Binding Of Aniline Research Articles

Selenium antagonism of cadmium-induced inhibition of hepatic drug metabolism in the male rat

Experiments were undertaken to examine the effect of selenium, an essential trace element, on cadmium-induced inhibition of drug metabolism in male, Sprague-Dawley derived rats. Prior administration of sodium selenite (1.6 mg Se/kg, ip) blocked the cadmium-induced (0.84 mg Cd/kg, ip) prolongation of hexobarbital-induced hypnosis and inhibition of hepatic microsomal biotransformation of ethylmorphine or aniline. Selenium also blocked cadmium-induced reduction in microsomal cytochrome P-450 content and the microsomal binding of both ethylmorphine and aniline. However, pretreatment of rats with selenium did not prevent the inhibitory effect of cadmium (10 −6 to 10 −3 m) added in vitro on either ethylmorphine or aniline biotransformation. In addition, the reduction in biotransformation of both substrates following in vivo cadmium administration was not reversed following the in vitro administration of selenium but, in fact, selenium produced further concentration-dependent decreases in drug metabolism. In additional in vitro experiments it was found that the inhibition in drug metabolism induced by in vitro additions of cadmium is not affected by similar additions of selenium when added to the incubation vessel either before or after the cadmium. Thus, for selenium to prevent the cadmium-induced inhibition of hepatic drug metabolism requires in vivo administration of selenium.

Tolerance Development to Cadmium-Induced Decrease in Hepatic Oxidative Xenobiotic Metabolism and Cytochrome P-450 Content in the Male Rat

Tolerance Development to Cadmium-Induced Decrease in Hepatic Oxidative Xenobiotic Metabolism and Cytochrome P-450 Content in the Male Rat. Roberts, Stanley A. and Schnell, R. Craig. (1981). Fundam. Appl. Toxicol 1:286–289. Cadmium, in dose of 0.84 mg/kg (ip), produces a marked inhibition in hepatic oxidative xenobiotic metabolism which results from a decreased cytochrome P-450 content in the male rat. The inhibitions in microsomal metabolism of aniline, ethylmorphine, or p-nitroanisole were not elicited by the 0.84 mg/kg cadmium dose in rats which had been pretreated (72 hours prior) with a lower dose of cadmium (0.21 mg Cd/kg, ip). Likewise, the decreases in both microsomal cytochrome P-450 content and in microsomal spectral binding of hexobarbital or aniline produced by the 0.84 mg/ kg cadmium dose were not elicited in the rats pretreated with the low cadmium dose. The 0.21 mg/kg cadmium dose produced an increase (370%) in the cadmium-binding capacity as well as an increase (395%) in total hepatic metallothionein. These data are consistent with the hypothesis that the pretreatment of animals with relatively low cadmium doses produces an increase in the cellular levels of metallothionein which can then bind and ameliorate the toxicological effects of a subsequently administered toxic dose of cadmium.

Inhibition of cytochrome P-448 mixed function oxidase activity following administration of 9-hydroxyellipticine to rats

The in vitro inhibitor of mixed-function oxidation, 9-hydroxyellipticine, non-competitively inhibited the binding of the type II substrate, aniline, to cytochrome P-448 of hepatic microsomal preparations from rats pretreated with 3-methylcholanthrene. In contrast, 9-hydroxyellipticine did not inhibit the binding of aniline to cytochrome P-450 of hepatic microsomal preparations from rats pretreated with phenobarbitone, nor did it inhibit the binding of the type I substrate, hexobarbitone to either cytochrome P-450 or cytochrome P-448. Following the pretreatment of rats intraperitoneally with 9-hydroxyellipticine and phenobarbitone, the cytochrome P-448-specific enzyme activity, ethoxyresorufin O-deethylase, was 50% inhibited in vitro but cytochrome P-450, cytochrome P-450 reductase, and other mixed function oxidase activities were unaffected. With rats pretreated with 9-hydroxyellipticine and 3-methylcholanthrene, inhibition of ethoxyresorufin O-deethylase was 90%, and cytochrome P-450 P-448 , cytochrome P-450 reductase, biphenyl 2- and 4-hydroxylase were inhibited by 30, 15, 50 and 40% respectively. It is concluded that 9-hydroxyellipticine administered in vivo markedly inhibits mixed-function oxidations which are specific to cytochrome P-448, but has no effect on cytochrome P-450-catalysed microsomal oxidation.

