Placental Microsomes Research Articles

Functional expression of transcoabalamin II cDNA In [formula omitted] oocytes

The products of in vitro transcription of human transcobalamin II (TC II) cDNA when microinjected into Xenopus laevis oocytes yielded a single secretory protein of 43 kDa. The mobility of the 43 kDa band did not change following digestion with peptide N-glycosidase F. [ 57Co]Cbl bound to the medium was immunoprecipitated with anti-serum to human TC II, but not to other Cbl binders. In addition, the [ 57Co]Cbl complex also bound to placental microsomes. These results suggest that TC II mRNA transcribed encodes TC II which contains both the Cbl and receptor binding domains. Furthermore, Xenopus oocytes can be used as a screening system to define structural elements important in TC II's secretion and binding reactions.

Analysis of coenzyme binding by human placental 3β-hydroxy-5-ene-steroid dehydrogenase and steroid 5→4-ene-isomerase using 5′-[ p-(fluorosulfonyl)benzoyl]adenosine, an affinity labeling cofactor analog

3β-Hydroxy-5-ene-steroid dehydrogenase and steroid 5→4-ene-isomerase copurify as a single, homogeneous protein from human placental microsomes. Affinity alkylation with 2α-bromoacetoxyprogesterone suggests that the dehydrogenase and isomerase substrate steroids bind at different sites on the same protein. However, the coenzyme, NADH, completely abolishes the alkylation of both enzyme activities by the progestin analog [Thomas J. L., Myers R. P., Rosik L. O. and Strickler R. C., J. Steroid Biochem. 36 (1990) 117–123]. Unlike bacterial 3-keto-5-ene-steroid isomerase, the human isomerase reaction is stimulated by diphosphopyridine nucleotides (NADH, NAD +). The affinity labeling nucleotide analog, 5′-[ p-(fluorosulfonyl)benzoyl]adenosine (FSA), inactivates the dehydrogenase and isomerase activities at similar rates in an irreversible manner which follows first order kinetics with respect to both time and alkylator concentration (0.2–0.6 mM). FSA is a cofactor site-directed reagent that binds with similar affinity as a competitive inhibitor of NAD + reduction by dehydrogenase ( K i = 162 μM) or as a stimulator of isomerase ( K m = 153 μM). Parallel plots derived from Kitz and Wilson analysis indicate that FSA inactivates the two enzyme activities with equal alkylation efficiency ( k 3 K i = 1/ slope = 0.5 l/mol-s for both). The 3β-hydroxy-steroid substrate, pregnenolone, protects isomerase as well as dehydrogenase from inactivation by FSA. These observations are evidence for a single cofactor binding region which services both enzyme activities.

Inhibition of estrone sulfatase enzyme in human placenta and human breast carcinoma

Estrone sulfatase is an important mechanism of local synthesis of biologically active estrogens in human breast cancer. The human placental microsome and breast carcinoma mitochondrial/microsomal estrone sulfatase activity were characterized and inhibition studies performed. The K m of the placental tissue enzyme was 6.83 μM, V max 0.015 nmol/min/mg, and for the breast carcinoma tissue K m was 8.91 μM and V max 0.022 nmol/min/mg. Danazol produced a significant inhibition of estrone sulfatase (20% with 50 μM danazol). No significant inhibition was seen in the presence of aminoglutethimide, rogletimide, tamoxifen, 4-hydroxyandrostenedione, stilboestrol, or any metabolites of danazol or tamoxifen. Studies with synthetic and naturally occuring steroids demonstrated that the presence of a sulfate group at the 3 position to be the most important factor in determining inhibition, and the most potent inhibitor was 5α-androstene-3β, 17β-diol-3-sulfate ( K i of 2.0 μM). The naturally occuring 3-sulfated steroids all demonstrated competitive inhibition. These studies could form the basis for the design of a potent estrone sulfatase inhibitor which would have potential therapeutic activity in the management of breast cancer.

