Enzymic catalysis of the accumulation of acetaldehyde from ethanol in human prenatal cephalic tissues: evaluation of the relative contributions of CYP2E1, alcohol dehydrogenase, and catalase/peroxidases.

BACKGROUND: The human prenatal brain is very sensitive to the toxic effects of ethanol, but very little information is available concerning the conversion of ethanol to the highly cytotoxic metabolite, acetaldehyde, in that organ. Thus, experiments were designed to investigate rates of accumulation of acetaldehyde from ethanol in the prenatal human brain. METHODS: Prenatal human cephalic tissue homogenates were used as enzyme sources and were compared with analogous preparations of adult rat livers. Generated acetaldehyde was derivatized with cyclohexane-1,3-dione to yield fluorescent decahydroacrizine-1,8-dione, which was readily separated, detected, and quantitated with HPLC. RESULTS: Detected rates of accumulation were unexpectedly high, even in the absence of added NADPH, NAD+, or H2O2, which are cofactors/cosubstrates for cytochrome P-450-, alcohol dehydrogenase- and catalase/peroxidase-catalyzed reactions, respectively. Without added cofactors/cosubstrates or other components and under linear reaction conditions, rates in human prenatal cephalic preparations were approximately 20% of those observed with analogous preparations of adult rat livers. Cofactor/cosubstrate-independent reactions were localized in the cytosolic (soluble) fraction and were strongly dependent on molecular oxygen (O2). They were not inhibited substantially by carbon monoxide (CO:O2 = 80:20 vs N2:O2 = 80:20) or by pyrazole in concentrations up to 10 mM and were only weakly inhibited by azide. Preincubations with excess catalase did not result in decreased activity. Reactions exhibited substrate saturation and heat inactivation indicating enzymic catalysis. CONCLUSIONS: Experiments indicated a relatively rapid accumulation of acetaldehyde from ethanol in human prenatal brain tissues and suggested that the observed cofactor/cosubstrate-independent reactions were largely independent of P-450 cytochromes, alcohol dehydrogenases, or catalase/peroxidases. Results were consistent with catalysis by an as yet unidentified cytosolic oxidase(s).

Catalysis of the 4-hydroxylation of retinoic acids by cyp3a7 in human fetal hepatic tissues.

Cytochrome P4503A7 (CYP3A7) is the primary CYP isoform expressed in human fetal hepatic microsomes, and its potential role in human embryotoxicity has attracted considerable investigative attention. In this study, we investigated the 4-hydroxylation of highly embryotoxic and teratogenic retinoic acids (RA) as catalyzed by human fetal liver microsomes (HFLM) and demonstrated that CYP3A7 is an efficient RA hydroxylase. When all-trans-retinoic acid (tRA), 9-cis-retinoic acid (9cRA), or 13-cis-retinoic acid (13cRA) were incubated with HFLM (54-109 gestational days) plus NADPH, each of these three retinoic acids was rapidly converted to its corresponding 4-hydroxy and 4-oxo metabolites. The reactions were strongly inhibited by CO (CO:O(2), 80:20) and were NADPH-dependent, indicating that the reactions were catalyzed by P450 isoenzymes. At 54 to 89 gestational days, 4-hydroxylase activities were relatively low. However, at gestational days 96 to 109, activities were much higher. Selective inhibitors were employed for elucidation of the roles of individual CYP isoenzymes in HFLM. alpha-Naphthoflavone, paclitaxel, and diethyldithiocarbamate showed little or no effects on HFLM-catalyzed reactions, indicating that CYP1A1, CYP1A2, CYP1B1, CYP2C8, and CYP2E1 did not play significant roles in the catalysis. By contrast, troleandomycin strongly inhibited the reaction (70-75% inhibition), suggesting that CYP3A7 was primarily responsible for the observed catalysis. It was also discovered that CYP3A7 SUPERSOMES efficiently catalyzed the 4-hydroxylations of tRA, 9cRA, and 13cRA. Because 4-hydroxylated metabolites of RA are much less potent embryotoxins and teratogens, the results indicated that the 4-hydroxylation of RA, catalyzed prenatally by CYP3A7, might play an important role in protecting the human fetus against RA-induced embryotoxicity.