Inhibition of rabbit liver microsomal oxidative metabolism and substrate binding by tamoxifen and the geometric isomers of clomiphene

The inhibitory effects of the triarylethylene antiestrogens tamoxifen (TAM) and enclomiphene (EC), and of the estrogenic geometric isomer of the latter compound (zuclomiphene, ZC) on the binding and metabolism of standard substrates in the presence of rabbit liver cytochrome P-450 have been studied. In the presence of microsomes at pH 7.4, the triarylethylenes interfered with the binding of β-diethylaminoethyl diphenylpropylacetate (SKF 525-A) and with the N-demethylation of ethylmorphine. Their inhibitory effects were less pronounced on the binding and metabolism (hydroxylation) of aniline. Kinetic studies showed that EC and TAM interfered with SKF 525-A binding by a mixed mechanism. These results suggest that the triarylethylenes affect preferentially the binding of ‘Type I’ substrates with cytochrome P-450.

Induction of rat hepatic microsomal enzymes by toxaphene pretreatment.

The effects of pretreatment of rats with toxaphene on hepatic drug metabolizing enzymes and several other parameters of the mixed function oxidase system were investigated. Adult male Sprague-Dawley rats were fed diets containing 0, 50, 100, 150 and 200 ppm of toxaphene for 14 days. The body weight gain was unaltered as well as the food consumption in all the toxaphene fed groups. There was no change in the weights of brain, kidney, heart, and testes but the liver weight was significantly increased. The thymus weight in all the toxaphene fed grups was decreased. Hydroxylation of pentobarbital and aniline was significantly enhanced in rats exposed to toxaphene. Ethylmorphine-N-demethylase activity in the toxaphene treated rats was also elevated. Enhanced hydroxylation of pentobarbital was also evident from the decreased sleeping time following pentobarbital administration. Exposure to toxaphene increased cytochrome P-450, NADPH-cytochrome c-reductase and dehydrogenase in hepatic microsomal fractions. The binding of aniline and hexobarbital to microsomes was also enhanced, suggesting that the intermediate steps in the electron-transfer system were increased. In conclusion, pretreatment of rats with toxaphene for fourteen days resulted in the induction of the hepatic mixed function oxidase system.

The inhibition and potentiation of procarbazine on hepatic mixed-function oxidases in phenobarbital tolerant and nontolerant mice.

The effect of procarbazine on mixed-function oxidases was investigated in naive and pentobarbital tolerant mice. In mice receiving procarbazine, 200 mg/kg, i.p. 1 hr earlier, metabolisms of pentobarbital, aniline and ethylmorphine in vitro and cytochrome P-450 content of hepatic microsomes were significantly decreased. The drug binding of either aniline or pentobarbital to cytochrome P-450 was also decreased. However, procarbazine failed to exert this effect after the enzymes had been induced by continuous administration of pentobarbital. Interestingly, procarbazine enhanced the barbiturate induced hepatic microsomal mixed-function oxidase activities when it was administered before the implantation of pentobarbital pellet. Both cytochrome P-450 and cytochrome b5 content after pentobarbital pellet implantation were further increased by pretreatment with procarbazine. This finding was further substantiated by the increase in pentobarbital and aniline binding to cytochrome P-450. The present studies may provide another model for studying the nature of hepatic mixed-function oxidase induction process.

Studies on the mechanism of cadmium-induced inhibition of the hepatic microsomal monooxygenase of the male rat

Cadmium is a potent inhibitor of hepatic drug metabolism but little is known about the biochemical mechanisms involved. Male rats receiving a single dose of cadmium acetate (2.0 mg Cd 2+/kg, ip) exhibited significant decreases in hepatic microsomal metabolism of hexobarbital (79%), ethylmorphine (71%), and aniline (47%), as well as decreased amounts of both microsomal cytochrome P-450 (39%) and cytochrome b 5 (30%) 72 hr after administration of the metal. In addition, the magnitudes of microsomal hexobarbital, ethylmorphine, and aniline binding spectra were significantly reduced. NADPH-cytochrome c reductase activity was not altered. In vitro addition of Cd 2+ (10 −6 to 10 −3 m) to liver microsomes produced a concentration-dependent inhibition of the metabolism of the substrates tested, a reduction in the level of cytochrome P-450 which was accompanied by an increase in the level of the inactive form of the hemo-protein, P-420, and decreases in the microsomal binding spectra of the three substrates. In kinetic studies, apparent V max values were significantly lowered for ethylmorphine N-demethylase (75%) and aniline hydroxylase (57%) by cadmium treatment. The apparent K m value for aniline hydroxylase was not altered, but that for ethylmorphine N-demethylase was significantly decreased following cadmium treatment. After in vitro cadmium addition, concentration-dependent decreases were observed in the apparent V max and K m values for both microsomal reactions.