Human placental estrogen synthetase (aromatase). Effect of environment on the kinetics of protein-protein and substrate-protein interactions and the production of 19-oxygenated androgen intermediates in the purified reconstituted cytochrome P450 enzyme system

Estrogen synthetase (aromatase) catalyzes the conversion of androgen into estrogen via two hydroxylations at C 19 and a subsequent C 19-10 lyase reaction. We report here the results of a reconstitution study using a highly purified aromatase cytochrome P450 monooxygenase enzyme system, with both protein components (cytochrome P450 and NADPH-cytochrome P450 reductase) obtained from human term placental microsomes. By varying one of the components (amounts of cytochrome P450, NADPH-cytochrome P450 reductase, or androgen substrate) as the other two were held constant in four different environments (phospholipid, non-ionic detergent, mixture of phospholipid and non-ionic detergent and buffer alone), we obtained evidence supporting the following conclusions. The reconstituted enzyme is more active and the protein components exhibit much lower apparent K m values in the detergent and/or lipid environment compared with buffer alone. Although the apparent K m and V max values for each aromatase protein component differ significantly in most cases with the particular limiting component and environment, the catalytic efficiency ( K cat/ K m ) was independent of the limiting protein component and varied with the environment only (highest in the lipid-detergent mixture and lowest in lipid alone). When the concentration of androgen substrate (androstenedione or testosterone) was varied at constant amounts of the aromatase protein components (NADPH-cytochrome P450 reductase saturating), the K m was lower and the V max was higher for adrostenedione. The specificity constant ( V max/ K m ) was a function of the reconstitution environment (highest in lipid alone and lowest in detergent alone) and was, on average, about 4-fold higher for androstenedione in a particular environment. The extent of production of 19-oxygenated androgen intermediates (19-hydroxy and 19-oxo androstenedione) was examined at three different levels of aromatase cytochrome P450 (subsaturating, saturating, super-saturating) relative to the NADPH-cytochrome P450 reductase component in the three different hydrophobic environments using androstenedione as substrate. Both 19-oxygenated androgens, each made in comparable amounts relative to control, were isolatable in greatest amounts under cytochrome P450 super-saturating conditions in the detergent-lipid mixed environment, and in least amounts under cytochrome P450 subsaturating conditions in the lipid-only environment. Based on these data, we propose that 19-oxygenated androgen intermediates are biosynthesized sequentially in a step-wise fashion as the cytochrome P450 and NADPH-cytochrome P450 reductase form transient complexes, and that the amount of isolatable 19-oxygenated androgen is proportional to the amount of excess cytochrome P450 component.

Synthesis and biochemical studies of 16- or 19-substituted androst-4-enes as aromatase inhibitors

Androst-4-en-17-one derivatives [19-acetoxide 4, 16-bromides 14 and 15, 19,19-difluoride 18, and (19R,S)-19-acetylenic alcohol 25] and androst-4-en-17 beta-ol derivatives 3, 5, 10, 12, and 19 were synthesized and tested for their ability to inhibit aromatase in human placental microsomes. All the 17-oxo steroids, except compound 25 and 17,19-diol 3 of this series, were effective competitive inhibitors with apparent Ki's ranging from 170 to 455 nM. 19,19-Difluoro steroid 18 and 19-acetylenic alcohol 25, a weak competitive inhibitor (Ki = 7.75 microM), caused a time-dependent, pseudo-first-order inactivation of aromatase activity with kinact's of 0.0213 and 0.1053 min-1 for compounds 18 and 25, respectively. NADPH and oxygen were required for the time-dependent inactivation, and the substrate, androst-4-ene-3,17-dione, prevented it, but a nucleophile, L-cysteine, did not in each case. The results strongly suggest that aromatase would attack the 19-carbon of steroids 18 and 25.

