult rat liver culture system as a model to explore the action of levels of alcohol below that which is necessary to produce intoxication in humans. The addition of 16 mM ethanol (70 mg/dl) to these hepatocytes results in a 49.5% decrease in cytochrome P-450 activity after 24 h, and a 3-fold increase in the activity of δ-aminolevulinate synthase, the rate-limiting enzyme in hepatic heme biosynthesis. Furthermore, ethanol treatment also causes a transient decrease in the level of intracellular heme. However, the diminished level of total heme does not appear to act as a repressor for δ-aminolevulinate synthase, since it occurs after the initial stimulation of the enzyme by ethanol.     

NADPH:cytochrome P-450(c) reductase: Biochemical characterization in rat brain and cultured neurons and evolution of activity during development

NADPH:cytochrome P-450 (c) reductase is a microsomal enzyme which is involved in the cytochrome P-450-dependent biotransformation of many exogenous agents as well as of some endogenous molecules. Using cytochromec as a substrate, the kinetic parameters of this enzyme were determined in brain microsomes. The comparison of the NADPH:cytochrome P-450 reductase's V_max values and cytochrome P-450 contents in both fractions, suggests a role of cerebral NADPH:cytochrome P-450 reductase in cytochrome P-450 independent pathways. This is also supported by the different developmental pattern of brain enzyme as compared to the liver enzyme, and by the presence of a relatively high NADPH:cytochrome P-450 reductase activity in immature rat brain and neuronal cultures, while cytochrome P-450 was hardly detectable in these preparations. The enzyme activity was not induced by a phenobarbital chronic treatment neither in the adult brain nor in cultured neurons, suggesting a different regulation of the brain enzyme expression.     

Multiplicity of cytochrome P-450 in microsomal membranes from the housefly, Musca domestica

The heterogeneity of cytochrome P-450 in abdominal microsomes from the CSMA, SBO, Fc, Rutgers and Baygon strains of the housefly was examined by three different methods. Examination of ‘apparent absolute absorption spectra’ indicated at least two types of cytochrome in all strains, one with an absorption maximum at about 394 nm, being present in greater quantity in the insecticide-resistant strains, while the other, with an absorption maximum at about 412 nm, predominates in the insecticide-susceptible strains.Controlled tryptic digestion of microsomes followed by spectral examination at various time intervals indicated a heterogeneous population of cytochromes P-450 in CSMA, Fc and Rutgers strains.Subfractionation of microsomes from houseflies of the CSMA and Fc strains by a two-step discontinuous sucrose gradient centrifugation method provided evidence for cytochromes P-450 of different spectral characteristics. The concentration of cytochrome P-450, as well as its spectral characteristics varied between fractions and strains.     

Induction of diamine oxidase activity in rat kidney during compensatory hypertrophy

Diamine oxidase (EC 1.4.3.6) activity, measured as [¹⁴C]Δ¹-pyrroline formation from [¹⁴C] putrescine, was studied in homogenates of rat kidney during compensatory hypertrophy after unilateral nephrectomy. Acetaldehyde and to a lesser degree phenobarbital, at concentrations which did not modify the activity of a preparation of hog kidney diamine oxidase, increased Δ¹-pyrroline formation in kidney homogenate, which suggests that aldehyde-metabolizing enzymes present in this tissue may interfere with yield of Δ¹-pyrroline and that the use of acetaldehyde may give better information on kidney diamine oxidase activity. Other inhibitors of aldehyde-metabolizing enzymes such as chloral hydrate, disulfiram, and pyrazole cannot be used for diamine oxidase determination since they stimulated or depressed this enzyme activity. In rat kidney undergoing compensatory hypertrophy the levels of putrescine, spermidine, and spermine increased rapidly and were followed by an increase in diamine oxidase activity that presented a first peak on day 2 and a second peak on day 6. The administration of cycloheximide or actinomycin D to nephrectomized rats prevented the increase in diamine oxidase activity. The study of the turnover rate of diamine oxidase with cycloheximide demonstrated that the half-life of this enzyme was about 14 h in normal and hypertrophic kidney. These results suggest that the increase in diamine oxidase activity in renal hypertrophy was due to the synthesis of new enzyme rather than to a slowing of its degradation.     

Diamine oxidase activity induction in regenerating rat liver

The synthesis and turnover of diamine oxidase (EC 1.4.3.6) activity was studied in regenerating rat liver after partial hepatectomy using inhibitors of protein and RNA syntheses. The administration to animals of cycloheximide or actinomycin D prevented the increase in diamine oxidase activity normally observed during the first hours after hepatectomy. The study of the turnover rate of diamine oxidase with cycloheximide demonstrated that the half-life of this enzyme was about 15 h in normal and regenerating liver. These results suggest that the rise in diamine oxidase activity in regenerating rat liver was due to the synthesis of new enzyme rather than to a lengthening of its turnover.     

