Phenobarbital-pretreated Animals Research Articles

Inhibition of aldo-keto reductases by phenobarbital alters metabolism, pharmacokinetics and toxicity of doxorubicin in rats.

Doxorubicin is an effective anticancer agent that is limited by numerous adverse effects, cardiotoxicity causing the most concern. Its alcohol metabolite, doxorubicinol, and free radicals have been implicated in the aetiology of this toxicity. This study was based on the premise that inhibition of aldo-keto reductases would improve the efficacy of doxorubicin by reducing its toxic metabolites and modifying its pharmacokinetics. We assessed the effect of in-vitro inhibition of aldo-keto reductases on the metabolism of doxorubicin in cytosolic fractions of heart and liver of rats in the presence of Na-phenobarbital. The inhibition was confirmed by a significant reduction in the formation of doxorubicinol. The results of the in-vitro study were further evaluated in-vivo. The concentrations of doxorubicin in plasma, bile and urine and its major metabolites in bile and urine were measured in Na-phenobarbital-pretreated rats. Each rat received 100 mg kg(-1)/day intraperitoneal injection of sodium phenobarbital for three days followed by a single intravenous dose of 10 mg kg(-1) [14C-14]doxorubicin (sp. act. 0.2 microCi mg(-1)) on the fourth day. The levels of drug in all biological samples were measured by HPLC. The pretreatment resulted in an increase in biological half-life (5.8 +/- 1.5 vs 3.7 +/- 0.93 h control group, P < 0.05) and area under plasma concentration-time curve (19.6 +/- 1.7 vs 14.65 +/- 1.68 mg h L(-1) control group, P < 0.05). The cumulative amount of doxorubicinol in the bile and urine of pretreated animals was reduced significantly. In terms of % dose, the amount in the bile declined from 4.2 +/- 0.8% in control to 2.4 +/- 0.3% and in urine from 0.18 +/- 0.08% to 0.12 +/- 0.07%. There were no significant changes in doxorubicin aglycone and doxorubicinol aglycone. Serum creatine kinase levels were measured as a biomarker of damage to cardiac muscle. The area under creatine kinase level-time curve was reduced by approximately 50% in phenobarbital-pretreated animals. The results indicate that the inhibition of aldo-keto reductase could provide a useful approach to improve the safety of doxorubicin by reducing its alcohol metabolite. Furthermore, if the reduction in the area under the serum creatine kinase-time curve represents a reduced damage to heart muscle, it can be concluded that doxorubicinol plays an important role in this injury.

Induction of megamitochondria by some chemicals inducing oxidative stress in primary cultured rat hepatocytes

Effects of hydrazine, hydrogen peroxide and bromobenzene, inducers of free radicals, and those of erythromycin and cycloheximide, inhibitors of protein synthesis on structural changes of mitochondria in primary monolayer culture of rat hepatocytes were examined using laser confocal microscope and electron microscope. After 22 h of incubation of hepatocytes with 0.2 mM hydrogen peroxide or 10 μg ml −1 of erythromycin, mitochondria became extremely enlarged. Mitochondria of hepatocytes isolated from control rats became slightly to moderately enlarged in the presence of 2 mM hydrazine, while those of hepatocytes isolated from phenobarbital-pretreated animals became extremely enlarged in the presence of 2 mM hydrazine. Cycloheximide (0.5–10.0 μg ml −1) and bromobenzene (0.1–1.0 mM) failed to induce structural changes of mitochondria. The level of cytochrome P-450 in freshly prepared hepatocytes from phenobarbital-treated rats was 2.5 times higher than that from the control rats, and remained about three times higher than the latter after 22 h of incubation with 2 mM hydrazine. The level of malondialdehyde was invariably elevated when megamitochondria were induced. These results may suggest that oxidative stress is intimately related to the mechanism of the formation of megamitochondria and that the inhibition of cytoplasmic protein synthesis seems not to contribute the phenomenon. However, the detailed mechanism by which free radicals may induce megamitochondria remains to be elucidated.