Patterns of CYP26 expression in human prenatal cephalic and hepatic tissues indicate an important role during early brain development.

CYP26 (P450RAI) catalyzes catabolic retinoic acid (RA) hydroxylation and thereby appears to play a critical role in retinoid signaling pathways during development. In this study, a quantitative competitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed for evaluation of CYP26 message levels in human prenatal tissues. Statistical analyses of transcription levels in 12 prenatal human brains and six prenatal human livers demonstrated good sensitivity and reproducibility. Quantitative profiles of CYP26 gene expression in early (gestational days 57-110) prenatal cephalic and hepatic tissues and comparisons with adult counterparts are reported for the first time. Prenatal cephalic tissues at days 57-67 exhibited values of 1950+/-420 (CYP26 molecules/10(6) GAPDH molecules) whereas prenatal cephalic tissues at days 105-110 exhibited values of 22300+/-4450 (CYP26 molecules/10(6) GAPDH molecules), indicating a sharp developmental increase (approximately 11-fold). Levels in human adult cephalic tissues were slightly less than the prenatal cephalic levels measured during the earliest stages of gestation and were approximately 3-fold lower than those measured in adult human hepatic tissues. Levels in human prenatal hepatic tissues at days 63-110 gestation were less than 800 (CYP26 molecules/10(6) GAPDH molecules) and did not exhibit developmental increases. Considered together, the data have strong implications for the importance of CYP26 in early development of the human brain.

Biosynthesis of all-trans-retinoic acid from all-trans-retinol: catalysis of all-trans-retinol oxidation by human P-450 cytochromes.

Oxidative conversion of all-trans-retinol (t-ROH) to all-trans-retinal (t-RAL) is recognized as the rate-limiting step for biosynthesis of all-trans-retinoic acid from t-ROH in mammalian hepatic tissues. The purpose of this study was to investigate the role of human cytochrome P-450 (CYP)-dependent monooxygenation in the conversion of t-ROH to t-RAL. Adult human liver microsomes (HLMS) were incubated with t-ROH, and retinoids generated were identified and quantified by liquid chromatography-mass spectroscopy, HPLC, and other methods. HLMS-catalyzed generation of t-RAL from t-ROH was primarily NADPH-dependent and was strongly inhibited by carbon monoxide. Rates of reactions increased linearly with time and concentrations of HLMS, and exhibited classical substrate saturation. These observations strongly indicated that the reaction proceeded via CYP-catalyzed monooxygenation. On the basis of responses to selective chemical inhibitors, isoforms from CYP family 1 and the CYP3A subfamily appeared to be very active. Members of the CYP2C subfamily and CYP2D6 exhibited lesser activities and CYP2A6, CYP2B6, and CYP2E1 were virtually inactive. cDNA-expressed human CYP enzymes (CYP SUPERSOMES) also were used to assess the capacity of individual CYP enzymes to catalyze the reaction. Based on responses to selective chemical inhibitors, specific activities, and levels present in adult human hepatic tissues, CYP1A2 and CYP3A4 strongly appeared to be the major CYP enzymes catalyzing hepatic oxidative conversion of t-ROH to t-RAL in the adult human liver. CYP1A1 and CYP1B1 SUPERSOMES both exhibited exceptionally high activities, and in extrahepatic tissues, these isoforms could play important roles in biosynthesis of all-trans-retinoic acid from t-ROH.

Catalysis of drug oxidation during embryogenesis in human hepatic tissues using imipramine as a model substrate.