Estrogen synthetase (aromatase). The cytochrome P-450 component of the human placental enzyme is a glycoprotein

Estrogen synthetase (aromatase) is a cytochrome P-450 enzyme system which converts androgens to estrogens. Although this enzyme has been purified and the cDNA-derived amino acid sequence elucidated, very little is known regarding post-translational modifications of this physiologically crucial enzyme. We show here that the cytochrome P-450 component, P-450 ES, purified from human term placental microsomes, is a glycoprotein based on the following evidence: its molecular weight is decreased following treatment with endoglycosidase F, concanavalin A-biotin specifically binds to this protein immobilized on nitrocellulose, and its oligosaccharide composition is consistent with a single N-linked fucosylated complex type carbohydrate chain. In a reconstitution system, the aromatase activity using the endoglycosidase F-treated P-450 ES was reduced by about 35–40% relative to the native form, regardless of whether androstenedione or testosterone was used as substrate.

Selection of 19-(ethyldithio)-androst-4-ene-3,17-dione (ORG 30958): A potent aromatase inhibitor in vivo

19-Mercaptoandrost-4-ene-3,17-dione (ORG 30365) has been reported to be both a competitive and irreversible inhibitor of aromatase. In comparison to the known aromatase inhibitors 4-hydroxy-androst-4-ene-3,17-dione (4OH-AD) and 1-methyl-1,4-androstadiene-3,17-dione (SH 489), ORG 30365 was found to be, respectively, about 16 and 8 times more active in vitro using human placental microsomes. Although the activity profile of ORG 30365 is very attractive, this compound was not selected for further development because it has limited pharmaceutical stability, which is probably due to its free —SH group and therefore a number of more stable dithio-derivatives of ORG 30365 have been synthesized. These derivatives are considered to be converted to ORG 30365 before they become active. The in vivo aromatase inhibiting activity of these derivatives was determined in hypophysectomized rats treated with the estrogen precursor dehydroepiandrosterone sulphate (DHEAS) using inhibition of cornification of vaginal epithelium as parameter. The 19-(ethyldithio)-derivative (ORG 30958) appeared to be the most active inhibitor in this series being twice as active as ORG 30365 and about 8 times as active as inhibitors like 4OH-AD and SH 489. Besides inhibition of cornification of vaginal epithelium ORG 30958 decreased ovarian aromatase and plasma E 2 levels in DHEAS-treated hypophysectomized rats. Plasma estradiol levels were also lowered by ORG 30958 in dogs which were treated with pregnant mare serum gonadotrophin in order to induce pro-estrus. ORG 30958 displayed much less than 1/400th of the androgenic activity of testosterone propionate in immature castrated rats and appeared to be devoid of estrogenic and anti-estrogenic activity in ovariectomized mature rats. A twice daily dose of 1.5 mg ORG 30958/kg postponed ovulation in mature female rats. In conclusion: ORG 30958 is a potent aromatase inhibitor in vivo. It probably becomes active after cleavage of the —Sz.sbnd;S— bond yielding ORG 30365 a poteny irreversible aromatase inhibitor. ORG 30958 does not display other hormonal activities making it an attractive candidate for the treatment of estrogen-dependent diseases.

Kinetic and immunologic evidence for the absence of glucose-6-phosphatase in early human chorionic villi and term placenta

The existence of the enzyme glucose-6-phosphatase (G6Pase) in early and term human placenta was investigated by comparing the characteristics of placental microsomal glucose 6-phosphate (G6P) hydrolytic activity and liver G6Pase. Placental microsomes exhibited similar apparent Km values for G6 P and β-glycerophosphate in intact and deoxycholate-treated microsomes, heat stability at acidic pH, low latency of mannose 6-phosphate hydrolysis, very low activity of pyrophosphate:glucose phosphotransferase, and undetectable [U-14C]G6 P transport into the placental microsomes, all of which indicated that specific G6Pase activity does not exist in placenta. Immunological evidence of the absence of both 36.5 kDa and T2 proteins, which represent the G6Pase catalytic protein and the phosphate / pyrophosphate transporter protein, respectively, confirmed that early and term human placenta are devoid of the multicomponent G6Pase enzyme.