Partial purification and properties of cytochrome P450 from homogenates of human fetal livers

Using ω-amino-n-octyl Sepharose 4B and hydroxylapatite columns, cytochrome P450 was purified to approx. 6.7 nmoles per mg of protein from the 105,000 g precipitate of homogenates of human fetal livers. The partially purified preparation of cytochrome P450 was free of detectable amounts of cytochrome b 5, NADPH-cytochrome P450 reductase and NADH-cytochrome b 5 reductase. The absolute spectrum of the preparation exhibited a peak at 417 nm in the Soret region, indicating that this cytochrome P450 is a low spin species. Binding of aniline and SKF 525-A to this partially purified preparation of cytochrome P450 produced a modified type II and a type I difference spectra, respectively. As judged by Ouchterlony double diffusion analysis, the cytochrome P450 preparation did not cross react with antibody against cytochrome P450 isolated from the livers of phenobarbital-pretreated rats. In reconstituted systems, the cytochrome exhibited considerable activity for aniline hydroxylation but only a low ethlymorphine N-demethylation activity compared to cytochrome P450 isolated from the livers of phenobarbital-pretreated rats.

Inhibition of rat hepatic microsomal cytochrome P-450 system by cobaltous chloride and reversal of inhibition by iron in vivo.

The effects of preexposure of rats to cobaltous chloride (CO) mixed in iron-sufficient (I.S) and -deficient (I.D) diets on hepatic microsomal electron transfer system was investigated. Male Sprague-Dawley rats were fed for 4 weeks on I.D diets mixed with 0, 100 and 200 ppm CO. At the end of 4 weeks three rats from each group were transferred to I.S diets mixed with the same amount of CO. Liver microsomal NADPH - Cytochrome C reductase, NADPH - dehydrogenase, cytochrome P-450 and aniline binding were determined in both batches of rats. The rats receiving CO in the I.D diets showed a 35, 60, 75 and 40% decrease in NADPH - Cyt. C reductase, dehydrogenase, Cyt. P-450 and aniline binding respectively. The rats transferred from I.D diet to I.S diet showed a complete recovery of the inhibition of the microsomal electron transfer system. The rats receiving 200 ppm mixed with I.S diets did not show any changes in any biochemical parameter measured.

The degradation of different forms of cytochrome P-450 [formula omitted] by fluroxene and allyl- [formula omitted]-propylacetamide

Treatment of uninduced, phenobarbital and 3-methylcholanthrene induced rats with fluroxene and allyl- iso -propylacetamide decreased hepatic microsomal cytochrome P-450 and equivalently decreased microsomal heme, aniline binding and p -nitroanisole demethylase. In contrast, ethylmorpnine demethylase, benzpyrene-3-hydroxylase and ethoxyresofurin deethylase were not in all cases decreased in proportion to the loss of cytochrome P-450. After phenobarbital induction fluroxene and allyl- iso -propylacetamide degrade multiple forms of cytochrome P-450, but degrade in the greatest amounts the form(s) of cytochrome P-450 inducible by phenobarbital. After 3-methylcholanthrene induction fluroxene preferentially degrades cytochrome P-448, while allyl- iso -propylacetamide is relatively specific for the form(s) of cytochrome P-450 inducible by phenobarbital.

Effect or oral administration of propylene glycol on the induction of enzymes and proteins in microsomes and cytosol of the rat liver.

Male Wistar rats were divided into 4 groups, receiving water, propylene glycol (PG), sodium phenobarbital (PB) and PB + PG, respectively. After oral administration of these materials for 7 days, various enzymes in hepatic microsomes and the binding capacity of Y and Z fractions in the cytosol were assayed. As compared with the water group, the aniline hydroxylase activity, the cytochrome b5 content and the amount of Z fraction increased in the PG group. As compared with the PB group, the microsomal protein increased but the activities of paranitrophenol-UDP-glucuronyltransferase (pNP-GT) and aminopyrine demethylase (AD) decreased in the PB + PG group. When the PB + PG group and the PG group were compared with the PB group and the water group respectively, there was no parallel change. Even though PG added to hepatic microsomes of untreated rats in vitro, there was no change in the difference spectrum produced by binding of cytochrome p-450 and aniline and also in the activities of AH and pNP-GT. Based on these observations, it can be concluded that PG affected the in vitro assay data of drug-metabolizing enzyme without changing the cytochrome P-450 content by its certain in vivo action rather than by its direct interaction with microsomes, and modified the effect of PB on the microsomal enzymes and the cytosol proteins. Therefore, it is not appropriate to investigate the effect of the drugs on enzymes and proteins of the liver cell by the use of PG as a solvent.

