Metabolic transformations of antitumor imidazoacridinone, C-1311, with microsomal fractions of rat and human liver

The imidazoacridinone derivative C-1311 is an antitumor agent in Phase II clinical trials. The molecular mechanism of enzymatic oxidation of this compound in a peroxidase model system was reported earlier. The present studies were performed to elucidate the role of rat and human liver enzymes in metabolic transformations of this drug. C-1311 was incubated with different fractions of liver cells and the reaction mixtures were analyzed by RP-HPLC. We showed that the drug was more sensitive to metabolism with microsomes than with cytosol or S9 fraction of rat liver cells. Incubation of C-1311 with microsomes revealed the presence of four metabolites. Their structures were identified as dealkylation product, M0, as well as a dimer-like molecule, M1. Furthermore, we speculate that the hydroxyl group was most likely substituted in metabolite M3. It is of note that a higher rate of transformation was observed for rat than for human microsomes. However, the differences in metabolite amounts were specific for each metabolite. The reactivity of C-1311 with rat microsomes overexpressing P450 isoenzymes, of CYP3A and CYP4A families was higher than that with CYP1A and CYP2B. Moreover, the M1 metabolite was selectively formed with CYP3A, whereas M3 with CYP4A. In conclusion, this study revealed that C-1311 varied in susceptibility to metabolic transformation in rat and human cells and showed selectivity in the metabolism with P450 isoenzymes. The obtained results could be useful for preparing the schedule of individual directed therapy with C-1311 in future patients.     

A comparison of aroclor 1254-induced and uninduced rat liver microsomes to human liver microsomes in phenytoin O-deethylation, coumarin 7-hydroxylation, tolbutamide 4-hydroxylation, S-mephenytoin 4'-hydroxylation, chloroxazone 6-hydroxylation and testosterone 6beta-hydroxylation

Aroclor 1254-induced rat liver homogenate supernatant (liver S-9) is routinely used as an exogenous metabolic activation system for the evaluation of mutagenicity of xenobiotics. The purpose of this study is to evaluate whether results obtained with Aroclor 1254-induced liver microsomes would be relevant to human. Aroclor 1254-induced and uninduced rat liver microsomes were compared to human liver microsomes in the metabolism of substrates which are known to be selectively metabolized by the major human cytochrome P450 (CYP) isoforms. The activities studied and the major CYP isoforms involved were as follows: phenacetin O-deethylation (CYP1A2); coumarin 7-hydroxylation, (CYP2A6); tolbutamide 4-hydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation (CYP2C19); dextromethorphan O-demethylation (CYP2D6); chloroxazone 6-hydroxylation (CYP2E1); and testosterone 6beta-hydroxylation (CYP3A4). We found that both induced and uninduced rat liver microsomes were active in all the pathways studied with the exception of coumarin 7-hydroxylation. Coumarin 7-hydroxylation was observed with human liver microsomes but not the rat liver microsomes. Aroclor-1254 was found to induce all activities measured, with the exception of coumarin 7-hydroxylation. Dextromethorphan O-deethylation activity was higher in the rat liver microsomes than the human liver microsomes. Testosterone 6beta-hydroxylation activity was found to be similar between the human liver microsomes and the induced rat liver microsomes. Our results suggest that experimental data obtained with Aroclor 1254-induced rat liver microsomes may not always be relevant to human.     

alpha-Zearalenol, a major hepatic metabolite in rats of zearalenone, an estrogenic mycotoxin of Fusarium species

The chemical and biological properties of the hepatic metabolite of zearalenone, an estrogenic and non-steroidal fungal toxin produced by Fusarium species, were investigated by employing TLC, GC/MS, high pressure liquid chromatography and fluorospectral analyses, as well as uterine weight bioassay in immature mice. All the chemical and physical data supported the view that the major metabolite, obtained by incubating zearalenone with S-9 and microsomes of rat liver in the presence of NADPH, is C-6'-alpha-hydroxylated zearalenone (alpha-zearalenol). The estrogenic activity of this metabolite was several times higher than that of the parent zearalenone, and the results of biological and toxicological evaluations of alpha-zearalenol are discussed.     

Formation of (4R)- and (4S)-4-hydroxyochratoxin A from ochratoxin A by liver microsomes from various species.