Comparison of Aromatase Activity in Human Prostatic, Testicular and Placental Tissues

The aromatase enzyme was quantified by the release of tritiated water from [1 beta-3H] androstenedione. Tritiated water was released by the crude homogenates in 4 of 18 samples of benign prostatic hyperplasia tissue and one of 5 samples of prostate carcinoma tissue. However, this apparent aromatase activity was not inhibited by 4-hydroxyandrostenedione (0.5 and 5.0 microM), and none of the particulate fractions (100,000 g pellet) prepared from each of the prostatic tissues exhibited aromatase activity. Particulate fractions from rat ovary (n = 3) and human testes (n = 6) displayed significant aromatase activity (mean values of 9.9 and 0.033 nmol estrone formed/g protein/h, respectively). The testicular aromatase was inhibited by aminoglutethimide, 4-hydroxyandrostenedione and CGS 16949A with IC50 values of 6.4, 0.17 and 0.0017 microM, respectively. These are of a similar order to values obtained with the aromatase enzyme from human placental microsomes (14, 0.43 and 0.0075 microM, respectively).

The effects of free radical scavengers on arachidonic acid metabolism by ovine placental microsomes

1. The effect of free radical (superoxide and hydroxyl) and hydrogen peroxide scavengers on the formation of prostaglandin E 2 by ovine placental microsomes was investigated. 2. Of the free radical scavengers tested only Mn Desferal, a low molecular weight (712 Da) superoxide scavenger, at concentrations of 0.5-5 mmol/1 significantly ( P < 0.001) stimulated PGE 2 formation by ovine placental microsomes. 3. This observation is consistent with the involvement of superoxide radicals in the inactivation of prostaglandin G/H synthase (PGHS). 4. The results obtained further suggest that if superoxide radicals are important regulators of PGHS activity in the ovine placenta, they are not accessible to large molecular weight free radical scavengers.

New non-steroidal aromatase inhibitors: Focus on R76713

R76713 is a novel triazole derivative which selectively blocks the cytochrome P450-dependent aromatase. In human placental microsomes, in FSH-stimulated rat and human granulosa cells and in human adipose stromal cells, 50% inhibition of estradiol biosynthesis was obtained at drug concentrations of 2–10 nM. In PMSG-injected female rats, R76713 lowered plasma estradiol levels by 50 and 90% 2 h after single oral doses of 0.005 and 0.05 mg/kg respectively. After 1 mg/kg, estradiol levels were suppressed by 90% for 16 h. In male cynomolgus monkeys, R76713 dose-dependently (0.03–10 μg/kg) inhibited peripheral aromatization with an ED 50 of 0.13 μg/kg without altering metabolic clearance rates and conversion ratios. In vitro R76713 had no effect on other P450-dependent steroidogenic enzymes up to 1000 nM at least. In rats, LHRH-, ACTH- and sodium-deprived diet stimulated plasma testosterone, corticosterone and aldosterone levels were not modified 2 h after single oral administrations of R76713 (up to 20 mg/kg). Furthermore, R76713 did not show any in vitro or in vivo estrogenic or antiestrogenic property. R76713 also induced regression of DMBA-induced mammary tumors after daily oral administration of 1 mg/kg b.i.d. In male volunteers ( n = 4), a single oral dose of 5 and 10 mg lowered median plasma estradiol levels from 70 pM to the detection limit of the assay (40 pM) 4, 8 and 24 h after intake whereas no changes were detected after placebo administration. In premenopausal women ( n = 15), receiving a single oral dose of 20 mg, median plasma estradiol levels decreased from 389 pM (before) to 168, 133 and 147 pM, 4, 8 and 24 h after intake whereas they remained above 420 pM after placebo ( n = 7).