Effect of preexposure to kepone on hepatic mixed-function oxidases in the female rat

The effects of preexposure of female rats to Kepone on hepatic drugmetabolizing enzymes and several other parameters of the multifunction oxidase system were investigated. Animals were exposed to 0, 50, 100, and 150 ppm Kepone in the daily ration for 16 days. There was a dose-related decline in body weight gained to 72, 55, and 22% of the controls for 50, 100, and 150 ppm Kepone, respectively. Liver weight was unaltered at all three levels of dietary Kepone. While hydroxylation of aniline, pentobarbital, and hexobarbital were all enhanced, the increase in metabolism of the latter two substrates was consistently higher than that of controls. Enhanced metabolism of pentobarbital was also evident from the dose-related decrease in the pentobarbital-induced sleeping time. Aminopyrine and ethylmorphine demethylase activities were increased three- to fourfold over the controls. Cytochrome P-450, NADPH-cytochrome c reductase, and aniline binding were all increased, suggesting that there were general increases in the intermediate steps in the electron-transfer system within the multifunction oxidase complex. Cytochrome b 5 was unaltered by Kepone and only a 100-ppm dose elicited a discernible increase in NADPH dehydrogenase activity. These effects in the female rats, along with previously published effects in the male rat, are suggestive of the potential of Kepone to modify hepatic drug-metabolizing function in animals of both sexes.

The nature of the rate-limiting step in aniline hydroxylation involving cytochrome P-450 rat liver microsomes

The kinetics of aniline hydroxylation was studied with: (1) rat liver microsomes involving NADPH and O 2 (system 1), (2) hepatic microsomes and tert-butylhydroperoxide (system 2) and (3) microsomes and cumyl hydroperoxide (system 3) at 15–37 °C. The reactions were characterized by the values of the aniline oxidation rate constants, k 2 = V/E 0, where E 0 is the initial concentration of cytochrome P-450: k 1 2 = 1.60 · 10 8 exp (−13 400/RT) sec −1, k 2 2 = 1.66 · 10 9 exp (−14500/RT) sec −1, k 3 2 = 6.83 · 10 9 exp (−15300/RT) sec −1. The values of ΔH 0 ∗ and ΔS 0 ∗, were calculated and compared for the three systems. The evidence suggests that oxygen insertion into the substrate molecule is the rate-limiting step in the reaction of aniline oxidation for the mentioned systems. The nature of aniline binding to cytochrome P-450 and that of the hydroxylating agent have been discussed.

Kepone induction of hepatic mixed function oxidases in the male rat

Effect of preexposure to kepone on hepatic drug metabolizing enzymes and several parameters of mixed function oxidase (MFO) system was investigated. Male rats were exposed to 0, 50, 100 and 150 ppm kepone in the daily ration for 16 days. Gain in body weight was declined after kepone to 86, 62 and 33% of controls for 50, 100 and 150 ppm kepone respectively. Liver weight was unaltered at all three levels of kepone. While hydroxylation of both aniline and pentobarbital was enhanced, the increased in aliphatic hydroxylation (i.e., pentobarbital) were greater at all levels of exposure to kepone. Aminopyrine demethylase was enhanced roughly 3 fold at 50 ppm kepone and was not further increased at higher levels of kepone. Cytochrome P 450, NADPH-cytochrome c reductase, and aniline binding were all increased suggesting that there were general increases with intermediate steps of the electron transfer and drug oxidation system. Cytochrome b 5 and NADPH dehydrogenase were unaltered after kepone treatment. These results suggest that kepone is an efficient inducer of hepatic MFO system.

Mechanism of inhibition by carbonyl cyanide m-chlorophenylhydrazone and sodium deoxycholate of cytochrome P-450-catalysed hepatic microsomal drug metabolism.