Two metabolic products were formed from ochratoxin A by human, pig, and rat liver microsomal fractions in the presence of reduced nicotinamide adenine dinucleotide phosphate. They were isolated from the incubation mixture in the presence of pig liver microsomes by extraction, thin-layer chromatography, and high-pressure liquid chromatography Their structures are suggested to be (4R)- and (4S)-4-hydroxyochratoxin A on the basis of mass and nuclear magnetic resonance spectroscopy. Km and the maximum velocity for the formation of the two metabolites by human, pig, and rat microsomes were determined. Their formation was inhibited by carbon monoxide and metyrapone. The results indicate that the microsomal hydroxylation system is a cytochrome P-450 and that different species are involved in the formation of the two epimeric forms of 4-hydroxyochratoxin A.     

Separation, quantitation and distribution of dolichol and dolichyl phosphate in rat and human tissues

Two procedures for quantitative determination of dolichol were studied and these were applied to analyze tissue and subcellular distribution. In the first procedure the dolichols were oxidized with Cr2O3 and reduced with NaB3H4. The radioactivity in the individual dolichols was measured using reversed-phase thin-layer chromatography. In the second procedure, dolichols were analyzed by high-pressure liquid chromatography. For determination of dolichyl phosphates the lipid extract was subjected to acid and alkaline hydrolysis, and after hydrolysis with acid phosphatase the distribution was determined by high-pressure liquid chromatography. Recovery was monitored by the addition of dolichol D15 and D23 phosphate to the homogenate. Rat spleen had the highest dolichol content (114 micrograms/g) followed by lower content in rat liver and brain. The distribution pattern was similar in all organs, with 18 and 19 isoprene residues as dominating components. Human organs contain considerably higher concentrations of dolichol, with the 19 and 20 isoprene residues as the main components. In rat liver, outer mitochondrial and Golgi membranes, lysosomes and plasma membranes contain considerable amounts of dolichol. A drastic increase in dolichol content was observed in rat liver hyperplastic nodules while human liver cirrhosis and hepatocarcinoma showed a marked decrease in dolichol. In the latter case, the distribution pattern was also changed. Of the total amount of dolichol present in the tissues, 2% was phosphorylated in human liver, 10% in human testis and 18% in rat liver. In rat liver mitochondria and in microsomes 4 and 31%, respectively, of the polyprenols were in activated form. The results demonstrated that dolichyl phosphate and dolichol concentrations were regulated by different mechanisms and that the two forms possessed an independent distribution.     

Metabolism of 12(R)-hydroxyeicosatetraenoic acid by rat liver microsomes

The in vitro metabolism of 12(R)-hydroxyeicosatetraenoic acid was studied using freshly isolated rat liver microsomes. Ten metabolites were isolated and identified by a combination of ultraviolet spectroscopy and gas chromatography/mass spectrometry. The two major metabolites were dihdroxyeicosatetraenoic acids generated by ω /ω − 1 hydroxylation. Oxidation at C-5 resulted in the formation of four leukotriene-like compounds, two of which differed from leukotriene B₄ in double-bond geometry alone. The other two differed from leukotriene B₄ in olefin geometry and C-5 configuration. Epoxidation at the 14,15-olefin resulted in the formation of two diastereomeric epoxy alcohols, while C-16 hydroxylation gave two diastereomeric dihydroxyeicosatetraenoic acids.     

Cytochrome P-450-catalyzed desaturation of valproic acid in vitro. Species differences, induction effects, and mechanistic studies

The cytochrome P-450-mediated desaturation of valproic acid (VPA) to its hepatotoxic metabolite, 2-n-propyl-4-pentenoic acid (4-ene-VPA), was examined in liver microsomes from rats, mice, rabbits and humans. The highest substrate turnover was found with microsomes from rabbits (44.2 +/- 2.7 pmol of product/nmol P-450/15 min), while lower activities were observed in preparations from human, mouse, and rat liver, in that order. Pretreatment of animals with phenobarbital led to enhanced rates of formation of 4-ene-VPA in vitro and yielded induction ratios for desaturation ranging from 2.5 to 8.4, depending upon the species. Comparative studies in the rat showed that phenobarbital is a more potent inducer of olefin formation than either phenytoin or carbamazepine. The mechanism of the desaturation reaction was studied by inter- and intramolecular deuterium isotope effect experiments, which demonstrated that removal of a hydrogen atom from the subterminal C-4 position of VPA is rate limiting in the formation of both 4-ene- and 4-hydroxy-VPA. Hydroxylation at the neighboring C-5 position, on the other hand, was highly sensitive to deuterium substitution at that site, but not to deuteration at C-4. Based on these findings, it is proposed that 4-ene- and 4-hydroxy-VPA are products of a common P-450-dependent metabolic pathway, in which a carbon-centered free radical at C-4 serves as the key intermediate. 5-Hydroxy-VPA, in contrast, derives from an independent hydroxylation reaction.     

