Catalysis of the dealkylation/debenzylation of phenoxazone ethers by hemoglobin in the absence of peroxides: implications for investigations of embryonic biotransformation.

Investigations of catalysis of the O-dealkylation and O-debenzylation of phenoxazone (resorufin) ethers in human and rodent embryonic tissue homogenates indicated that, with few exceptions, each conceptal tissue investigated contained enzymes capable of catalyzing each of the reactions under study. All observable reactions exhibited NADPH dependence and strong inhibition by carbon monoxide, ketoconazole, alternate electron acceptors, and by hypoxic incubation conditions; but, they were not strongly inhibited by several other classical cytochrome P450 (P450) inhibitors. Cyanide, azide, superoxide dismutase/catalase, and glutathione/glutathione peroxidase each also failed to inhibit the reactions significantly. Subcellular fractionation experiments revealed that cytosolic fractions contained a preponderance of the observable monooxygenase activities. Attempts to identify components responsible for the cytosolic catalytic activity indicated that cytosolic nitric oxide synthases did not contribute significantly. Column fractionation of the cytosol indicated that significant catalytic activity coeluted with fractions containing hemoglobin (Hgb), and experiments with purified Hgb as enzyme source showed that Hgb would catalyze all reactions under study at very slow rates in the absence of added reductases or peroxides. Additions of either reductases or peroxides, however, resulted in marked increases in rates of Hgb-catalyzed reactions. Further investigations strongly suggested that virtually all dealkylation or debenzylation of phenoxazone ethers catalyzed by embryonic cytosolic fractions could be accounted for by the presence of Hgb in those fractions. Conceptal microsomal fractions, however, exhibited definitive, P450-dependent monooxygenase activities attributable to specific individual, identifiable P450 isoforms.

Effects of ethanol on biotransformation of all-trans-retinol and all-trans-retinal to all-trans-retinoic acid in rat conceptal cytosol.

Enzymatic catalysis of the oxidations of ethanol, all-trans-retinol (tretinol) and all-trans-retinal (t-retinal) were demonstrated in the cytosolic fractions of rat conceptal homogenates at day 12 of gestation. Products of the retinoid oxidation reactions were identified with HPLC by comparing elution times with those of authentic standard retinoids. NAD-dependent oxidations of each of the three substrates were demonstrable with assay conditions used; t-retinol and t-retinal each were converted to readily detectable quantities of all-trans-retinoic acid (t-RA). At 1.0 mM or higher concentrations, ethanol effectively inhibited the synthesis of t-RA from both t-retinol and t-retinal when adult hepatic cytosol was used as enzyme source. Approximately 70% and 40% inhibitions, respectively, were observed at 10 mM ethanol concentrations. By contrast, for the reactions catalyzed by rat conceptal cytosol (RCC) under the same experimental conditions, ethanol falled to inhibit significantly the conversion of either t-retinol or t-retinal to t-RA at concentrations up to 1,000 mM. For the RCC-catalyzed conversion of t-retinal to t-RA, increasing concentrations of ethanol (0 to 1.0 M) resulted in linear increases rather than decreases in quantities of t-RA generated. At a 2.0 M concentration of ethanol, the quantity of t-RA increased by > 50%. Significant inhibition of t-RA generation from t-retinal occurred only at extremely high (> 4.0 M) concentrations. The results indicated that ethanol was a very ineffective inhibitor of RCC-catalyzed synthesis of t-RA from either t-retinol or t-retinal. This contrasted strongly with effective inhibitory effects with adult hepatic cytosol as enzyme source. The results supported the concept that competitive inhibition of conversion of t-retinol to t-RA in conceptal tissues is not a significant factor in ethanol-elicited embryotoxicity and dysmorphogenesis, at least in rodents. Mechanisms for the ethanol-induced increases in conversion of t-retinal to t-RA remain to be elucidated.

Interactive dysmorphogenic effects of all-trans-retinol and ethanol on cultured whole rat embryos during organogenesis.