Interlaboratory comparison of microsomal ethoxyresorufin and pentoxyresorufin O-dealkylation determinations: standardization of assay conditions

Assay conditions and results of cytochrome P-450 dependent 7-ethoxyresorufin (ER) and 7-pentoxyresorufin (PR) O-dealkylation (OD) by rat liver microsomes were compared by four laboratories in the Netherlands. Microsomal mixtures were prepared from control, 3-methylcholanthrene and phenobarbital pretreated animals, resulting in different levels of cytochrome P-450 isozymes. EROD and PROD activities were determined in each laboratory according to their own protocols. Considerable variability was found both between and within laboratories. Further studies demonstrated that protocol differences are important factors causing this interlaboratory variation. Main factors of influence were buffer type, batch of resorufin used for calibration, substrate solvent and substrate concentration. Based on the results obtained, standardized protocols for optimized measurement of microsomal EROD and PROD activities were developed. Additional experiments demonstrated that the use of these standardized protocols reduced intralaboratory variation in both the EROD and the PROD assay, whereas it also reduced the interlaboratory variability for the PROD determinations. The interlaboratory variation for measurement of microsomal EROD activities was only reduced for the laboratories using a Cobas-Bio analyzer. The results of the present study demonstrate clearly that data obtained with EROD and PROD activity measurements are highly sensitive to factors frequently varying from one laboratory to another. In addition, they demonstrate the necessity to be careful with absolute values presented in the literature for these activities, unless well characterized assay conditions are applied.

Inverse Relationship Between Total Glutathione S‐Transferase Content and Bile Acid Release in Isolated Hepatocytes from Untreated, Phenobarbital Pretreated and Hypothyroid Rats*

The role of cytosolic anion binding proteins (glutathione S-transferases) in the hepatic transport of bile acids remains controversial. To investigate whether increased levels of the hepatocyte total glutathione S-transferase content were associated with changes in the release of bile acids from the hepatocyte, we measured the rate of release of radioactive bile acids in isolated hepatocytes from thyroidectomized, phenobarbital pretreated and untreated rats. The isolated hepatocytes were preincubated with either 14C-cholic acid or 14C-taurocholic acid, and the release rate of radiolabeled bile acids was determined. Hepatocyte total glutathione S-transferase content was measured by rocket immunoelectrophoresis. The release rate of the radiolabeled bile acids was significantly (P less than 0.005) decreased in both hypothyroid and phenobarbital pretreated hepatocytes. The levels of total glutathione S-transferase content were significantly (P less than 0.001) increased in the hepatocytes from both hypothyroid and phenobarbital pretreated animals. Our findings reveal a striking inverse relationship between the total glutathione S-transferase content of the hepatocyte and the release rate of radiolabeled bile acids in isolated hepatocytes from two independent animal models. These observations support the hypothesis that cytosolic anion binding proteins (glutathione S-transferases) may influence the net flux across the hepatocyte plasma membrane largely by limiting efflux.

Different effects of 8-OH-DPAT, a 5-HT 1A receptor agonist, on cortical acetylcholine release, electrocortigram and body temperature in guinea pigs and rats

8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) dose dependently increased cortical acetylcholine (ACh) release and the body temperature of freely moving guinea pigs. Moreover, it evoked an electroencephalographic (EEG) arousal reaction in phenobarbital-pretreated animals. 8-OH-DPAT was about 10 times less effective in increasing cortical ACh release in the rat than in the guinea pig, and did not modify the phenobarbital-synchronized EEG but dose dependently decreased body temperature. The possibility that 5-HT 1A receptors play different roles, depending on the animal species, is discussed.