High-performance liquid chromatography method for determination of N-glucuronidation of 4-aminobiphenyl by mouse, rat, and human liver microsomes

A simple and sensitive method for determination of the N-glucuronidation activity of mouse, rat, and human liver microsomes toward the carcinogenic arylamine 4-aminobiphenyl (4-ABP) using high-performance liquid chromatography with ultraviolet detection has been developed. The method uses chemically synthesized 4-ABP-N-glucuronide (4-ABP-G) as a standard for method validation. Validation was done with respect to specificity, linearity, precision, accuracy, and lower limits of detection. The method was specific since there were no interference peaks from the reaction matrix. The calibration curve for 4-ABP-G was linear from 50 to 5000 pmol/200 microl with R2=0.999. The newly developed method has good precision and accuracy. The intra- and interday precisions were less than 5 and 10%, respectively, and the highest values for intra- and interday accuracies were -4.6 and -12%, respectively. The lower limit of detection was 10 pmol/200 microl. The developed method was used to determine the glucuronidation activity of mouse, rat, and human liver microsomes. Human liver microsomes were the most active in 4-ABP glucuronidation (344.1 pmol/min/mg) followed by rats (30.6 pmol/min/mg) and then mice (12.3 pmol/min/mg). Human UGT1A4 supersomes were much more active than UGT1A9 (184.4 mol/min/mg versus 25.2 mol/min/mg). These results are consistent with those of earlier studies that used the radioactive [C14]UDPGA.     

Metabolism of prostaglandin endoperoxide by microsomes from human lung parenchyma and comparison with metabolites produced by pig, bovine, rat, mouse and guinea-pig

The metabolism of PGH2 by human lung parenchymal microsomes was characterized by radiometric high performance liquid chromatography and compared with metabolism by pig, bovine, rat, mouse, and guinea pig lung microsomes. Microsomes from human lung synthesized 0.74 nmoles/mg protein and 0.72 nmoles/mg protein, PGI2 (6-Keto-PGF1 alpha) and TxA2 (TxB2) respectively, upon incubation with 4.0 nmoles of PGH2. Pig, bovine, rat, mouse, and guinea pig microsomes respectively synthesized 1.0, 1.0, 0.9, 0.4, and 0.1 nmoles of PGI2/mg protein, and 0.9, 1.0, 0.7, 0.3, 1.8 nmoles of TxA2/mg protein, and preparations formed some PGE2, PGF2 alpha, and PGD2. Mouse lung microsomes were unique in synthesizing PGE2 as the major prostaglandin. The thromboxane synthetase inhibitor 1-benzylimidazole was a specific inhibitor in these six species.     

Isolation and characterization of active metabolites of tryptophan-pyrolysate mutagen,TRP-P-2, formed by rat liver microsomes

The mutagenic compound derived from the pyrolysis of tryptophan, 3-amino-1-methyl-5H-pyrido-[4,3b]indole (Trp-P-2) was metabolized by rat liver microsomes to more than four metabolites, separable by high performance liquid chromatography. Among these metabolites, two metabolites, M-3 and M-4 were directly active in increasing the frequency of mutation in Salmonella typhimurium TA98. Treatments of rats with polychlorinated biphenyl (PCB) or 3-methylcholanthrene dramatically induced the activity of liver microsomes to form these active metabolites, while treatment with phenobarbital was without effect. A major active metabolite (M-3) formed the pentacyano-ammine ferroate, which is known to be formed by reaction of sodium pentacyano-ammine ferroate with some hydroxylamines. Further this metabolite was oxidized to the minor active metabolite (M-4) with potassium ferricyanide or γ-manganese dioxide, and was reduced back to Trp-P-2 with titanium trichloride. These results indicated that the major active metabolite of Trp-P-2, which is formed by cytochrome P-450, is the 3-hydroxyamino derivative.     