Whole rat conceptuses (10.5 gestational days) were explanted into a culture medium containing all-trans-retinol (t-retinol, vitamin A1), ethanol, or combinations of the two alcohols at various concentrations, and were cultured at 37 degrees C for 24 hr. Parameters emphasized in morphological analyses were branchial arch development, closure of neural tube, axial rotation, and development of otic vesicles and of optic cup. Additions of t-retinol alone to the culture medium resulted in significant decreases in viability at concentrations of 7.0 microM and above. A primary target site affected by t-retinol was the second branchial arch. With initial culture medium concentrations of 3.5 microM, 28% of embryos exhibited an underdeveloped second branchial arch, and the effect was concentration dependent. Incubations with t-retinol alone also caused failure of closure of neural tubes, underdevelopment/absence of otic and optic vesicles, and failure of normal axial rotation, but these effects were statistically significant only at the higher concentrations (10.5-14.0 microM). Incubations of conceptuses with ethanol alone resulted in statistically significant decreases in viability and increases of incidence of embryonic abnormalities at 50 mM but not at 10- or 20-mM concentrations. The embryotoxicity of ethanol appeared less site-specific than that of t-retinol. However, ethanol-elicited developmental abnormalities included underdevelopment of the first and second branchial arches, abnormally open neural tubes, abnormally small or absent otic and optic vesicles, and incomplete axial rotation in common with effects elicited by t-retinol. In general, embryos incubated with combinations of t-retinol and ethanol showed lower survival rates and higher incidences of developmental abnormalities when compared to the calculated values expected for simple additive effects; i.e., interactive effects were most frequently greater than additive and probably synergistic but not antagonistic. To assist in the elucidation of possible mechanism(s) for the greater than additive/synergistic dysmorphogenic effects observed, concentrations of all-trans-retinoic acid (t-RA) and all-trans-retinal(t-retinal) in cultured conceptal tissues were determined by high-performance liquid chromatography (HPLC). HPLC analysis showed increases in conceptal tissue levels of both t-RA and t-retinal after conceptuses were exposed to t-retinol (10.5 microM) plus various quantities of ethanol for 24 hr. These observations, in combination with those of previous studies, suggested that the observed greater-than-additive/synergistic dysmorphogenic effects were not due to the inhibition by ethanol of conceptal biosynthesis of t-RA. Whether the increased levels of t-RA and t-retinal caused the observed greater than additive/synergistic dysmorphogenic effects remains to be elucidated.

Biotransformation of all-trans-retinol and all-trans-retinal to all-trans-retinoic acid in rat conceptal homogenates.

Catalysis of the oxidation of all-trans-retinol (vitamin A1) or of all-trans-retinal to all-trans-retinoic acid (all-trans-RA) by rat conceptal enzymes was investigated during organogenesis. Products of the reaction were identified and quantified with HPLC by comparing their elution times with those of authentic standard retinoids. Under the incubation and assay conditions utilized, all-trans-retinol and all-trans-retinal were converted to readily detectable quantities of all-trans-RA. Rat conceptal homogenates from gestational days 10.5, 11.5 and 12.5 each exhibited enzymatic activity for oxidation of all-trans-retinol and all-trans-retinal to all-trans-RA. Enzymatic catalysis was verified by showing that: (1) both reactions were coenzyme dependent; (2) the rates of reactions increased as concentrations of conceptal protein increased; (3) both reactions were abolished by heating the tissue homogenates (100 degrees, 5 min); and (4) both reactions exhibited substrate saturation. Under the same experimental conditions, formation of all-trans-RA from all-trans-retinol was much slower than from all-trans-retinal, suggesting that oxidation of all-trans-retinol to all-trans-retinal was the rate-limiting step for biotransformation of all-trans-retinol to all-trans-RA in embryonic tissues. When NAD or NADP were replaced by NADH or NADPH, the rate of oxidation of all-trans-retinol was reduced markedly, indicating that the reaction was catalyzed primarily by an NAD/NADP-dependent dehydrogenase(s). Carbon monoxide (CO:O2 = 90:10) did not inhibit the reaction. NAD appeared to be a more effective cofactor than NADP in catalyzing oxidation of all-trans-retinal to all-trans-RA. When NAD was omitted, formation of all-trans-RA from all-trans-retinal was reduced by approximately 55%. Replacing NAD by NADH or NADPH also reduced the conversion of all-trans-retinal to all-trans-RA by about 60%. These observations suggested at least two pathways for the generation of all-trans-RA from all-trans-retinal in embryos: oxidation catalyzed by an NAD/NADP-dependent dehydrogenase(s) and oxidation catalyzed by an oxidase(s) that did not require NAD, NADH, NADP or NADPH. Conversion of all-trans-retinol to all-trans-RA was inhibited strongly by low concentrations of citral, but not by high concentrations of sodium azide, 4-methylpyrazole, or metyrapone. Similarly, oxidation of all-trans-retinal was inhibited strongly by citral but not by metyrapone.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of functional cytochrome P4501A1 in human embryonic hepatic tissues during organogenesis.