We investigated the catalysis of drug monooxygenation by human embryonic hepatic tissues at a very early stage of gestation (days 52-59). Imipramine was used as a model substrate and the metabolites generated were identified and quantified by electrospray mass spectroscopy and HPLC. The primary metabolite generated was desipramine. It was reported previously from this and other laboratories that cytochrome P-450 monooxygenase (CYP) 1A1, 1B1, 2E1, and 3A7 are each expressed in human embryonic hepatic tissues, and selective inhibitors were therefore used to elucidate their respective roles. Furafylline did not inhibit the reaction, supporting that CYP1A2 was not expressed in human embryonic hepatic tissues. Diethyldithiocarbamate also failed to inhibit the same reaction, suggesting that CYP2E1 did not play a significant role in catalyzing the reaction. Triacetyloleandomycin inhibited the reaction by approximately 90%, suggesting that CYP3A7 was primarily responsible for catalyzing the reaction. However, alpha-naphthoflavone inhibited the same reaction by approximately 70%, suggesting that CYP1A1 and/or CYP1B1 may also catalyze the reaction substantially. To explore this issue more, a cDNA-expressed human CYP3A7 (CYP3A7 SUPERSOMES) was incubated with alpha-naphthoflavone (1 microM). Generation of desipramine was inhibited by approximately 40 to 50%. The addition of the CYP3A subfamily selective inhibitor triacetyloleandomycin (1 microM) produced no statistically significant inhibition in reactions catalyzed by CYP1A1 or 1B1 SUPERSOMES. Taken together, the results indicated that CYP3A7 was the major if not sole isoform responsible for catalysis of the N-demethylation of imipramine in human hepatic tissues during embryogenesis.

Catalytic activity and quantitation of cytochrome P-450 2E1 in prenatal human brain.

Cytochrome P-450 2E1 (CYP2E1) is a readily inducible hemoprotein that catalyzes the oxidation of endogenous compounds and many low molecular weight xenobiotics. As the major component of the microsomal ethanol oxidizing system, it contributes significantly to ethanol metabolism and the formation of the highly reactive metabolite acetaldehyde. The leaky property of this enzyme results in the generation of reactive oxygen species that can induce oxidative stress and cytotoxic conditions deleterious to development. To further investigate the proposed role of CYP2E1 in the etiology of alcohol teratogenesis, the current study focused on the quantification of CYP2E1 in prenatal human brain, a tissue that is highly vulnerable to the damaging effects of ethanol throughout gestation. In microsomal samples prepared from pools of brain tissues, immunoreactive protein was detected by Western blot analysis using enhanced chemiluminescence, whereas functional protein was estimated with an enzymatic assay using p-nitrophenol and an electrochemical detection system. CYP2E1 transcript was consistently detected in RNA samples prepared from individual brain tissues using the ribonuclease protection assay. Quantitative data were collected by scanning densitometry and phosphorimaging technology. There was a dramatic increase in human brain CYP2E1 content around gestational day 50 and a fairly constant level was maintained throughout the early fetal period, until at least day 113. The relatively low levels of the P-450 isoform present in conceptal brain may be sufficient to generate reactive intermediates that elicit neuroembryotoxicity following maternal alcohol consumption.

Inhibition of human prenatal biosynthesis of all-trans-retinoic acid by ethanol, ethanol metabolites, and products of lipid peroxidation reactions: a possible role for CYP2E1.

Biotransformation of all-trans-retinol (t-ROH) and all-trans-retinal (t-RAL) to all-trans-retinoic acid (t-RA) in human prenatal hepatic tissues (53-84 gestational days) was investigated with HPLC using human adult hepatic tissues as positive controls. Catalysis of the biotransformation of t-ROH by prenatal human cytosolic fractions resulted in accumulation of t-RAL with minimal t-RA. Oxidations of t-ROH catalyzed by prenatal cytosol were supported by both NAD+ and NADP+, although NAD+ was a much better cofactor. In contrast, catalysis of the oxidation of t-RAL to t-RA appeared to be solely NAD+ dependent. Substrate Km values for conversions of t-ROH to t-RAL and of t-RAL to t-RA were 82.4 and 65.8 microM, respectively. At concentrations of 10 and 90 mM, ethanol inhibited the conversion of t-ROH to t-RAL by 25 and 43%, respectively, but did not inhibit the conversion of t-RAL to t-RA significantly. In contrast, acetaldehyde reduced the conversion of t-RAL to t-RA by 25 and 87% at 0.1 and 10 mM respective concentrations. Several alcohols and aldehydes known to be generated from lipid peroxides also exhibited significant inhibition of t-RA biosynthesis in human prenatal hepatic tissues. Among the compounds tested, 4-hydroxy-2-nonenal (4-HNE) was highly effective in inhibiting the conversion of t-RAL to t-RA. A 20% inhibition was observed at a concentration of only 0.001 mM, and nearly complete inhibition was produced at 0.1 mM. Human fetal and embryonic hepatic tissues each exhibited significant CYP2E1 expression as assessed with chlorzoxazone 6-hydroxylation, a highly sensitive western blotting technique, and reverse transcriptase-polymerase chain reaction (PCR) (RT-PCR), suggesting that lipid peroxidation can be initiated via CYP2E1-catalyzed ethanol oxidation in human embryonic hepatic tissues. In summary, these studies suggest that ethanol may affect the biosynthesis of t-RA in human prenatal hepatic tissues directly and indirectly. Ethanol and its major oxidative metabolite, acetaldehyde, both inhibit the generation of t-RA. Concurrently, the CYP2E1-catalyzed oxidation of ethanol can initiate lipid peroxidation via generation of a variety of free radicals. The lipid peroxides thereby generated could then be further converted via CYP2E1-catalyzed reactions to alcohols and aldehydes, including 4-HNE, that act as potent inhibitors of t-RA synthesis.