1. Treatment of liver microsomal fraction with 0.03-0.12% sodium deoxycholate and 0.005-0.06 mM carbonyl cyanide m-chlorophenylhydrazone decreases phospholipid-dependent hydrophobicity of the microsomal membrane, assayed by the kinetics of 8-anilinonaphthalene-1-sulphonate binding and ethyl isocyanide difference spectra. 2. Sodium deoxycholate at a concentration of 0.01% lacks its detergent properties, but competitively inhibits aminopyrine binding and activates the initial rate of NADPH-cytochrome P-450 reductase. In the presence of 0.03-0.09% sodium deoxycholate the rate-limiting factor in p-hydroxylation of aniline is the content of cytochrome P-450. and that for N-demethylation of aminopyrine is the activity of NADPH-cytochrome P-450 reductase. 3. Carbonyl cyanide m-chlorophenylhydrazone has no effect on the binding and metabolism of aniline; investigation of its inhibiting effect on aminopyrine N-demethylase established that the rate-limiting reaction is the dissociation of the enzyme-substrate complex in the microsomal preparations. 4. In the mechanism of action of carbonyl cyanide m-chlorophenylhydrazone the key step may be the electrostatic interaction of its protonated form and one of the forms of activated oxygen at the catalytic centre of cytochrome P-450. 5. at least two different phospholipid-dependent hydrophobic zones are assumed to exist in the microsomal membrane, both coupled with cytochrome P-450. One of them reveals selective sensitivity to the protonation action of carbonyl cyanide m-chlorophenylhydrazone and contains the 'binding protein' for type I substrates and NADPH-cytochrome P-450 reductase; the other contains the cytochrome P-450 haem group and binding sites for type II substrates.

Haem accessibility in cytochrome P-450 from rabbit liver. A proton magnetic relaxation study by stereochemical probes

Cytochrome P-450 was solubilized from phenobarbital induced rabbit liver and purified by affinity chromatography. The longitudinal proton magnetic relaxation rates of this ferric, low-spin sample (as confirmed by ESR) in 20% glycerol aqueous solution are very large compared with low-spin methaemoglobin and myoglobin derivatives. Similarly high rates were measured in a deuterated solution using the aliphatic protons of glycerol as stereochemical markers, which strongly suggests that the haem iron in cytochrome P-450 is much more accessible to the solvent than in haemoglobin or myoglobin. Type I substrate (Spasman ®) produced small but significant increases in NMR rates both in the H 2O and in the 2H 2O solution, while binding of aniline (Type II substrate) doubled the rates.

Influence of the oral contraceptive, menstranol, on drug-metabolizing enzymes of female rats in thiamin-supplemented and deficiency states.

The administration of a diet deficient in thiamin results in elevated hepatic microsomal activity of aniline hydroxylase, ethylmorphone demethylase, NADPH cytochrome c reductase and cytochrome P-450 when compared to similar female rats fed diets supplemented with thiamin. Accompanying these differences in enzyme activity are increased concentrations of microsomal docosahexaenoic acid and arachidonic acid. Binding of aniline to microsomes from rats fed high levels of thiamin is decreased due to a decrease in cytochrome P-450. On the other hand, the binding of ethylmorphine to P-450 is decreased by feeding high levels of thiamin. The daily administration of mestranol enhanced ethylmorphine and aniline metabolism to a greater extent in rats fed thiamin-rich diet than in rats fed thiamin-deficient diet or laboratory chow. This treatment did not increase cytochrome P-450 cytochrome c reductase or microsomal protein nor does it appear to affect the binding of aniline to cytochrome P-450. Ethylmorphine binding is generally decreased by this treatment. Alterations in the Michaelis constants for these reactions were limited to an increase in the Ks for aniline in pair-fed animals and in the Ks for ethylmorphine in thiamin-deficient rats receiving 1.0 mg mestranol per day.

Cytochrome components of plant microsomes.