Investigations with chemical inhibitors and with inhibitory antibodies specific for cytochrome P4501A-catalyzed ethoxyresorufin (ethoxyphenoxazone) O-deethylation and 2-acetylaminofluorene (N-2-fluorenylacetamide) ring hydroxylation indicated that cytochrome(s) P450 of the 1A subfamily was functionally expressed in human embryonic hepatic tissues at very early stages (days 50-60) of gestation. Lack of detectable capacity of hepatic microsomal enzymes to catalyze either N-hydroxylation of 2-acetylaminofluorene or O-demethylation of methoxyresorufin indicated that functional cytochrome P4501A2 is expressed minimally or negligibly in human embryonic hepatic tissues. By contrast, profound inhibition of the ring hydroxylation of 2-acetylaminofluorene and of the O-deethylation of ethoxyresorufin by 7,8-benzoflavone as well as by anti-cytochrome P4501A1 antibodies indicated the presence of significant levels of functional cytochrome P4501A1 in hepatic microsomes of human embryos. Using the reverse transcriptase-linked polymerase chain reaction with specific oligonucleotide primers, we also detected significant expression of cytochrome P4501A1 mRNA in human embryonic livers. Polymerase chain reaction amplification, cloning and sequencing of the corresponding cDNA provided evidence that the cytochrome P4501A1 mRNA expressed in human embryonic tissues was identical to that expressed in adult human tissues. The results of the study have important implications in terms of the embryotoxic effects of chemicals that are known to be substrates, inhibitors or inducers of cytochrome P4501A1 and to which pregnant women are exposed.

Benzene and benzene metabolites as embryotoxic agents: effects on cultured rat embryos.

Benzene and several of its metabolites were investigated for dysmorphogenic and embryotoxic effects after direct exposures of cultured whole rat conceptuses. Benzene produced no statistically significant effects at concentrations up to 1.6 mM. Inclusion with 1.6 mM benzene of an hepatic xenobiotic-biotransforming system (S9) resulted in only minor decreases in embryonic growth parameters and no detectable dysmorphogenesis. Phenol, a major benzene metabolite, also elicited only minimal embryotoxicity at 1.6 mM concentrations. However, inclusion of an S9 system with phenol resulted in significant dysmorphogenic and embryotoxic effects at concentrations as low as 0.01 mM. For phenol bioactivation, S9 from phenobarbital-induced rats was the most effective, with induction by pregnenolone-16 alpha-carbonitrile, isopropanol, Aroclor 1254, no inducer, and 3-methylcholanthrene following in order of effectiveness. Bioactivating activity resided solely in the microsomal fraction. Metabolites coeluting on HPLC with hydroquinone and catechol were the major metabolites generated from phenol by each S9 system, but no significant correlation between specific metabolite generation and embryotoxicity was apparent. Of the benzene metabolites studied, trans, trans-muconaldehyde exhibited the highest embryotoxic potency but was not detectably generated by any of the S9 systems. Hydroquinone, catechol, and benzoquinone were approximately equipotent, each producing 100% lethality at 0.1 mM. Combined additions to the culture medium of hydroquinone together with phenol resulted in greater than additive effects, indicating a possible synergistic interaction between these metabolites and suggesting that peroxidase activity may be important to the mechanism of phenol-elicited embryotoxicity.

Cytochrome P-450-dependent biotransformation of 2-acetylaminofluorene in cell-free preparations of human embryonic hepatic, adrenal, renal, pulmonary, and cardiac tissues.

Human embryonic hepatic, renal, adrenal, pulmonary, and cardiac tissues (gestational age = 50-60 days) were probed for functional P-450 isoforms with 2-acetylaminofluorene (AAF) in cell-free preparations. Each of these tissues exhibited P-450-dependent hydroxylation at several positions on the AAF molecule, although activities in renal, pulmonary, and particularly cardiac preparations were generally low. N-hydroxylation activities were marginal to undetectable in all five tissues, but 7-hydroxylation was detectable in each tissue. Highest aromatic ring-hydroxylation activities were observed in hepatic tissues, and adrenal tissues also exhibited relatively high activities for ring-hydroxylation, particularly at carbon-7. The 9-hydroxylated AAF metabolite (9-OH-AAF) was the predominant metabolite for all human embryonic tissues, but generation via catalysis by P-450 isoforms appeared to be minimal/negligible. Activity profiles for human embryonic tissues (days 50-60 of gestation) were compared with those of 12 separate, vector-expressed human P-450 isoforms, with those of human fetal tissues (days 72-140 days of gestation), with those of various rodent embryonic tissues, and with those of adult rhesus monkey and adult rat tissues preexposed to inducing agents. These analyses suggested that each of the human embryonic tissues studied expresses functional, xenobiotic-biotransforming P-450 isoforms, but contrasted with previous investigations with phenoxazone ethers as functional P-450 probes. Resolution of these apparent differences will require further research. Early prenatal expression of functional P-450 isoforms in organogenesis-stage human embryonic tissues has important implications for our understanding and predicting of teratogenic/embryotoxic and other biologic effects of exposures to drugs and other environmental chemicals during human pregnancy.

In vitro embryotoxicity of N-methyl-N-(7-propoxynaphthalene-2-ethyl)hydroxylamine (QAB): evidence for N-dehydroxylated metabolite as a proximate dysmorphogen.