Recombinant human glutathione S-transferases catalyse enzymic isomerization of 13-cis-retinoic acid to all-trans-retinoic acid in vitro.

The steric conversion of 13-cis-retinoic acid (13-cRA) to all-trans-retinoic acid (t-RA) has been proposed as an activation mechanism for the observed therapeutic and teratogenic activities of 13-cRA. Here we have investigated the catalysis of isomerization of 13-cRA to t-RA by recombinant human glutathione S-transferases (GSTs). Substrate was incubated with GST in 0.1 M sodium phosphate buffer, pH 7.5, at 37 degrees C in total darkness. The t-RA generated was measured quantitatively by HPLC. Under the reaction conditions used, GSTP1-1 was far more effective than human GSTM1-1 or human GSTA1-1 in catalysing the isomerization reaction. The reaction catalysed by GSTP1-1 showed substrate saturation and the Km and Vmax values for the reaction were approx. 7 microM and 650 pmol/min per nmol respectively. The reaction rate increased linearly with increasing enzyme concentration. The reaction was inhibited both by heat treatment and by S-decylglutathione (a potent inhibitor of transferase activity associated with GST). Additions of polyclonal rabbit antiserum for human GSTP1-1 to the reaction resulted in a significant decrease in generation of t-RA (70-80%). In addition, ethacrynic acid, a selective substrate for Pi isoforms of GST, also inhibited the isomerization of 13-cRA to t-RA catalysed by GSTP1-1. Under the same reaction conditions, GSTP1-1 was much less effective in catalysing the steric conversion of 9-cis-retinoic acid to t-RA, indicating that the enzyme was stereospecific for the conversion of 13-cRA to t-RA. These observations suggest that enzymic catalysis was the primary mechanism for the GSTP1-1-dependent conversion of 13-cRA to t-RA. Reactions catalysed by a purified rat hepatic GST Pi isoenzyme proceeded more slowly than reactions catalysed by human GSTP1-1. Comparative studies also showed that there were marked species differences in catalytic activities between various purified mammalian hepatic GST mixtures.

Expression of cytochrome P450RAI (CYP26) in human fetal hepatic and cephalic tissues.

PCR amplifications with two sets of degenerate primers that were targeted to CYP26-specific regions were performed with cDNAs from human fetal liver and brain as templates. PCR products were purified, cloned, sequenced and analyzed with the BLAST program. Our results revealed expression of CYP26 in both human fetal liver and brain. Furthermore, human fetal CYP26 cDNA exhibited 99.2%-100% nucleotide sequence identity to its adult counterpart. Novel isoforms, that would have indicated additional CYP26 genes, were not found. A Northern blot containing poly(A+)RNAs from 43 human adult and 7 human fetal tissues was tested for CYP26 expression. We were able to detect CYP26 message in most tissues but hybridization signals varied in intensity. Highest levels of transcription were in adult liver, heart, pituitary gland, adrenal gland, placenta and regions of the brain. CYP26 expression in fetal tissues was strongest in the brain and comparable with message levels in adult tissues.

