Developmental expression of cytochrome CYP26B1 (P450RAI-2) in human cephalic tissues.

Expression levels of cytochrome CYP26B1, which is involved in the specific all-trans-RA inactivation, were evaluated in human cephalic tissues at gestational days 57-110 and exhibited values approximately 10-fold higher than later gestational days 112-224 and adult tissues. CYP26B1 mRNA levels in the adult cerebellum were approximately 2-fold higher than in the adult whole brain tissue. Considered together, these data suggest a unique role for CYP26B1 during human brain development.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

Apoptosis induced in cultured rat embryos by intra-amniotically microinjected sodium nitroprusside.

Previously, we reported that massive cell death was induced in the mesencephalic area of cultured rat embryos after embryos of gestational day 10.5 were intra-amniotically microinjected with sodium nitroprusside (SNP, 800 microM) and cultured for 24 hr at 37 degrees C. The massive cell death apparently was the result of NO-mediated embryotoxicity.

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.

Xenopus laevis: a model system for the study of embryonic retinoid metabolism. III. Isomerization and metabolism of all-trans-retinoic acid and 9-cis-retinoic acid and their dysmorphogenic effects in embryos during neurulation.

These investigations provide data pertaining to the metabolism and disposition of exogenous 9-cis-retinoic acid and all-trans-retinoic acid during neurulation in Xenopus embryos. Each isomer elicited malformations of the heart, eye, and brain, but approximately 2-fold higher concentrations of all-trans-retinoic acid than 9-cis-retinoic acid were required to produce qualitatively and quantitatively similar dysmorphogenic effects. The dymorphogenic effects of all-trans-retinoic acid could not be attributed to the isomerization of all-trans-retinoic acid to 9-cis-retinoic acid.