The rat conceptus biotransforms N-methyl-N-(7-propoxynaphthalene-2-ethyl)hydroxylamine (QAB) in vitro to 7-propoxynaphthalen-2-ylacetic acid (QAA) and six more (M1 to M6) metabolites. Thus far, M4 has been identified as N-demethyl-QAB and M6 as N-desoxy-QAB. We investigated which of these two metabolites might be involved in QAB-embryotoxicity in vitro. Conceptuses were cultured from day 9.5 to 11.5 post-coitum, and were exposed to N-demethyl-QAB or N-desoxy-QAB either in the culture medium or by microinjection directly into the amniotic cavity. When added to the culture medium, N-demethyl-QAB (No Observed Adverse Effect Level, NOAEL, for growth 122 microM and for differentiation 41 microM) was less active than QAB itself (NOAEL for growth and differentiation 12 microM). N-desoxy-QAB caused severe growth retardation and an impairment of differentiation at a concentration of 11 microM (NOAEL 3.6 microM). As regards causing anomalies, the NOAEL of N-demethyl-QAB (41 microM) was 10-fold higher than that of QAB (NOAEL 3.9 microM) and that of N-desoxy-QAB (NOAEL 3.6 microM). At an intraamniotic concentration of 0.7 mM, N-demethyl-QAB caused no effects on growth and differentiation and no increase of anomalies was observed, whereas QAB and N-desoxy-QAB each elicited an increase in dysmorphogenic embryos at equimolar concentrations without affecting growth and differentiation. It is, therefore, concluded that N-desoxy-QAB, but not N-demethyl-QAB, could be a proximate dysmorphogen responsible for the embryotoxicity/teratogenicity of QAB in vitro.

cAMP-dependent regulation of P450-catalyzed dealkylation in rat conceptal tissues.

Exposures of cultured whole rat conceptuses to varying concentrations of dibutyryl cyclic AMP or isobutylmethylxanthine, alone or in combination, resulted in significant increases in rates of cytochrome P450-dependent depentylation of pentoxyphenoxazone in cell-free preparations. Lesser increases in rates of debenzylation of benzyloxyphenoxazone were also observed. In cultured whole conceptuses, basal depentylase and debenzylase activities in the visceral yolk sac were approximately sixfold higher than in the embryo. The ectoplacental cone and decidual tissues exhibited no detectable depentylase activity. Only the visceral yolk sac exhibited increased depentylase activity in response to dibutyryl cyclic AMP and isobutylmethylxanthine. Inhibitory antibodies raised against adult hepatic P450s IIB1, IIC11, and IA1 failed to significantly inhibit the yolk sac depentylase activities of noncultured conceptuses. The results suggested that the conceptal depentylation reaction may be catalyzed by a unique P450 isoform(s) that is not expressed during adult life.

Cytochrome P450-dependent bioactivation of prodysmorphogens in cultured conceptuses.

These investigations were undertaken to determine the extent to which tissues of cultured rat conceptuses contain cytochrome P450 isoforms in sufficient quantities to significantly influence the capacity of certain chemicals to elicit dysmorphogenic effects in vitro. Investigations with highly sensitive probe substrates/inhibitors and with immunologic methods enabled the detection of at least four separate P450 isoforms in tissues of the visceral yolk sac, ectoplacental cone, and embryo proper. One of the isoforms was identified as P450IA1 and was found to be inducible by polycyclic aromatic hydrocarbons in all three tissues. Other isoforms exhibited properties differing from characterized adult rat hepatic isoforms. Each of the isoforms was detectable in conceptuses on gestational days 10, 11, 12, and 14 and was present in the highest concentrations in the visceral yolk sac. Conceptal P450IA1 catalyzed the conversion of dysmorphogenically inactive 2-acetylaminofluorene to 7-hydroxy-2-acetylaminofluorene, a proximate dysmorphogen. Investigations with microinjections suggested that visceral yolk sac hydroxylation was largely responsible for the bioactivation reaction in vitro. The same isoform exhibited no capacity to influence the dysmorphogenic activity of cyclophosphamide. The results demonstrated that tissues of cultured rat conceptuses may contain P450 isoforms in sufficient amounts to markedly influence the dysmorphogenic activity of substrates of the corresponding isoforms.

Dysmorphogenesis elicited by microinjected acetaminophen analogs and metabolites in rat embryos cultured in vitro.