Simultaneous determination of blood concentrations of methohexital and its hydroxy metabolite by gas chromatography and identification of 4′-hydroxymethohexital by combined gas—liquid chromatography—mass spectrometry

A simple, sensitive and selective method is described for the simultaneous determination of low concentrations (less than 50 ng/ml) of underivatized methohexital and its hydroxy metabolite in small (0.1 ml) samples of human and rat plasma or whole blood by gas chromatography with nitrogen-selective detection.Moreover, the main metabolite in rat and man was identified as 4′-hydroxymethohexital by comparison of chromatograms from gas—liquid chromatography (GLC) with data obtained from GLC—mass spectrometry and ¹H-nuclear magnetic resonance spectrometry of this metabolite, produced both by incubating methohexital with isolated rat liver microsomes and by isolating this metabolite from rat urine.     

High-performance liquid chromatographic assay detects pentamidine metabolism by Fisher rat liver microsomes

Fisher rat liver microsomes metabolized the antimicrobial drug pentamidine to four new compounds detected by gradient elution reversed-phase high-performance liquid chromatography with variable wavelength detection. Coelution experiments with pentamidine metabolite standards determined the new peaks to be previously identified hydroxylated metabolites of pentamidine, with 1,5-bis(4′-amidinophenoxy)-3-pentanol and 1,5-di-(4′-amidinophenoxy)-2-pentanol formed in the greatest amount. The data contradict a previous report that Fisher rat liver homogenates do not metabolize pentamidine. Pentamidine and its known primary metabolites have almost identical absorption spectra; thus, pentamidine metabolism must be evaluated using gradient elution HPLC to resolve pentamidine from its metabolites. The current assay has now been used to demonstrate that Fisher and Sprague-Dawley rat, mouse, rabbit and human liver microsomes all metabolize pentamidine in vitro.     

High-performance liquid chromatographic assay for N-glucuronidation of nicotine and cotinine in human liver microsomes

A method for the determination of N-glucuronidation of nicotine and cotinine in human liver microsomes by high-performance liquid chromatography was developed. Nicotine or cotinine was incubated with human liver microsomes and UDP-glucuronic acid in a 200-microl incubation mixture. The nicotine N-glucuronide (Nic-glu) and cotinine N-glucuronide (Cot-glu) formed were analyzed by ion-pair chromatography with a C-18 column. The sensitivity of quantification at 260 nm absorption was improved by using a noise-base clean Uni-3, and the limit of quantification was 10 pmol/200 microl mixture for both Nic-glu and Cot-glu. Linear standard curves were obtained within the concentration ranges 25-1000 pmol/200 microl mixture for Nic-glu and 100-5000 pmol/200 microl mixture for Cot-glu. The intraassay precision and accuracy were < or =11.1% coefficient of variation (CV) and 97.5-106.6% for Nic-glu and < or =4.6% CV and 96.7-100.4% for Cot-glu. The interassay precision and accuracy were < or =7.2% CV and 98.2-106.1% for Nic-glu and < or =4.6% CV and 96.8-99.3% for Cot-glu. This is the first report of the in vitro determination of Nic-glu and Cot-glu in human liver microsomes. Furthermore, this highly sensitive HPLC method can be used for the determination of Nic-glu and Cot-glu in biological specimens in vivo.     

Determination of cytochrome P450 1A activities in mammalian liver microsomes by high-performance liquid chromatography with fluorescence detection

A sensitive method for the determination of cytochrome P450 (P450 or CYP) 1A activities such as ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) in liver microsomes from human, monkey, rat and mouse by high-performance liquid chromatography with fluorescence detection is reported. The newly developed method was found to be more sensitive than previous methods using a spectrofluorimeter and fluorescence plate reader. The detection limit for resorufin (signal-to-noise ratio of 3) was 0.80 pmol/assay. Intra-day and inter-day precisions (expressed as relative standard deviation) were less than 6% for both enzyme activities. With this improved sensitivity, the kinetics of EROD and MROD activities in mammalian liver microsomes could be determined more precisely. EROD activities in human and monkey liver microsomes, and MROD activities in liver microsomes from all animal species exhibited a monophasic kinetic pattern, whereas the pattern of EROD activities in rat and mouse liver microsomes was biphasic. In addition, the method could determine the non-inducible and 3-methylcholanthrene-inducible activities of EROD and MROD in rat and mouse liver microsomes under the same assay conditions. Therefore, this method is applicable to in vivo and in vitro studies on the interaction of xenobiotic chemicals with cytochrome CYP1A isoforms in mammals.     