Alteration of liver microsomal monooxygenases and substrate competition with aniline hydroxylase from rats chronically fed low-fat and high-fat-containing alcohol diets

Male Sprague-Dawley rats fed ethanol (EtOH) 36% of total calories for four weeks in a liquid diet containing either 34% (HF) or 12% (LF) of calories as fat were studied with respect to induction of microsomal monooxygenases (MFO) and substrate competition with EtOH-inducible aniline hydroxylase. The specific activity and turnover of aniline hydroxylase were induced to similar extents by HF-EtOH and LF-EtOH diets. Whereas, both LF-EtOH and HF-EtOH caused a decrease in the turnover of arylhydrocarbon (benzo[a]pyrene) hydroxylase (AHH) and aldrin epoxidase compared to pair-fed (PF) controls, LF-EtOH but not HF-EtOH increased the turnover of ethoxycoumarin and ethoxyresorufin O-deethylase (ECOD and EROD). The increase in ECOD and EROD and the decrease in AHH by EtOH is contrary to the parallel induction of these activities by J-methylcholanthrene (3-MC) and Aroclor 1254 (Aroclor). Benzo(a)pyrene (BaP) stimulated aniline hydroxylase in the HF-EtOH and PF systems, whereas with LF diet, stimulation was seen only in the EtOH group. Ethoxycoumarin (EC) inhibited aniline hydroxylase by microsomes from EtOH- and pyrazole-treated rats, whereas it stimulated aniline hydroxylase by control microsomes, suggesting that the EC effects were associated with EtOH-inducible cytochrome P-450. Ethoxyresorufin (ER) inhibited aniline hydroxylase in EtOH and PF groups, thus the differential effects of EC were not nonspecific O-deethylase effects. The effects of EtOH feeding on ECOD, EROD, and AHH (ie, substrates for 3-MC-inducible cytochrome P-450) displayed a greater differential between the experimental and control group with the LF- than with the HF-containing diet. The findings suggest that the alteration of certain MFO activities by chronic EtOH ingestion can be modified by the content of dietary fat. Moreover, the competition dynamics of MFO substrates toward EtOH-inducible aniline hydroxylase are altered by EtOH feeding and, in turn, modified by dietary fat.     

Absorption spectra of highly purified liver microsomal cytochrome P-450 in non-equilibrium conformational states at low temperatures

Absorption spectra of highly purified liver microsomal cytochrome P-450 in non-equilibrium states were obtained at 77 K by reduction with trapped electrons, formed by gamma-irradiation of the water-glycerol matrix. In contrast to the equilibrium form of ferrous cytochrome P-450 with the heme iron in the high-spin state the non-equilibrium ferrous state has a low-spin heme iron. The absorption spectrum of the non-equilibrium ferrous cytochrome P-450 is characterized by two bands at 564 (-band) and 530 nm (-band). When the temperature is increased to about 278 K this non-equilibrium form of the reduced enzyme is relaxed to the corresponding equilibrium form with a single absorption band at 548 nm in the visible region characteristic for a high-spin heme iron.     

Changes in immunohistochemical distribution of cytochrome MC-P-448 in the rat liver during acclimation to cold

Immunohistochemical localization of cytochrome MC-P-448 (form of cytochrome P-450 induced by methylcholanthrene) in rat hepatic lobule and the changes in their distribution pattern in response to cold exposure at 4°C were investigated. The distribution of hepatocytes expressing immunoreactivity to cytochrome MC-P-448 was demonstrated with rabbit anti-MC-P-448 serum using a microphotomeasurement system P1 (Nikon). A duration of cold exposure for 1, 2, 3 or 4 weeks at 4°C was applied to study the effect of cold adaptation of cytochrome MC-P-448. In control rats housed at 24°C, hepatocytes showing high immunoreactivity to cytochrome MC-P-448 were located in the centrilobular areas of the hepatic lobules, whereas they disappeared markedly in the 4-week cold-exposed rats. In 1-week and 2-week cold-exposed rats, only a slight decrease in the expression of MC-P-448 positive hepatocytes was observed. These changes were clearly seen by visual inspection of the distribution topography as determined by a microphotomeasurement technique. In conclusion, cytochrome MC-P-448 forms which were predominantly located in centrilobular areas in the hepatic lobule decreased in 4-week cold-exposed rats. This was in contrast to our early report which showed an increasing tendency of cytochrome PB-P-450 forms in 4-week cold-exposed rats.     

Induction of hepatic drug-metabolising enzymes and tamoxifen metabolite profile in relation to administration route during low-dose treatment in nude rats

Tamoxifen is the most used anticancer drug and is approved for chemoprevention. Little is known about the enzyme inducing properties of low-dose regimens and the influence of route of administration. In this study, nude rats received 5 mg/kg/day of tamoxifen orally or a 50 mg continuous-release pellet subcutaneously. The mRNAs for cytochrome P450-enzymes (CYPs), flavin-containing monooxygenase 1 (FMO1) and phase II drug-metabolising enzymes were quantified by real-time RT-PCR. Tamoxifen and metabolite concentrations were measured using HPLC. We observed a significant increase in CYP3A18 and FMO1 mRNA expression levels in the orally treated animals, whereas the increase in CYP3A2 expression did not reach statistical significance (p = 0.057). No significant induction of enzyme expression was observed in rats that received subcutaneous (S.c.) treatment. After 33 days the serum levels of 4-hydroxytamoxifen (4OHtam), tamoxifen and N-desmethyltamoxifen (NDtam) in orally treated animals were 1.8 ± 0.7, 11.1 ± 3.2 and 11.4 ± 3.8 ng/ml, respectively. In subcutaneously treated animals, tamoxifen and N-desmethyltamoxifen were detected in tissues, but not in serum. These data demonstrate that in contrast to the subcutaneous administration, low-dose oral tamoxifen induced tamoxifen-metabolising enzymes. Furthermore, the different routes of administration resulted in different serum and tissue levels of tamoxifen and metabolites.