Direct additions of acetaminophen (APAP), 3,5-dimethylacetaminophen, 3-hydroxyacetaminophen or 3-methoxyacetaminophen to the medium of cultured embryos each produced an increased incidence of morphologically similar, abnormally open anterior neuropores. Approximate concentrations required to produce an equal incidence were 0.5 mM, 1.0 mM, 0.1 mM and 0.75 mM, respectively. In contrast, 2.6-dimethylacetaminophen and N-acetyl-p-benzoquinoneimine failed to produce elevated incidences of abnormal neurulation unaccompanied by marked growth retardation. However, with intra-amniotic microinjections, 3-hydroxyacetaminophen and N-acetyl-p-benzoquinoneimine were roughly equipotent for eliciting abnormal neurulation, whereas 3-methoxyacetaminophen required greater than 30-fold higher concentrations. This suggests that N-acetyl-p-benzoquinoneimine does not readily transit the visceral yolk sac and would likely not be a major factor in APAP-elicited neural tube abnormalities unless generated in target tissues. The differential effects produced by two dimethylated (2.6 and 3.5) APAP analogs further suggest that sulfhydryl oxidation is associated more closely than sulfhydryl conjugation with the neurulation defect. Intra-amniotic microinjections of large quantities (3500 ng) of 7-hydroxy-2-acetylaminofluorene (7-OH-AAF) or APAP failed to produce the specific neurulation defect. Microinjections of 7-OH-AAF into the exocoelomic cavity effected the characteristic abnormal neurulation. Conversion by conceptal homogenates of 7-OH-AAF was roughly 7- to 8-fold more rapid than conversion of APAP to respective catechol metabolites, and specific activities in yolk sac tissues were greater than those in the embryo. Rates of conceptal conversion to the quinoneimine were approximately 2- to 3-fold lower than catechol generation.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunodetection, immunoinhibition, immunoquantitation and biochemical analyses of cytochrome P-450IA1 in tissues of the ratoffceptus during the progression of organogenesis.

Polyclonal antibodies raised against the adult form of rat hepatic cytochrome P-450IA1 were used to immunologically detect, inhibit and quantitate an analogous isozymic form(s) in various tissues of the rat conceptus during the progression of organogenesis. Tissues investigated were the embryo proper, the visceral yolk sac and the ectoplacental cone/chorioallantoic placenta. Studies were performed on conceptuses from day 10 (day of conception = day 0) to day 14 of gestation. Ethoxyphenoxazone deethylation, benzo[a]pyrene (BaP) hydroxylation, and ring- and N-hydroxylation of 2-acetylamino-fluorene (AAF) were utilized in assessments of cytochrome P-450IA1-dependent monooxygenase activities during the same gestational period. In untreated conceptuses, cytochrome P-450IA1 could not be detected immunologically in any of the three tissues at any stage of gestation investigated. The deethylation reaction was quantifiable in embryos and yolk sacs of untreated conceptuses, but was not inhibited by cytochrome anti-P-450IA1 antibodies, alpha-naphthoflavone or metyrapone. Treatment of pregnant rats with 40 mg/kg of 3-methylcholanthrene 48 hr prior to removal of the conceptuses resulted in marked increases in measured enzymatic activities as well as in readily immunodetectable cytochrome P-450IA1. Inducibility for the deethylase was greatest in the visceral yolk sac (3-8x), was evident in the embryo proper (2-3x) but was minimal in the ectoplacental cone (1.5-2x). Much greater induction (up to 70x) was observed with BaP and AAF as substrates. Induced activities were inhibited effectively (70-100% inhibition) by cytochrome anti-P-450IA1 antibodies and by alpha-naphthoflavone but not by metyrapone. Inducibility increased as a function of gestational age in the ectoplacental cone/chorioallantoic placenta but reached maxima on day 12 in the embryo and visceral yolk sac. A good correlation between antibody/alpha-naphthoflavone-inhibited enzymatic activities and quantities of immuno-detectable cytochrome P-450IA1 was also apparent. The results indicate that cytochrome P-450IA1, or a very closely related isoform(s), is both inducible and enzymatically functional in tissues of the conceptus throughout organogenesis and have important implications for the potential effects of bioactivatable proteratogens.

On the substrate specificity of cytochrome P450IIIA1.

The instability of the solubilized/purified form, the lack of catalytic activity of the stabilized, macrolide-complexed form, and the compromised catalytic activity of the decomplexed form of steroid-inducible cytochrome P450IIIA1 motivated further investigations of the substrate specificity of this isozyme. A major complementary goal was to identify reactions utilizable as sensitive, specific diagnostic probes for the detection and partial characterization of this isozyme in tissues for which isolation and purification are not practical (e.g., extrahepatic, embryonic tissues, etc.). The approach utilized a combination of a specific, purified inducer, specific inhibitors including triacetyloleandomycin and inhibitory antibodies, and diagnostic probe substrates including the phenoxazone ethers, testosterone, warfarin, 2-acetylaminofluorene, estradiol-17 beta and benzo[a]pyrene. The results obtained indicated that steroid-inducible, rat hepatic P450IIIA1 would catalyze minimal or no O-dealkylation of methoxy-, ethoxy- or pentoxyphenoxazone but catalyzed rapid O-debenzylation of benzyloxyphenoxazone. Hydroxylation of testosterone was specific for the beta face of the molecule at the 2-, 6-, 15- and 16-positions with no detectable conversion to androstenedione and minimal hydroxylation on the alpha face. Both the R- and S-enantiomers of warfarin were attacked at positions 9 and 10, and these reactions appeared to be specific to isozymes of the IIIA family. Aromatic hydroxylation of estradiol-17 beta was efficiently catalyzed, particularly at the 2-position. Hydroxylations of 2-acetylaminofluorene at positions 5 and 7 were catalyzed at relatively rapid rates, but N-hydroxylation of the same substrate was not catalyzed effectively. Hydroxylation of benzo[a]pyrene occurred preferentially at carbon 3 with much lesser activity at carbon 9 and little or no detectable attack at positions 7 or 1. The results indicated that the 2 beta- and 15 beta-hydroxylation of testosterone and the 10-hydroxylation of warfarin would serve as the most useful probes thus far available for detection of the presence of functional P450IIIA1 isozymes in tissues for which isolation and purification are impractical. The results also indicated a very broad, yet selective substrate specificity for the steroid-inducible P450IIIA1.