Biochemical and pharmacological development of steroidal inhibitors of aromatase

Androstenedione analogs containing 7α-substituents have proven to be potent inhibitors of aromatase both in vitro and in vivo. Several of these agents have exhibited higher affinity for the enzyme complex than the substrate. In order to examine further the interaction(s) of 7-substituted steroids with aromatase, biochemical and pharmacological studies were performed on 7α-thiosubstituted androstenediones and 7-substituted 4,6-androstadiene-3,17-diones. Potent inhibition of aromatase activity in human placental microsomes has been observed with several new 7α-thiosubstituted androsenediones. 7-Benzyl-and 7-phenethyl-4,6-androstadiene-3,17-diones effectively inhibited microsomal aromatase, with apparent K i s ranging from 61 to 174 nM. On the other hand, 7-phenyl-4,6-androstadiene-3,17-dione exhibited poor activity, with an apparent K i of 1.42 μM. Similar inhibitory activity was observed with reconstituted, purified cytochrome P450 Arom preparations. Additionally, these agents were evaluated for inhibition of aromatase activity in two human carcinoma cell lines, the MCF-7 human mammary cancer line and the JAr choriocarcinoma line. The 7α-thiosubstituted androstenediones and 7-substituted 4,6-androstadiene-3,17-diones produced dose-dependent inhibitions of aromatase activity in the cell cultures. The most effective inhibitors were the 7α-substituted androstenediones, with EC 50 values ranging from 7.3 to 105 nM. Finally, the JAr cell culture system exhibited prolonged inhibition of aromatase activity following exposure to 7α-APTADD, suggesting enzyme inactivation by this inhibitor. Thus, these agents are effective aromatase inhibitors, and the results encourage further development of this group of medicinal agents for the treatment of estrogen-dependent mammary carcinoma.

Aromatase inhibition by flavonoids

Several synthetic flavones were found to inhibit the aromatization of androstenedione to estrone catalyzed by human placental microsomes. Twenty-one compounds were tested and the IC 50 of the most active were: flavone, 10 μM; 7-hydroxyflavone, 0.5 μM; 7,4′-dihydroxyflavone, 2.0 μM; flavanone, 8.0 μM; and 4′-hydroxyflavanone, 10 μM. Most of the others had IC 50 values ranging from 80 to 200 μM. These findings show that 4′-hydroxylation results in either no change or very little change in IC 50 for flavanone, isoflavone and isoflavanone as well as other ring A hydroxylated flavones. Derivatives of flavone with a hydroxyl substituent at position 5, 6 and 7 were also screened. 7-Hydroxy-flavone ( 11) was the most effective competitive inhibitor (IC 50=0.5 μM) with an apparent K i value of 0.25 μM. Compound 11 also induced a change in the absorption spectrum of the aromatase cytochrome P-450 which is indicative of substrate displacement. The relative binding affinities of the flavonoid analogs were determined and only ring A and ring B dihydroxylated analogs were found to bind to the estrogen receptor.

R 76713 and enantiomers: Selective, nonsteroidal inhibitors of the cytochrome P450-dependent oestrogen synthesis

The triazole derivative, R 76713 and its enantiomers R 83839(−) and R 83842(+) are effective inhibitors of the aromatization of androstenedione. For human placental microsomes, the (+) enantiomer (R 83824) is about 1.9- and 32-times more active than the racemate ( ic 50 2.6 nM) and the (−) enantiomer, respectively. R 83842 is about 30- and 1029-times more active than 4-hydroxy-androstene-3, 17-dione and aminoglutethimide. This potency might originate from its high affinity for the microsomal cytochrome P450 (P450). Indeed, R 83842, compared to R 76713 and R 83839, forms a more stable P450-drug complex. Difference spectral measurements indicate that the triazole nitrogen N-4 coordinates to the haem iron. The reversed type I spectral changes suggest that R 76713 is able to displace the substrate from its binding place and the stable complex formed in particular with the (+) enantiomer suggests that its N-1-substituent occupies a lipophilic region of the apoprotein moiety. Kinetic analysis implies that there is a competitive part in the inhibition of the human placental aromatase by R 76713. The K i , values for R 76713, R 83842 and R 83839 are 1.3 nM, 0.7 nM and 18 nM, respectively. These results are indicative of stereospecificity for binding. Up to 10 μM, R 76713 and its enantiomers have no statistically significant effect on the regio- and stereoselective oxidations of testosterone in male rat liver microsomes. All three compounds have no effect on the P450-dependent cholesterol synthesis, cholesterol side-chain cleavage and 7α-hydroxylation and 21-hydroxylase. At 10 μM, R 76713 has a slight effect on the bovine adrenal 11β-hydroxylase. This effect originates mainly from R 83839, the less potent aromatase inhibitor. On the other hand, the inhibition of the 17,20-lyase of rat testis observed at concentrations ⩾ 0.5 μM, originates rather from R 83842. However, 50% inhibition is only achieved at 1.8μM R 83842, i.e. at a concentration about 1300-times higher than that needed to reach 50% inhibition of the human placental aromatase.