Cytochrome-P450-dependent biotransformation of xenobiotics in human and rodent embryonic tissues.

Profound species differences and developmental stage differences as well as a lack of solid data prevent broad, sweeping generalizations in terms of statements that can be made concerning the prenatal expression of individual P450 isoforms. It is clear, however, that several of such isoforms are expressed at levels that can be toxicologically significant. At present, the greatest interest appears to be in P450s 1A1, 1B1, 2E1, and 3A7, each of which has been reported to be expressed at toxicologically significant levels or at least at potentially toxicologically significant levels during organogenesis. Reports of the expression of other P450 isoforms at later stages of gestation also have appeared in the recent literature.

Biotransformation of 13-cis- and 9-cis-retinoic acid to all-trans-retinoic acid in rat conceptal homogenates. Evidence for catalysis by a conceptal isomerase.

The purpose of this study was to investigate whether and to what extent the steric isomerization of retinoic acids in conceptal tissues can be attributed to enzymatic catalysis in addition to thiol-dependent, nonenzymatic catalysis. Conversions of 13-cis-retinoic acid and 9-cis-retinoic acid to all-trans-retinoic acid catalyzed by cell-free preparations of conceptal rat tissues (gestational day 12.5) were investigated. Substrates and rat conceptal homogenates (RCH) were incubated in sodium phosphate buffer (0.1 M, pH 7.5) at 37 degrees C in the dark. Incubation mixtures were quantitatively analyzed by HPLC. In RCH-catalyzed reactions, conversions of 13-cis-retinoic acid or 9-cis-retinoic acid to all-trans-retinoic acid were very rapid, in comparison with uncatalyzed isomerization reactions (incubations without RCH). Comparisons of the rates of reactions catalyzed by freshly prepared vs. freshly prepared/dialyzed RCH showed no significant differences, indicating that small, suflhydryl-containing molecules such as reduced glutathione did not significantly contribute to the RCH-catalyzed reactions. Furthermore, at physiological concentrations (2.5-10 mM), reduced glutathione exhibited very low specific catalytic activities, indicating that nonenzymatic, sulfhydryl-dependent catalysis was not a major mechanism in catalyzing interconversions of retinoic acids in vivo. Enzymatic catalysis by RCH of the conversion of 13-cis-retinoic acid to all-trans-retinoic acid was further characterized by showing 1) substrate saturation kinetics, 2) reaction rates that increased proportionally with protein concentrations, and (3) much greater sensitivity of the reactions to heat inactivation and denaturation by urea, compared with nonenzymatic, glutathione-catalyzed reactions. Thus, isomerization of retinoids in conceptal tissues appeared to be under enzymatic control.

Inhibition of embryonic retinoic acid synthesis by aldehydes of lipid peroxidation and prevention of inhibition by reduced glutathione and glutathione S-transferases.

Inhibition of conceptal biosynthesis of all-trans-retinoic acid (t-RA) by aldehydes generated from lipid peroxidation was investigated. Oxidative conversion of all-trans-retinal (t-RAL, 18 microM) to t-RA catalyzed by rat conceptal cytosol (RCC) was sensitive to inhibition by trans-2-nonenal (tNE), nonyl aldehyde (NA), 4-hydroxy-2-nonenal (4HNE), and hexanal. With an initial molar ratio of aldehyde/t-RAL of 2:1, tNE, NA, and 4HNE caused 70, 65, and 40% reductions of t-RA synthesis, respectively. Hexanal reduced generation of t-RA by approximately 50% as the ratio of aldehyde/t-RAL was raised to 20:1. tNE significantly increased the Km of the reaction and kinetic analyses indicated a mixed competitive/noncompetitive inhibition. By contrast, analogous reactions catalyzed by adult rat hepatic cytosol (ARHC) were highly resistant to inhibition by the same aldehydes. Significant inhibition (> 40% reduction of t-RA generation) by 4HNE, NA, and tNE were achieved at high molar ratios of aldehyde/t-RAL (> 175:1). Hexanal did not inhibit the reaction significantly even at very high ratios of aldehyde/t-RAL (> 2,000:1). Interestingly, when reduced glutathione (GSH, 10 mM) alone or GSH plus glutathione S-transferase (GST) were added to RCC-catalyzed reactions, additions of tNE or 4HNE showed either no significant inhibition or a partial lack of inhibition. Results suggested that GSH-dependent conjugation with 4HNE proceeded slowly compared to conjugation with tNE. To test the hypothesis that GST-catalyzed GSH conjugation can effectively prevent inhibition of t-RA synthesis by aldehydic products of lipid peroxidation, triethyltin bromide (TEB, a potent inhibitor of GST, 20 microM) was added to ARHC-catalyzed reactions when hexanal or tNE were present in the incubations. Eighty and 60% of hexanal and tNE inhibition, respectively, were observed. This was apparently due to TEB blockage of GST-catalyzed GSH conjugation reactions and thus strongly supported the stated hypothesis.