Cytochrome P-450-dependent biotransformation of a series of phenoxazone ethers in the rat conceptus during early organogenesis: evidence for multiple P-450 isoenzymes.

Using highly sensitive probe-substrate analyses, investigations of drug biotransformation in tissues of the rat conceptus during an early stage of organogenesis revealed that three separate tissue components each contained P-450 isozymes capable of catalyzing the monooxygenation of foreign organic chemicals. Tissues of the embryo proper contained constitutive P450(s) that catalyzed readily measurable O-depentylation and O-debenzylation of pentoxyphenoxazone and benzyloxyphenoxazone, respectively, but no measurable O-demethylation of methoxyphenoxazone and barely detectable O-deethylation of ethoxyphenoxazone. Higher specific activities for the O-depentylation and O-debenzylation reactions were measured in preparations of the yolk sac and this organ also appeared to contain constitutive P450(s) for the readily detectable O-deethylation of ethoxyphenoxazone. The O-demethylation of methoxyphenoxazone could not be detected in the yolk sac. Only the O-debenzylation reaction could be detected in tissues of the ectoplacental cone. Treatment of conceptuses in utero with 3-methycholantherene (MC) resulted in significantly increased rates of O-deethylation reactions in preparations of yolk sac and embryo but not ectoplacental cone. Demethylation was not detectable in the same preparations. Treatment with phenobarbital, pregnenolone-16 alpha-carbonitrile, or isosafrole produced no observable effect on any of the reactions studied. Carbon monoxide (CO:O2 = 80:20 versus N2:O2 = 80:20) markedly inhibited all reaction rates and inhibition could be reversed by replacement of CO with N2. Deethylation and debenzylation were inhibited by anti-P450IA1 IgG after MC induction but were not affected by the same IgG fraction in untreated conceptuses. Depentylation reactions were not inhibited by anti-P450IA1 or anti-P450IIB1/2 antibodies under any of the conditions used. Deethylation was strongly inhibited by 1.0 microM 7,8-benzoflavone in tissues from MC-treated but not untreated conceptus. Metyrapone (0.1 mM) failed to significantly inhibit any of the measurable conceptus-catalyzed depentylation reaction. The results indicated the presence of four (or more) functional P450 isozymes in tissues of the conceptus during organogenesis, a constitutive depentylase(s) in the yolk sac and embryo, a constitutive deethylase(s) present in the yolk sac, an MC-inducible deethylase(s) in the embryo and yolk sac, and constitutive debenzylase(s) present in all three tissues. No O-demethylation was detectable in any of the three tissues, even after in utero exposure to inducers.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of intracellular glutathione in rat embryos and visceral yolk sacs and its effect on 2-nitrosofluorene-induced malformations in the whole embryo culture system.

The dysmorphogenic effects of 2-nitrosofluorene (NF) in vitro were modulated in Day 10 rat embryos by agents which regulate intracellular glutathione (GSH) levels. The incidence of abnormal axial rotation caused by NF alone increased in a dose-dependent manner at NF concentrations in excess of 25 microM. No effects were observed at 15 microM NF and doses of 100 microM resulted in a 100% incidence of mortality. L-Buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH synthesis, produced malformations (50%) in embryos exposed to 15 microM NF but produced no additional effects on embryos at higher NF concentrations. BSO treatment alone resulted in a greater than 50% decrease in GSH content in visceral yolk sacs and had a lesser but likewise significant effect (15% decrease) on the GSH content of embryos. Protein content was inversely affected as embryonic levels were increased by 20% and yolk sac levels were unchanged. When BSO was added in combination with NF at the onset of the culture period, embryonic GSH decreased in a dose-dependent manner, suggesting a relatively low rate of embryonic GSH turnover that could be increased by addition of an exogenous substrate capable of forming adducts with and removing GSH from the cells. 2-Oxothiazolidine-4-carboxylate (OTC), a compound which is enzymatically modified to provide an additional source of intracellular cysteine and increase GSH synthesis, produced no significant changes in embryonic or yolk sac GSH when added alone to the culture medium. When OTC (5 mM) was added in combination with NF, however, NF-elicited malformations were eliminated. This was also the case at 100 microM NF in which OTC not only prevented malformations but completely protected embryos against the loss in viability. The GSH and protein levels were indistinguishable from controls when OTC and NF were added simultaneously except for the 41 microM NF dose at which a highly significant increase in both embryonic and yolk sac protein was observed. This study clearly demonstrates the potential importance of GSH in the modulation of chemical dysmorphogenesis and provides an important new tool for the study of mechanisms of developmental toxicity.