The excretion of chlorophenylmercapturic acid, chlorophenols and a guanine adduct in the urine of chlorobenzene-treated rats after phenobarbital pretreatment

Chlorobenzene (CB) was administered to male Wistar rats. Twenty-four-hour urine samples were collected over a period of 7 days. p-Chlorophenylmercapturic acid, chlorophenols and a guanine adduct were determined by chromatographic methods. The excretion pattern of p-chlorophenylmercapturic acid did not seem to be significantly affected by phenobarbital. To analyze for the chlorophenols and guanine adducts, diluted urine was subjected to cation exchange chromatography using UV-detection. Fractions were found containing chlorophenols and a compound showing chromatographic properties similar to those of the synthetic N7-phenylguanine. The results of the HPLC analyses suggested the presence of a guanine adduct excreted on days 1 and 2 and between days 4 and 6 post-administration. There were no compounds detected which were identical with p-, m- or o-isomers of N7-chlorophenylguanine. The excretion of p- and m-chlorophenols by phenobarbital-pretreated animals was twice as high as that in untreated rats. A 4-fold increase was detected for o-chlorophenol. It is assumed that it is mainly direct hydroxylation that can be induced by phenobarbital. Dehalogenated phenolic metabolites may be capable of covalent binding to DNA.

Role of P-450 activity and glutathione levels in 1,2-dibromo-3-chloropropane tissue distribution, renal necrosis and in vivo DNA damage

Treatments known to alter P-450 activity and glutathione levels were used to elucidate the involvement of P-450 and glutathione S-transferase metabolism in 1,2-dibromo-3-chloropropane (DBCP) organ toxicity in the rat. Phenobarbital pretreatment abolished DBCP-induced renal necrosis, whereas it had only a small effect on initial renal DNA damage. The DBCP levels in plasma and tissues were markedly reduced by phenobarbital pretreatment. Perdeuterated DBCP had much higher plasma and tissue levels than protio-DBCP in phenobarbital-pretreated animals, but perdeuteration was without effect in uninduced animals. This indicates that P-450 metabolism of DBCP is of major importance only in phenobarbital-pretreated animals. In order to study the effects of decreased glutathione levels on renal distribution and toxicity, rats were pretreated with either diethyl maleate or buthionine sulfoximine. The DBCP levels in plasma and tissues showed transitory elevations after diethyl maleate and buthionine sulfoximine pretreatment compared to the control situation. Despite the fact that diethyl maleate and buthionine sulfoximine pretreatments are known to block DBCP-induced DNA damage in vitro, these pretreatments did not significantly alter DBCP-induced renal necrosis nor DNA damage. Thus, a role for glutathione conjugation in DBCP-induced in vivo renal toxicity could not be established in the present study.

Biochemical and immunochemical evidence for the induction of an ethanol-inducible cytochrome p-450 isozyme in male syrian golden hamsters

The effects of ethanol and of phenobarbital pretreatment on hamster microsomal metabolism of aniline and p-nitrophenol have been investigated. Hydroxylation of both compounds was increased over 2-fold by ethanol pretreatment, whereas phenobarbital pretreatment had little effect on either activity. Ethanol pretreatment had no effect on the specific content of total cytochrome P-450, while phenobarbital pretreatment increased the specific content 1.6-fold. Comparison of the specific activities for aniline hydroxylation and p-nitrophenol hydroxylation of individual microsomal samples from control, ethanol-pretreated and phenobarbital-pretreated animals showed a high degree of correlation r 2 = 0.98) consistent with the involvement of the same site for catalysis of these two compounds. Antibody to rabbit ethanol-inducible cytochrome P-450 (isozyme 3a) inhibited over 80% of the aniline (high affinity) and p-nitrophenol hydroxylase activities of microsomes from ethanol-treated hamsters. A comparison of the antibody-inhibitable rates for both hydroxylase activities with microsomes from untreated, ethanol- or phenobarbital-pretreated hamsters suggested that an isozyme homologous to rabbit isozyme 3a (hamster cytochrome P-450alc) was induced in hamsters about 3.5-fold by ethanol and was unaffected by phenobarbital. The induction of hamster cytochrome P-450alc was confirmed by immunoblot analysis of hamster microsomes. A single protein with a molecular weight of approximately 54,000 was recognized by antibody to the rabbit isozyme. Quantification of the immunoblots demonstrated that the hamster isozyme was increased about 3-fold, in good agreement with the induction determined by a comparison of the antibody-inhibitable rates. The results indicated that, although there was no change in the total spectrally observable cytochrome P-450, there was a marked change in the distribution of the isozymes of cytochrome P-450, with an increase in the alcohol-inducible form after 28-day ethanol consumption by chow-fed hamsters. This isozyme can be readily monitored by either high-affinity aniline or p-nitrophenol hydroxylation or by Western immunoblot analysis and appears to be the ethanol-inducible form of cytochrome P-450 in hamsters.