Metabolism of cocaine by human placentas: Implications for fetal exposure

To assess placental metabolism of cocaine, placentas were obtained at the time of delivery and the microsomes were extracted by ultracentrifligation within 2 hours. Placental microsomes were cultured with cocaine at physiologic plasma concentrations similar to those of cocaine users (0.75 µg/ml). Two control groups were established. In the first group an anticholinesterase was added to the culture to suppress enzyme activity, and in the second cocaine was cultured alone without placental microsomes to obtain baseline spontaneous conversion of the drug. The results indicate that cocaine is biotransformed by the human placenta, presumably by cholinesterase activity. This suggests that the placenta may provide a moderate degree of protection from cocaine-induced morbidity, such as abruptio placentae and fetal growth retardation, by converting cocaine into less active metabolites. These results also have pharmacogenetic implications because cholinesterase activity varies among individuals. Hence placentas that cannot transform the drug may place the conceptus at greater risk of developmental abnormalities.

7-Substituted 1,4,6-androstatriene-3,17-diones as enzyme-activated irreversible inhibitors of aromatase

7-Phenyl-1,4,6-androstatriene-3,17-dione (4), 7-benzyl-1,4,6-androstatriene-3,17-dione (5) and 7-phenethyl-1,4,6-androstatriene-3,17-dione (6) were synthesized and evaluated in vitro in human placental microsomes as enzyme-activated irreversible inhibitors of aromatase. The compounds were synthesized from appropriate 7-substituted 4,6-androstadiene-3,17-diones by reaction with DDQ under neutral conditions. All the compounds produced a first order inactivation of aromatase in the presence of NADPH but not in the absence of NADPH. Substrate 4-androstene-3,17-dione protected the enzyme from inactivation by the inhibitors. Furthermore, cysteine failed to protect aromatase from inactivation by compounds 5 and 6. In contrast, cysteine partially protected aromatase from inactivation by compound 4. Irreversibility studies illustrated the covalent nature of the inactivation by 4, 5 and 6. The above experimental evidence demonstrated that compounds 5 and 6 are effective enzyme-activated irreversible inhibitors of aromatase.

Determination of aromatization of 19-oxygenated 16α-hydroxyandrostenedione with human placental microsomes by high-performance liquid chromatography coupled with coulometric detection

A sensitive assay of aromatization of 16α-hydroxylated androgens, 16α-hydroxyandrostenedione (16α-OHA), 16α,19-dihydroxyandrostenedione [16α,19-(OH) 2A], and 16α hydroxy-19-oxo androstenedione (16α-OH-19-oxo A), was developed using reversed phase high-performance liquid chromatography with a coulometric detector. The estrogens, estriol and 16α-hydroxyestrone, were simultaneously detected in quantities as low as 300 pg of the estrogens formed in an assay by an internal standard method. Apparent K m and V max of the microsomal aromatase for 16α-OHA, 16α,19-(OH) 2A or 16α-OH-19-oxo A were 1.06, 4.00 or 571 μM and 0.014, 0.087 or 1.67 pmol/min/μg protein, respectively. The results show that the 19-oxo steroid has extremely low affinity for aromatase relative to the other substrates.