Modulation of the embryotoxicity in vitro of reactive metabolites of 2-acetylaminofluorene by reduced glutathione and ascorbate and via sulfation.

To provide insights into mechanisms whereby reactive intermediary metabolites of foreign chemicals elicit teratogenic and embryotoxic effects, we initiated investigations of the capacity of physiologic factors to modulate the effects of embryotoxic metabolites of 2-acetylaminofluorene (AAF). The whole embryo culture system was utilized in order to avoid potentially confounding maternal factors. Reduced glutathione (GSH) effectively protected cultured embryos from the embryolethal effects of N-acetoxy-2-AAF (AAAF) and also reduced the severity of AAAF-elicited malformations although the percentage of embryos exhibiting malformations was not affected significantly. GSH also reduced the embryolethality of 2-nitrosofluorene (NF) as well as the percentage of NF-elicited axial rotation defects. Ascorbate protected against the embryolethality of both AAAF and NF and exhibited significant protection in terms of the capacity of NF to cause flexure abnormalities. However, significant protection against NF-elicited prosencephalic deformities was not detected. N-Acetylcysteine and alpha-tocopherol each failed to produce significant protection, even at the highest concentrations utilized. Enzymatic sulfation of N-hydroxy-AAF (N-OH-AAF) markedly increased the incidence of observable malformations. Synthesized N-sulfonyloxy-AAF also elicited a high incidence of malformations at relatively low concentrations when added to the culture medium. Malformations elicited, however, resembled those produced by NF rather than by AAAF. The results suggest that endogenous metabolic systems can play a critical role as determinants of both the quantitative and the qualitative capacity of foreign organic chemicals to produce embryotoxic or teratogenic effects via the generation of reactive intermediates.

Phase II biotransformation of carcinogens/atherogens in cultured aortic tissues and cells. I. Sulfation of 3-hydroxy-benzo(a)pyrene.

The capacity of polynuclear aromatic hydrocarbons to elicit profound effects on the development of avian aortic atheromata has raised questions regarding the biotransformation of polynuclear aromatic hydrocarbons in the target (aortic) tissue. Results of this investigation demonstrate the capacity of aortic enzymes to affect the sulfoconjugation of 3-hydroxy-benzo(a)pyrene and describe several characteristics of the aortic sulfotransferase activity. Conjugating activities measured in avian aortic tissues were approximately 10-20% of those assayed in corresponding preparations of avian hepatic tissues under the same reaction conditions. Activities were measured in homogenates, in a series of homogenate subfractions, in whole organ cultures, in cultured aortic endothelial cells, and in cultured aortic smooth muscle cell preparations. Sulfoconjugation was localized in the cytosolic fraction and kinetics in this fraction yielded a range of apparent Km values from 9 to 16 microM (mean = 11.8 +/- 3.1, n = 4) and a range of apparent Vmax values from 281 to 457 pmol/mg of protein/30 min (mean = 360 +/- 49, n = 4). Abdominal and thoracic segments of the aorta exhibited virtually identical specific activities. Also, activities assayed in cultured aortic smooth muscle cells were similar to those measured in cultured aortic endothelial cells. Capacity to generate adenosine 3'-phosphate 5'-phosphosulfate (PAPS) appeared to limit the reaction rate as judged by comparative investigations with PAPS and a PAPS-generating system. Aortic sulfatases actively hydrolyzed benzo(a)pyrene-3-O-sulfate. The sulfatase activity appeared to partially mask sulfotransferase activities measured in organ and cell culture preparations and in particulate subfractions of cellular homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)

Phase II biotransformation of carcinogens/atherogens in cultured aortic tissues and cells. II. Glucuronidation of 3-hydroxy-benzo(a)pyrene.