Demonstration of H2O2productionin vivoduring aminopyrine metabolism and phenobarbital induction

It has been shown previously that, by using methanol and a catalase inhibitor, 3-amino-1, 2, 4,-triazole, changes in hepatic H2O2 production in vivo can be detected. Using this method in guinea pigs and rats we could demonstrate increased H2O2 production during metabolism in vivo of aminopyrine, especially in phenobarbital-pretreated animals. In contrast, administration of antipyrine does not lead to H2O2 production. In the guinea pigs, phenobarbital induction also stimulates the H2O2 production in vivo without administration of exogenous substrates. The rate and extent of this additional H2O2 production depend on the induction state, drug metabolism and species; the major findings are in agreement with and extend previous research in vitro on microsomes, isolated hepatocytes and perfused liver.

Inhibition of δ-aminolevulinic acid dehydratase by trichloroethylene

Male Wistar rats (8 animals/group; 180–200 g) were exposed continuously to trichloroethylene (TRI) for 48 or 240 h or methylchloroform (1,1,1-trichloroethane: MC for 48 h at 50, 400 and 800 ppm. The inhibition of δ-aminolevulinic acid dehydratase (ALA-D) was examined in liver, blood and bone marrow of naive and phenobarbital pretreated animals exposed to TRI. A clear cut dose-effect relationship between the exposure concentration or duration of exposure and the inhibition of ALA-D activity was seen for rats exposed to TRI. In addition to this finding, significant interaction between TRI exposure and phenobarbital treatment was observed in the inhibition of ALA-D in liver and blood. MC did not produce inhibition. Trichloroacetic acid and trichloroethanol failed to inhibit the ALA-D activity in vitro. It seems that a metabolite(s) of TRI other than the above 2 substances may play a role in the inhibition of ALA-D. The inhibition of ALA-D (38% or 48% of the control in liver or in blood, respectively) observed after the 240 h exposure at 400 ppm to TRI was accompanied by the significant elevation of δ-aminolevulinic acid synthase (186% of the control) in liver and the increase in excretion of δ-aminolevulinic acid in urine (142% of the control). This occurred without an apparent weight loss, liver injury or hematological changes.

Acetylhydrazine hepatotoxicity: The role of covalent binding

Acetyhydrazine, a human metabolite of isoniazid and a hepatotoxin, binds covalently to rat liver protein and to the proteins of other organs in vivo. The covalent binding to macromolecules in the liver, but not to those of other organs, was increased by pretreatment with phenobarbital. The time course of binding to total hepatic protein and to hepatic subcellular fractions was also altered by phenobarbital pretreatment. There was a concommitant time-dependent decline in cytochrome P-450 levels in the phenobarbital pretreated animals, but not controls. Acetylhydrazine did not produce detectable lesions in organs other than the liver, despite the occurrence of covalent binding to protein in those organs. The results suggest that only a critical fraction of the covalent binding to specific subcellular components is related to the subsequent development of tissue lesions.