The electron transport components of the microsomal fraction of cauliflower buds and mung bean hypocotyls were investigated using split-beam and dual wavelength spectrophotometry under a variety of reducing conditions. Cauliflower microsomes were found to contain an ascorbate-reducible component, termed cytochrome b-559.5 [E'0 = +135 +/- 20 mV; lambdamax (reduced minus oxidised) = 559.5, 527 and 429 nm at 23 degrees C], cytochrome b5 [E'0 = -20 +/- 20 mV; lambdamax (reduced minus oxidised) = 556, 526 and 425 nm at 23 degrees C], cytochromes P-450 and P-420. On the basis of binding studies with ethyl isocyanide, degradation of cytochrome P-450 to P-420, redox potential, aniline binding, and relative rates of reduction by NADPH and NADH, it is suggested that the cytochrome P-450 system is analogous to that mammalian microsomes. Other components, reducible only by dithionite, may also be present. Mung bean microsomes were found to contain an ascorbate-reducible component, termed cytochrome b-562 [E'0 = +120 +/- 20 mV; lambdamax (reduced minus oxidised) = 562, 528 and 430 nm at 23 degrees C], cytochrome b5, and a low potential component which was reducible only by sodium dithionite. No cytochrome P-450 or P-420 could be detected. A general method of analysis of the cytochromes was developed and applied to the microsomes from a variety of plant sources. The results indicate that large variations, both in type and amount of components, occur between the microsomes from different plant materials.

Effect of protein deficiency on the inducibility of the hepatic microsomal drug-metabolizing enzyme system—III: Effect of 3-methylcholanthrene induction on activity and binding kinetics

Male, weanling rats divided into three groups were maintained for 15 days on a semipurified diet containing either 5% casein fed ad lib. (group 1), 20% casein pair-fed to group 1 (group 2), or 20% casein fed ad lib. (group 3). After each group was further subdivided, animals were injected i.p. on days 13 and 14 with either corn oil or 3-methylcholanthrene (3-MC) (20 mg/kg) in corn oil. Twenty-four hr after the last injection, animals were decapitated and liver microsomes were prepared. Contents of microsomal protein, phosphatidylcholine and hemoprotein (cytochrome P-450 or cytochrome P-448 or both) were measured and used as the basis of expression for spectral and enzyme kinetic constants; the activities of cytochrome c and P-450 reductases were also determined. Protein deficiency reduced microsomal protein and hemoprotein, but did not alter phosphatidylcholine (PC); 3-MC treatment produced increases in protein, hemoprotein and PC/mg of protein, but a decrease in PC/hemoprotein. In general, 3-MC produced greater percentage increases in the above parameters with protein deficiency, although the absolute amounts remained considerably below that achieved with groups 2 and 3. The 3-MC-induced groups showed the characteristic spectral shift for the P-450-CO complex to 448 nm. Ethylmorphine (EM) metabolism was decreased by both protein deficiency and 3-MC induction, whereas aniline (AN) metabolism was decreased by protein deficiency but increased by 3-MC treatment. Spectral binding ( Δ A max) of EM was decreased by protein deficiency and unchanged by 3-MC when expressed on a protein basis, but was reduced when expressed on a hemoprotein basis, indicating the loss of the type I site in the 3-MC-induced hemoprotein. Aniline binding, when based on protein, was decreased by protein deficiency but remained unchanged when expressed on a hemoprotein basis, whereas 3-MC treatment increased the Δ A max when expressed on a protein basis, but did not change this parameter when expressed on a hemoprotein basis, indicating the retention of the type II site after 3-MC induction. Protein deficiency reduced both cytochrome c and P-450 reductase activities, whereas 3-MC treatment did not alter these parameters. Based on these and previous data, a role for the type I binding site as a “substrate effector site” was proposed.

Effects of thiamin antagonists on drug hydroxylation and properties of cytochrome P-450 in the rat

The administration of small amounts of thiamin (0.3 μg day or more, i.p., for 21 days) depressed musomal cytochrome P-450 content and the V max of aniline hydroxylase when compared to values obtained from rats fed a thiamin-deficient diet (approximately 0.1 μg of thiamine/day in basal diet). The concurrent administration of neopyrithiamin (50 μg day , i.p.) eliminated the depressant effect of 10.0 μig of thiamin/ day, but was without significant effect in rats receiving more than 100 μg of thiamin/ day. In contrast, 100 μg of oxythiamin/day had no thiamin-opposing effect on cytochrome P-450 content and only partially counteracted the effects of 1 μg of thiamin/day on aniline hydroxylase activity. These treatments were without significant effect on the K m for this reaction. Using ethyl isocyanide as the ligand, there appears to be a qualitative change induced in the cytochrome P-450 from thiamin-deficient rats. The absorption peak height ratios indicate that cytochrome P 1-450 is increased in a manner analogous to that produced by the administration of 3-methylcholanthrene. This was supported by the fact that aniline binding, as evidenced by increased ΔA max, is enchanced in musomes from thiamin-deficient animals, whereas the hexobarbital spectral shift was unaltered.