Production and secretion of C-19 steroids by rat and guinea pig adrenals

The concentrations of C-19 steroids were measured in guinea pig and rat adrenals before and after castration as well as after stimulation with adrenocorticotropin hormone (ACTH). Characterization of adrenal C-19 steroids was also carried out by isolation with high-performance liquid chromatography and gas chromatography/mass spectrometry (GC/MS). From radioimmunoassay (RIA) data, androstenedione (4-DIONE) and 11 beta hydroxyandrostenedione (11 beta-DIONE) were the major C-19 steroids found in guinea pig adrenals, and castration induced a decrease of 4-DIONE levels only while all other C-19 steroids remained unchanged. In rat adrenals, the major C-19 steroids were 4-DIONE and testosterone, and they were also markedly inhibited after castration. With the exception of 11 beta-DIONE, all other C-19 steroids in circulation were eliminated after castration in both animals species. After ACTH administration in the guinea pig, adrenal 4-DIONE and 11 beta-DIONE levels were markedly stimulated, while an increase of only 11 beta-DIONE was observed in plasma. In the rat, ACTH had a small stimulatory effect on adrenal 52-androstane-3 alpha, 17 beta-diol (3 alpha-DIOL) and plasma 11 beta-DIONE levels. Analysis of guinea pig adrenal steroids by GC/MS confirmed the presence of C-19 steroids in adrenals (namely, 4-DIONE and 11 beta-DIONE) while, in the rat, this could not be confirmed. Our data indicate that production of C-19 steroids occurs in guinea pig adrenals, and 11 beta-DIONE is the major C-19 steroid as well as the only C-19 steroid secreted into the circulation. In the rat, the production of C-19 steroids detected by RIA is not supported by GC/MS data.     

Purification of cytochrome P-450, NADPH-cytochrome P-450 reductase,and epoxide hydratase from a single preparation of rat liver microsomes

A simplified procedure is presented for the simultaneous purification of the enzymes cytochrome P-450, epoxide hydratase (EC 3.3.2.3), and NADPH-cytochrome P-450 reductase (EC 1.6.2.4) from a single preparation of rat liver microsomes. All three enzymes can be recovered after chromatography of detergent-solubilized microsomes on a column of n-octylamino-Sepharose 4B. The major form of cytochrome P-450 (of phenobarbitaltreated rats) is purified by subsequent DEAE-cellulose chromatography, epoxide hydratase is purified by DEAE- and O-(carboxymethyl)-cellulose chromatography, and NADPH-cyto-chrome P-450 reductase is purified using 2′,5′-ADP agarose chromatography. The nonionic detergent Lubrol PX and the ionic detergents sodium cholate and deoxycholate are used in these procedures to permit utilization of uv-absorbance measurements in monitoring protein during purification. Overall yields of the three enzymes are approximately 20, 25, and 60%, respectively. All three enzymes are apparently homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and are functionally active. The same procedure can be used to obtain the major cytochrome P-450 present in liver microsomes isolated from β-naphthoflavone (5,6-benzoflavone)- or 3-methylcholanthrene-treated rats. Thus, the described procedures permit the rapid and reproducible purification of three major rat liver microsomal enzymes which can be coupled to study bioactivation and detoxification of a variety of xenobiotics in reconstituted systems.     

Fungal metabolism of acenaphthene by Cunninghamella elegans.

The filamentous fungus Cunninghamella elegans ATCC 36112 metabolized within 72 h of incubation approximately 64% of the [1,8-14C]acenaphthene added. The radioactive metabolites were extracted with ethyl acetate and separated by thin-layer chromatography and reversed-phase high-performance liquid chromatography. Seven metabolites were identified by 1H nuclear magnetic resonance, UV, and mass spectral techniques as 6-hydroxyacenaphthenone (24.8%), 1,2-acenaphthenedione (19.9%), trans-1,2-dihydroxyacenaphthene (10.3%), 1,5-dihydroxyacenaphthene (2.7%), 1-acenaphthenol (2.4%), 1-acenaphthenone (2.1%), and cis-1,2-dihydroxyacenaphthene (1.8%). Parallel experiments with rat liver microsomes indicated that the major metabolite formed from acenaphthene by rat liver microsomes was 1-acenaphthenone. The fungal metabolism of acenaphthene was similar to bacterial and mammalian metabolism, since the primary site of enzymatic attack was on the two carbons of the five-member ring.     