An enzyme-linked immunoadsorbent assay for measuring cytochrome b5 and NADPH-cytochrome P-450 reductase in rat liver microsomal fractions. Evidence for functionally inactive protein.

Immunoreactive cytochrome b5 and NADPH-cytochrome P-450 reductase (EC 1.6.2.4) from rat liver microsomal fractions were measured by using an enzyme-linked immunoadsorbent assay (e.l.i.s.a.) as a function of age, sex and type of inducer (phenobarbital or 3-methylcholanthrene), and the values were compared with those obtained by spectral measurement (for cytochrome b5) or enzymic assay (for reductase). In untreated animals, there was more cytochrome b5 and NADPH-cytochrome P-450 reductase when measured by an e.l.i.s.a. than was seen spectrally or enzymically. However, for microsomal preparations from phenobarbital-pretreated animals, spectrally obtained values for cytochrome b5 and immunoreactive-cytochrome b5 values were similar. Values from control animals suggest that there is about 20-30% more immunoreactive cytochrome b5 than that which is spectrally detectable.

CCl 4-induced alterations in Ca ++ homeostasis in chlordecone and phenobarbital pretreated animals

Male S-D rats were maintained on normal powered diet or on the same diet containing 10 ppm chlordecone or 225 ppm phenobarbital for 15 days. On day 15, all the animals received a single ip injection of either corn oil or a subtoxic dose of CCl 4 (25–200 μl/kg) in corn oil vehicle (1 ml/kg). The animals were sacrificed 12 hrs later. Liver microsomal cytochrome P-450 and Ca ++ levels in whole liver, mitochondria, microsomes and cytosol were determined. Cytochrome P-450 induction was greater with phenobarbital pretreatment than with chlordecone but the CCl 4 induced destruction of cytochrome P-450 was almost similar in both groups and progressive with the dose of CCl 4. CCl 4 given to animals on normal diet in a dose range of 25–200 μ l/kg did not significantly alter the cytochrome P-450 levels. These findings are consistent with greater bioactivation of CCl 4 after the above two pretreatments. There was a massive accumulation of Ca ++ in chlordecone and phenobarbital pretreated animals after CCl 4 administration. Cytosolic Ca ++ levels remained high despite the mitochondrial and microsomal sequestration. This perturbation of hepatocellular Ca ++ homeostasis might lead to hepatic lesion and hepatic failure. Chlordecone or phenobarbital alone do not alter hepatic Ca ++ levels. These findings suggest that excessive accumulation of Ca ++ may be causally related to the progression of hepatotoxic response due to CCl 4 in chlordecone treated animals.

Metabolism of paraldehyde to acetaldehyde in liver microsomes: Evidence for the involvement of cytochrome p-450

A concentration-dependent acetaldehyde (AcH) generation was observed when paraldehyde was incubated with the mouse liver microsomal fraction. The process, which exhibited a requirement for oxygen and NADPH and was inhibited by carbon monoxide, was found to have a K m of 17.9mM with respect to paraldehyde and a V max of 40.1 nmoles/mg protein/min with respect to AcH formation. NADH was much less effective as an electron donor than NADPH, though a more than additive increase in AcH generation was observed when both of these nucleotides were added to the incubation. The rate of microsomal AcH generation from paraldehyde was increased 2.5-fold by pretreatment of the mice with phenobarbital but only 0.6-fold by pretreatment with 3-methylcholanthrene. Pretreatment with 2-diethylaminoethyl-2, 2-diphenylvalerate hydrochloride (SKF-525A) resulted in 54% inhibition of the reaction rate. Addition of metopirone to the incubation inhibited AcH generation in a concentration-related fashion, the inhibition being greatest, proportionately, in microsomes from phenobarbital-pretreated animals. The above results conclusively indicate the involvement of cytochrome P-540 mixed function oxidase in the formation of AcH from paraldehyde by mouse liver microsomes. It is also postulated that this process may be accomplished in the reaction analogous to O-dealkylation.