ChemInform Abstract: Synthesis and Biochemical Studies of 7‐Substituted 4,6‐Androstadiene‐3,17‐diones as Aromatase Inhibitors.

AbstractAllylic oxidation of the protected dione (I) yields the androsten‐7‐one (II) which is treated with the organometallics (III).

6α-Flurotestosterone: a nonaromatizable androgen inhibotor of aromatase cytochrome P450

In an early survey of steroids which might serve as estrogen precursors, Gual et al. reported that 6α-fluorotestosterone is not aromatiz.ed by human placental microsomes. Subsequently, 6α-fluorotestosterone has been used to distinguish between androgen- and estrogen-mediated physiologic effects. We have reexamined the interaction of 6α-fluorotestosterone with human placental and rat ovarian microsomes and with reconstituted purified aromatase cytochrome P450. Under conditions in which testosterone was readily aromatized, no aromatiwtion of 6α-fluorotestosterone was observed using either fluorescence detection of dansyl-estrogens separated by high-performance liquid chromatography or estrogen radioimmunoassay methods. The lack of aromatiwtion is not due to failure of6a-fluorotestosterone to bind to P450 arom, because 6α-fluorotestosterone acts as a competitive inhibitor of the enzyme, and it exhibits a binding affinity similar to that of testosterone. Moreover, the addition of 6α-fluorotestosterone to human placental microsomes elicits a spectral shift indicative of conversion of the heme from a low to a high spin state as observed for androgen substrates, consistent with its binding to the substrate site. The mechanism by which substitution of a fluorine at the 6α-position interferes with the aromatization reaction remains to be determined, but the inhibitory action on estrogen formation may potentiate the androgenic properties of 6α-fluorotestosterone in vivo due to a lowering of estrogen levels . ( Steroids 55:242–246, 1990).

Immunochemical and molecular biological studies on human placental cigarette smoke-inducible cytochrome P-450-dependent monooxygenase activities

The induction of specific forms of cytochrome P-450 and P-450-associated xenobiotic-metabolizing monooxygenase activities by maternal cigarette smoking was characterized in human placenta employing polyclonal and monoclonal antibodies and recombinant DNA probes. The anti-BNF-B 2 (prepared against rat liver P-450 induced by beta-naphthoflavone) inhibited about 60 per cent of aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin O-deethylase activities (ERDE) in placental tissues from smoking mothers, whereas the anti-PB-B 2 (to phenobarbital-induced rat liver P-450) was without significant inhibitory effect. Inhibition of 7-ethoxycoumarin O-deethylase (ECDE) by the anti-BNF-B 2 was dependent on maternal smoking: the enzyme from non-smokers was not significantly inhibited, whereas the enzyme from smokers was variably inhibited by 15–60 per cent. The monoclonal antibodies towards the major 3-methylcholanthrene-inducible and phenobarbital-inducible rat liver P-450s (Mab 1-7-1 and 2-66-3, respectively) behaved similarly, except the inhibition was somewhat stronger if present. Antibody raised against rat liver NADPH-cytochrome P-450 oxido-reductase did not inhibit any activity studied. In immunoblotting experiments, the anti-reductase recognized the protein in human placental microsomes. However, neither anti-BNF-B 2, anti-PB-B 2 or Mab 1-7-1 or Mab 2-66-3 detected any proteins in human placental microsomes, regardless of smoking status. Northern blot hybridization analysis of placental RNA samples showed that only P-450IA1 mRNA existed in the placentas of smoking mothers with detectable ERDE activity. Despite the discrepancy between protein blotting and immunoinhibition data all other findings support the conclusion that maternal cigarette smoking induces the expression of CYPIA1 gene (and not CYPIA2), resulting in an increased synthesis of P-450IA1 protein and increased AHH, ERDE and ECDE activities in human placenta.