Moses J. Namkung

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.

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.

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.

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.

A comparison of the capacity of fetal and adult liver, lung, and brain to convert polycyclic aromatic hydrocarbons to mutagenic and cytotoxic metabolites in mice and rats

Preparations of S-9 (supernatant fractions of tissue homogenates centrifuged at 9000g for 10 min) fractions from the fetal brains of rats displayed a high capacity to convert 7,12-dimethylbenz(a)anthracene to metabolites mutagenic to Salmonella typhimurium tester strains TA-98, TA-100, and TA-1538. The same tissue was only minimally active or inactive in converting benzo(a)pyrene or N-2-fluorenylacetamide to mutagenic metabolites. Fetal brain tissues of mice were virtually inactive with respect to the bioactivation of each of the three procarcinogens but fetal pulmonary tissues of mice produced mutagen-generating activities that were five- to ninefold above background with respect to 7,12-dimethylbenz(a)anthracene. Fetal hepatic and brain tissues of mice also catalyzed the conversion of each of the three promutagens to cytotoxic intermediates but this phenomenon was not observed with fetal hepatic or brain tissues of rats. Analyses with high-pressure liquid chromatography demonstrated that brain tissues of fetal mice were very active in converting 7,12-dimethylbenz(a)anthracene to oxygenated metabolites whereas the fetal brain tissues of rats were only minimally active. The chromatographic patterns observed also indicated that different metabolites were formed in the presence of S-9 fractions from rats vs mice. The data are consistent with the hypothesis that the previously observed species difference in susceptibility to transplacental tumorigenesis by polycyclic hydrocarbons is related to differences in target organ biotransformation of these compounds.

Transplacental induction of carcinogen-hydroxylating systems with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The effects of a highly toxic herbicide contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) upon rates of hydroxylation of N-2-fluorenylacetamide (FAA) and of benzo[a]-pyrene (BP) were studied in fetal livers, in placentas, maternal livers, and adrenal glands of pregnant Sprague-Dawley rats. Aryl hydrocarbon hydroxylase (AHH) activity was increased 14- and 100-fold in maternal and fetal livers, respectively, but only minimal increases were observed in placentas and adrenal glands. Similar results were obtained with respect to rates of 7-, 5-, N-, 3-, and 1-hydroxylations of FAA. Histologic examinations revealed extensive cellular damage in the placentas as well as pathologic changes in fetal and maternal livers. Electron microscopy indicated enlarged mitochondria and extensive glycogen deposition in maternal and fetal livers and increases in the amounts of rough endoplasmic reticulum in fetal livers. Analyses of BP metabolism with high-pressure liquid chromatography also revealed that the formation of diols (relative to phenols) was markedly increased in fetal and maternal livers following treatments with TCDD. The results suggested that the relative lack of response of fetal hepatic mixed-function oxidase systems to commonly employed inducing agents (polycyclic aromatic hydrocarbons) was not due to a lack of appropriate structural and/or regulatory genes required for the expression of enzyme induction during fetal life.

Immunodetection, immunoinhibition, immunoquantitation and biochemical analyses of cytochrome P-450IA1 in tissues of the rat conceptus 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.

Teratogenicity in vitro of two deacetylated metabolites of N-hydroxy-2-acetylaminofluorene

In previous studies [E. Faustman-Watts, J. C. Greenaway, M. J. Namkung, A. G. Fantel, and M. R. Juchau (1983) Teratology 27, 19-28] an embryo culture system was utilized to investigate the role of biotransformation in the embryotoxicity of 2-acetylaminofluorene. For this investigation, the capacity of two deacetylated metabolites of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to produce malformations in cultured whole rat embryos is reported. The relative capacities of N-hydroxy-2-aminofluorene (N-OH-AF) and 2-nitrosofluorene (NF) to elicit embryotoxic effects, including embryolethality, malformations, growth retardation, and alterations in macromolecular content, were assessed and compared with effects produced by N-OH-AAF and bioactivated 2-acetylaminofluorene (AAF). Qualitatively similar patterns of malformations were produced by NF and N-OH-AF. At initial concentrations greater than 60 microM, both deacetylated compounds caused abnormalities in axial rotation (flexure), decreased viability, and decreases in embryonic DNA and protein content. Both chemicals were active in the absence of a bioactivating system. AAF produced a different spectrum of defects, and was active only in the presence of a complete monooxygenase system. The malformations produced by bioactivated AAF included abnormally open neural tubes; flexure abnormalities were rarely observed. The primary defect elicited by N-OH-AAF was prosencephalic hypoplasia. This chemical was active without an added bioactivating system. Temporal studies demonstrated that exposure of embryos to NF (128 microM) for as little as 2 hr was sufficient to elicit embryotoxic effects. None of the individual metabolites appeared to be solely responsible for the interruptions of neural tube closure produced by bioactivated AAF.

Regulation of aromatic oxidation of estradiol-17β in maternal hepatic, fetal hepatic and placental tissues: Comparative effects of a series of inducing agents

The effects of nine separate inducers of cytochrome P-450-dependent monooxygenases on the hydroxylation of estradiol-17 beta (E2) were investigated in near-term pregnant rats. Isosafrole exhibited highly effective inducing properties in the maternal liver (20-fold and 5-fold increases in 4- and 2-hydroxylase activities respectively). Pregnenolone 16 alpha-carbonitrile produced approx 20- and 30-fold increases in measured respective rates of 4- and 2-hydroxylase activities in fetal hepatic tissues; isosafrole produced only 2-fold increases in the same reaction. Only minor changes or slight increases in estrogen hydroxylation rates were observed in maternal hepatic, fetal hepatic or placental tissues following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or other potent 3-methylcholanthrene (MC)-like inducing agents (beta-naphthoflavone, MC, caffeine). Phenobarbital exhibited relatively weak inducing properties and exposure of pregnant rats to ethanol from days 3-19 of gestation was without statistically significant effects on the parameters investigated. Rat placentas exhibited extremely low estrogen hydroxylase activities irrespective of pre-exposure of pregnant rats to the inducers studied. The results suggested separate regulatory controls for estrogen 2- and 4-monooxygenase activities even though relatively high correlation between the two reaction were generally observed in all three tissues.