Expression of CYP2E1 during embryogenesis and fetogenesis in human cephalic tissues: implications for the fetal alcohol syndrome.

Reverse transcription and the polymerase chain reaction (RT-PCR) with oligonucleotide primers designed to target cDNA nucleotides 1241-1357 corresponding to exons 8 (3' end) and 9 (5' end) in human genomic CYP2E1 detected consistently strong signals in 9 of 10 prenatal human brains. Cephalic tissues analyzed were between 54 and 78 days of gestation. RT-PCR signals for expression of CYP2E1 in corresponding human hepatic or adrenal tissues were weaker or, with only 2 exceptions, undetectable. Attempts to approximate levels of P4502E1 mRNA with Northern blots and RNase protection assays indicated that levels in human prenatal whole brain tissues tended to increase as a function of gestational age but, at the early stages investigated, were far lower than the constitutive levels in hepatic tissues of adult humans or male rats. Localized, P4502E1-dependent cephalic bioactivation of ethanol, with associated generation of several reactive chemical species, could contribute significantly to the etiology of neuroembryotoxic effects of prenatal ethanol exposure.

Biotransformation of all-trans-retinal, 13-cis-retinal, and 9-cis-retinal catalyzed by conceptal cytosol and microsomes.

Oxidative conversions of all-trans-retinal (t-RAL), 13-cis-retinal (13-cRAL), and 9-cis-retinal (9-cRAL) to their corresponding retinoic acids (RAs) catalyzed by rat conceptal cytosol (RCC) or microsomes (RCM) were studied. The primary product of RCC-catalyzed oxidations of both t-RAL and 13-cRAL was t-RA, with only trace amounts of 13-cRA and 9-cRA. In the RCC-catalyzed oxidation of 9-cRAL, generated t-RA, 9-cRA, and 13-cRA constituted approximately 56, 34, and 10%, respectively, of the total RAs. For all RCC-catalyzed retinal oxidations, NAD was a much more effective cofactor than NADP. And t-RAL and 13-cRAL were much better substrates than 9-cRAL. Formaldehyde, acetaldehyde, citral, and disulfiram were investigated as inhibitors, but only citral and disulfiram effectively inhibited the RCC-catalyzed conversion of t-RAL or 13-cRAL to t-RA. Methanol and ethanol failed to inhibit either reaction even at very high concentrations (> or = 10 mM). RCM exhibited lower specific enzymatic activities than RCC in catalyzing oxidations of t-RAL, 13-cRAL, and 9-cRAL, indicating that the cytosolic fraction was dominant for converting retinals to RAs. The predominant RA produced from RCM-catalyzed oxidations of t-RAL, 13-cRAL, or 9-cRAL was t-RA for each substrate, and again NAD was a much more effective cofactor than NADP in all cases. For RCM-catalyzed oxidations of RALs, 13-cRAL was a much better substrate than t-RAL or 9-cRAL. Methanol and ethanol were not effective inhibitors for RCM-catalyzed oxidations of t-RAL or 13-cRAL. In RCM-catalyzed reactions, citral (10 mM) completely inhibited oxidation of t-RAL but showed only a minor effect on oxidation of 13-cRAL. 13-cRA was converted almost completely to t-RA after 2 hr of incubation with RCC.

Chemical teratogenesis in humans: biochemical and molecular mechanisms.