Fungal metabolism of acenaphthene by Cunninghamella elegans.

The filamentous fungus Cunninghamella elegans ATCC 36112 metabolized within 72 h of incubation approximately 64% of the [1,8-14C]acenaphthene added. The radioactive metabolites were extracted with ethyl acetate and separated by thin-layer chromatography and reversed-phase high-performance liquid chromatography. Seven metabolites were identified by 1H nuclear magnetic resonance, UV, and mass spectral techniques as 6-hydroxyacenaphthenone (24.8%), 1,2-acenaphthenedione (19.9%), trans-1,2-dihydroxyacenaphthene (10.3%), 1,5-dihydroxyacenaphthene (2.7%), 1-acenaphthenol (2.4%), 1-acenaphthenone (2.1%), and cis-1,2-dihydroxyacenaphthene (1.8%). Parallel experiments with rat liver microsomes indicated that the major metabolite formed from acenaphthene by rat liver microsomes was 1-acenaphthenone. The fungal metabolism of acenaphthene was similar to bacterial and mammalian metabolism, since the primary site of enzymatic attack was on the two carbons of the five-member ring.     

Identification of an arene oxide metabolite of 2,2',5-5'-tetrachlorobiphenyl by gas chromatography-mass spectroscopy

Tritiated 2,2'5,5'-tetrachlorobiphenyl (3H-TCB) was incubated with phenobarbital(PB)-induced rat liver microsomes in the presence of an epoxide hydrase inhibitor and brominated analog (BrAO) of the expected metabolic intermediate, 2,2',5,5'-tetrachlorobiphenyl-3,4-oxide (TCBAO). A putative arene oxide intermediate (3H-AO), which was radiolabeled, was separated from 3H-TCB and BrAO by column chromatography, high pressure liquid chromatography (HPLC) and thin-layer chromatography (TLC), and was analyzed for TCBAO by methods that were independent of radiometric techniques. The retention times (Rt's) of TCBAO and 3H-AO on two gas chromatography (GC) columns were the same, both before and after acid catalyzed rearrangement. 3H-AO was further characterized by rearrangement to a mixture of 3- and 4-hydroxy-TCB that was identified by gas chromatography-mass spectroscopy (GC-MS). The rate of TCB metabolism and the production of 3H-AO by liver microsomes from a PB-induced, adult male rhesus monkey was less than that observed with rat microsomes. The 3H-AO from the monkey was also characterized as TCBAO by rearrangement to the characteristic TCB phenols that were analyzed by GC-MS using selective ion monitoring. This study is the first in which an arene oxide of a polychlorinated biphenyl (PCB) was actually isolated as a mammalian metabolite and subjected to direct chemical analysis.     

Unusual patterns of benzo[a]pyrene metabolites and DNA-benzo[a]pyrene adducts produced by human placental microsomes in vitro

Human placental microsomes were incubated with [³H]benzo[a]pyrene (BP) and Salmon sperm DNA and the resulting metabolite-nucleoside complexes resolved by Sephadex LH-20 chromatography. The metabolite pattern was analyzed by high-pressure liquid chromatography (HPLC). The incubates were also co-chromatographed with extracts obtained from incubates with rat liver microsomes and [¹⁴C]BP. Phenols, quinones and 7,8-dihydrodiol were detected in the placental incubates. Both 9,10- and 4,5-dihydrodiols were very low as compared with control rat liver samples. Placental microsomes catalyzed the binding of BP metabolites to DNA in vitro, giving rise to two main complexes which co-chromatographed with rat liver-produced peaks attributable to 7,8-diol-9,10-epoxide and 7,8-oxide and/or quinones when metabolized further. The nucleoside metabolite peaks attributable to 4,5-oxide and 9-phenol-4,5-oxide were lacking when compared with the binding pattern catalyzed by rat liver. Both the total binding and specific metabolite-nucleoside adducts in the placenta correlated with fluorometrically measured aryl hydrocarbon hydroxylase (AHH) activity and with the amount of dihydrodiol formed. The results demonstrate that both the metabolite pattern and the nucleoside-metabolite complexes formed by the placental microsomes in vitro differed greatly from those produced by rat liver microsomes. These studies also suggest that it is not possible to predict specific patterns of DNA binding from AHH measurements or even from BP metabolite patterns, especially when comparing different tissues and species.