Avian aortic tissues contain active UDP-glucuronosyltransferase(s) (EC 2.4.1.17) which catalyze(s) the glucuronidation of 3-hydroxybenzo(a)pyrene and p-nitrophenol. Activities were compared in abdominal segments (susceptible to the atheroma-promoting effects of polynuclear aromatic hydrocarbons) vs. thoracic segments (susceptible to the atheroma-initiating effects of polynuclear aromatic hydrocarbons). Activities measured in the abdominal segments were approximately 8-9-fold higher than those measured in thoracic segments from the same cockerels. Surprisingly, pretreatment of cockerels with phenobarbital, but not 3-methylcholanthrene, resulted in small (60-120%) but consistent increases in glucuronosyltransferase activities in both aortic segments. Activities were readily detectable in microsomal fractions of both segments and in cultured smooth muscle cells derived from aortic segments, but were not detectable in cultured endothelial cells. Avian aortic microsomal glucuronosyltransferases exhibited minimal or no response to the effects of several common activators of hepatic microsomal glucuronosyltransferases.

Cytosolic activation of hematin-dependent microsomal monooxygenase activity in the lung.

Additions of micromolar concentrations of hematin to washed rat pulmonary microsomal preparations resulted in marked (5-7-fold) increases in the NADPH-dependent generation of phenolic metabolites of benzo[a]pyrene (BaP). 9-Hydroxy-BaP was identified as the major reaction product. Additions of pulmonary cytosolic fractions to microsomes produced no measurable effect but cytosol and hematin added together elicited 25-30-fold increases in total phenolic products. Cytosolic fractions from other tissues, including rat kidneys and perfused rat livers, were also highly effective in enhancing the hematin-mediated increases in monooxygenase activity. However, cytosol from human placental tissues was only minimally effective when either pulmonary or placental microsomes were utilized as enzyme source. Superoxide dismutase and catalase (alone or in combination) had no measurable effect on hematin-mediated increases. Horseradish peroxidase effectively inhibited the hematin-dependent reactions but hematin-independent reactions were inhibited with equal effectiveness. Carbon monoxide profoundly inhibited all hematin-mediated increases in metabolite formation. The activating cytosolic component was non-dialyzable, inactivated by trypsin and heat, and eluted in the void volume from Sephadex G-150 columns. This suggested that the cytosolic factor(s) responsible for the increased hematin-dependent oxidation was a protein(s) with a high molecular weight or perhaps an aggregate or oligomer of proteinaceous material. HPLC profiles indicated a major effect on the generation of phenolics; quinones were also increased but only minimal increases in diols were observed. Results were consistent with the hypothesis that hematin-mediated increases in pulmonary monooxygenase activity result from an increased association of a small pool of pulmonary P-450-apoprotein(s) with the hematin prosthetic group to result in increased levels of an unidentified holocytochrome(s) with a relatively high substrate turnover number. The current data suggest a quaternary interaction among P-450 apoprotein(s), heme prosthetic group, reaction products (particularly 3-hydroxy-BaP) and a cytosolic protein(s). We postulate that the mechanism of action of the cytosolic factor is to facilitate the interaction of hematin with the apocytochrome.

Analysis of metabolites of 2-acetylaminofluorene generated in an embryo culture system. Relationship of biotransformation to teratogenicity in vitro.

2-Acetylaminofluorene (AAF) produced abnormal, open neural tubes in cultured whole rat embryos only in the presence of an added, NADPH-dependent monooxygenase system. Reactive intermediary metabolites, including N-hydroxy-AAF, N-hydroxy-2-aminofluorene, 2-nitrosofluorene and N-acetoxy-AAF, each elicited embryonic malformations under culture conditions, but a statistically significant increase in the incidence of abnormal neurulation was not observed. Using [14C]AAF and high pressure liquid chromatography (HPLC) separation techniques, the biotransformation of AAF was studied under conditions in which embryos and the monooxygenase system were coincubated. The major metabolites produced cochromatographed with 5-hydroxy-AAF, 7-hydroxy-AAF, 9-hydroxy-AAF and 3-hydroxy-AAF. Other metabolic products also were detected. The embryonic effects of these major AAF metabolites were tested singly and in combination in the embryo culture system. Addition of 7-hydroxy-AAF to the embryo culture system resulted in open neural tubes in the absence of an added monooxygenase system. Other individual ring-hydroxylated metabolites produced retarded growth, but neurulation appeared normal. Ring-hydroxylated metabolites, added to the embryo culture system in combination in the same proportions as were formed during biotransformation in culture, also produced a marked increase in incidence of neural tube defects in the absence of an exogenous (added) biotransforming system. In combination with 3-, 5- and 9-hydroxy-AAF, 7-hydroxy-AAF exposure (86 microM) resulted in a 47% incidence of abnormal, open neural tubes. When tested individually, higher concentrations of 7-hydroxy-AAF (104 microM) produced a lower percentage of malformed embryos (13%). The results suggested that 7-hydroxy-AAF was principally responsible for the neural tube defects caused by AAF following monooxygenase-dependent bioactivation, but that other metabolites also appeared to contribute to the observed effect.