Serine and glycine-induced catalepsy in porphyric rats: An animal model for psychosis?

It was investigated whether an increased demand for glycine, as postulated to occur in patients who have suffered from episodic psychoses accompanied by multiple perceptual distortions, could evoke psychotic reactions. Catalepsy was used as a measure for psychosis and was observed after injection of serine or glycine in porphyric rats. Catalpesy was shown to occur after serine as well as glycine administration in 2-allyl-2-isopropylacetamide (AIA) pretreated rats, while in lead (Pb) + phenobarbital pretreated rats only glycine was effective. Administration of AIA to rats resulted in a strongly enhanced excretion of porphobilinogen (PBG) in urine, while Pb + phenobarbital pretreated rats showed increased excretion of σ-aminolevulinic acid (σ-ALA). The Pb + phenobarbital pretreated animals showed elevated serine plasma levels and lowered glycine plasma levels 18 hours after injection, while no significant differences in plasma levels of these amino acids were found 24 hours after AIA administration. In AIA or saline pretreated animals, but not in those pretreated with Pb + phenobarbital, glycine formation from serine was elevated. It is concluded that the present animal model can be used to investigate episodic psychoses.

Phenobarbital-digitoxin interaction in the guinea pig liver.

The influence of phenobarbital pretreatment on liver concentration of digitoxin and its metabolites was studied in guinea-pigs after i.p. administration of the cardiac glycoside. During the first hour an increase in liver uptake was observed in pretreated animals. The differences detected in the hepatic subcellular distribution do not seem to explain the higher concentrations found in the liver of phenobarbital pretreated animals. About 80% of the liver radioactivity was found in the supernatants. Inhibition of digitoxin biotransformation by phenobarbital was demonstrated by chromatographic analysis of the organic soluble compounds present in the supernatants. The possible binding of digitoxin and its metabolites to soluble proteins of liver cytosol was excluded by thin-layer gel filtration. The decrease in digitoxin biotransformation seems to be the reason for the increase in liver uptake and for the decrease in bile concentrations, observed in phenobarbital pretreated animals.

Hexose-6-phosphate dehydrogenase of rat liver microsomes. Isolation by affinity chromatography and properties.

Hexose-6-phosphate dehydrogenase was purified from rat liver microsomal fraction more than 500-fold with a 45% recovery using DEAE-cellulose and 2',5'-ADP-Sepharose 4B columns. The purified enzyme appeared to be immunologically and electrophoretically homogeneous and had broad substrate and cofactor specificities. The enzyme activity was not inhibited by p-chloromercuribenzoate. The purified enzyme was a glycoprotein in nature, having a Stokes radius of about 55 A, a sedimentation coefficient of about 8.2 s, and an isoelectric point of about 6.4. Minimum molecular weight of the enzyme was about 108,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the product cross-linked with glutaraldehyde or dimethyl suberimidate had Mr approximately equal to 220,000, suggesting that the active enzyme existed as a dimer of identical subunits. Antiserum raised against the purified enzyme inhibited the activity of the solubilized enzyme but did not inhibit the cytosol glucose-6-phosphate dehydrogenase activity. The antigenic sites of the enzyme were latent in intact microsomes. Comparison was also made between the enzymes isolated from untreated and phenobarbital-pretreated animals.

Conversion of N-hydroxyamphetamine to phenylacetone oxime by rat liver microsomes

These in vitro studies indicate that N-oxidation of N-hydroxyamphetamine (NOHA) by rat liver homogenates yields phenylacetone oxime (PAOx) as the major metabolite. This oxidation was NADPH and oxygen dependent but was not appreciably increased in microsomes from phenobarbital-pretreated animals. The addition to microsomal incubations of Superoxide dismutase (SOD), catalase (CAT), azide or mannitol did not alter the rate of oxidation, suggesting that O 2su−. H 2O 2, or OH . are not involved in this process. The reaction was minimally inhibited by a 2:1 ratio of CO/O 2, and there was no significant reduction in the formation of product by the presence of diethylaminoethyl diphenylvalerate (SKF-525A) or 2,4-dichloro-6-phenylphenoxyethylamine (DPEA) in micromolar concentrations. Thus, although this NADPH-dependent N-oxidation pathway was catalyzed by rat hepatic microsomes, the data suggest that it was not a cytochrome P-450 mediated monooxygenase reaction.