In this review, an attempt has been made to summarize our current understanding of the mechanisms whereby certain chemicals cause birth defects. The chemicals selected for consideration were those that have been designated as established or recognized human teratogens. It is clear that our current understanding of mechanisms whereby these agents cause teratogenic effects (birth defects) can vary dramatically from one agent to the next. Extremes include the folic acid antagonists, which are now well established as agents that produce birth defects by virtue of potent inhibition of dihydrofolate reductase as a primary biochemical mechanism. An example at the other extreme is ethanol, for which very few definitive statements can be made with regard to teratogenic mechanisms, and the probability exists that a large number of interacting, contributory mechanisms can be invoked. For nearly all chemical teratogens, the critical links in the chains of events between the initial, primary biochemical and molecular mechanistic event (e.g. dihydrofolate reductase inhibition) and the manifestations of specific abnormalities (pathogenic mechanisms) remain to be delineated. This will provide an enormous challenge for investigators for years to come.

Glutathione S-transferases act as isomerases in isomerization of 13-cis-retinoic acid to all-trans-retinoic acid in vitro.

A discovery that rapid enzymic isomerization of 13-cis-retinoic acid (13-cRA) to all-trans-retinoic acid (t-RA) can be catalysed by purified hepatic glutathione S-transferases (GSTs; EC 2.5.1.18) from rat is now reported. Rates of cis-trans isomerization were determined quantitatively by HPLC. GST-catalysed reactions reached equilibrium rapidly, in marked contrast with uncatalysed or GSH-catalysed isomerizations. The GST-catalysed reaction exhibited substrate saturation kinetics with a Km of approx. 8 microM. The maximal velocity of the reaction and the catalytic efficiency of GSTs were determined. The initial rate of the reaction increased linearly as a function of enzyme concentration. Catalysis by GSTs was independent of the presence of GSH, indicating that GSTs act as GSH-independent isomerases as well as transferases. Incubation with guanidine (7-8 M) or heat-inactivation of GSTs (100 degrees C for 3 min) decreased isomerase activities by approx. 50% and 75% respectively. The same heat treatment did not significantly inhibit isomerization catalysed by GSH and apoferritin, indicating that the observed decrease in isomerase activity by heat inactivation was not primarily due to oxidation of protein thiol groups in the GSTs. The specific activity of GSTs was approx. 23- and 340-fold those of GSH and apoferritin respectively when comparisons were made on the basis of free thiol concentrations, indicating that free thiol in GSTs cannot account for the majority of observed isomerase activities and suggesting that specific conformations of GSTs are important for such activities. Complete inhibition of the reaction by low concentrations of N-ethylmaleimide (10 microM) demonstrated that intact protein thiols are required for the isomerase activities of GSTs.

Drug metabolic enzymes in developmental toxicology.

Although much is known about the metabolism of environmental toxicants in adult organisms, little information exists on the role of cytochrome P450 (CYP) enzymes during development. The developing organism is remarkably dynamic, presenting a constantly changing metabolic profile as various enzyme systems are activated or repressed. This may explain the markedly different sensitivities to various toxicants that are exhibited throughout the developmental period. The application of molecular biological methods has provided important information on the roles of these enzymes in modulating the response of the developing organism to toxicological exposures. The first talk will focus on the identification and role of CYPs during early organogenesis, particularly on how these enzymes influence the response of the conceptus and early embryo to toxic chemicals. The second presentation will discuss the identification of CYPs expressed during human development, as many of the enzymes present in adults are not expressed in the fetus. The third speaker will discuss the developmental consequences of loss of expression of particular metabolic enzymes, focusing on recent studies employing knockout mice to examine the role of drug metabolic enzymes during development. The last two talks will discuss some of the short- and long-term consequences of in utero exposures to toxic chemicals and the role of CYP in modulating the toxic response of the developing organism. The first of these will focus on the role of CYP2E1 in human fetuses during late gestation and the response of this enzyme to inducing agents such as alcohol. The last talk will discuss the role of CYP1A1 in the activation of the Ki-ras oncogene following in utero exposure to carcinogens as a mechanism for lung tumor formation in a pharmacogenetic mouse model.

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.