Body residue and metabolism of adriamycin and daunorubicin in control and phenobarbital-pretreated mice.

1. Metabolism of adriamycin (ADR) and daunorubicin (DR) was investigated in DBA/2Ha mice after i.p. injection and compared with that in phenobarbital-pretreated animals. At 24 h after drug administration 83% of ADR and 67% of DR derived fluorescence were recovered from cadavers and excreta. Destruction of anthracyclines in excreta to non-fluorescent materials could be partly responsible for the low recoveries. 2. ADR was metabolized to a limited extent in mice. At 24 h 70% dose was detected as the unchanged drug and only 13% as its deoxyaglycones. 3. DR was metabolized more extensively. At 24 h 29% of the dose was as the unchanged drug, while 32% was present as daunorubicinol (DROL) and 16% as aglycones. Phenobarbital did not significantly affect these parameters. 4. In phenobarbital-pretreated mice there was a marked increase in the amount of ADR or DR aglycones in the liver, and a decrease in the levels of unchanged ADR or DR.

The comparison of in vivo plasma radioactivity clearance and 14CO 2 breath elimination of model drugs in the rat: A study in regional hepatocyte function

We systematically related the in vivo plasma radioactivity clearance and the 14CO 2 breath elimination of two drugs ([4- dimethyl- 14C]aminopyrine and [1- methyl- 14C]caffeine), whose major sites of hepatic metabolism correspond to the microsomal mixed-function oxidase system (cytochromes P-450 and P-448, respectively), and two compounds ([1- 14C]ethanol and [ 14C]methanol], whose major sites of hepatic metabolism correspond to the cytosol. All studies were carried out in the freely moving rat. Measurements of plasma radioactivity clearance and 14CO 2 breath elimination were done with normal animals as well as with animals pretreated with agents that either stimulate or inhibit the model drugs' in vivo metabolism. Following pretreatment with phenobarbital, the aminopyrine plasma radioactivity clearance was significantly increased over normal, and this was paralleled by a significant increase of the 14CO 2 breath elimination as well. The ratio (plasma elimination/breath elimination) was unchanged between the normal and phenobarbital-pretreated animals and approached unity. by contrast, neither the aminopyrine plasma radioactivity clearance nor the 14CO 2 breath elimination was significantly altered by pretreatment with 3-methylcholanthrene. When the plasma radioactivity clearance and the 14CO 2 breath elimination were studied after administration of [ 14C]caffeine, both the plasma radioactivity clearance and 14CO 2 breath elimination showed a significant increase in the 3-methylcholanthrene-treated animals as compared to normal. The ratio (plasma elimination/breath elimination) was unchanged, however, between the normal and 3-methylcho-lanthrene-pretreated animals. Neither the caffeine plasma radioactivity clearance nor 14CO 2 breath elimination was significantly altered by pretreatment with phenobarbital. In animals pretreated with pyrazole, the ethanol plasma radioactivity clearance and 14CO 2 breath elimination were both significantly decreased. However, neither measurement was significantly altered after pretreatment with 3-amino-1,2,4-triazole. By contrast, the methanol plasma radioactivity clearance and 14CO 2 breath elimination were significantly decreased in both the pyrazole and 3-aminio-1,2,4-triazole-pretreated animals. These studies illustrate that for the model compounds selected and the dosages utilized, the 14CO 2 breath elimination rate closely reflected the plasma radioactivity elimination rate.