Analysis of metabolites of 2-acetylaminofluorene generated in an embryo culture system

Faustman-Watts, Elaine M.; Namkung, Moses J.; Greenaway, Jean C.; Juchau, Mont R.

doi:10.1016/0006-2952(85)90021-8

Cited by 37 publications

(5 citation statements)

References 20 publications

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“…In contrast to this, peroxidase occurs in abundant quantities in the human placentas of nonsmokers [I91 and the purified enzyme was found to avidly oxidize a wide range of model chemicals [19][20][21]. Previous mechanistic studies on teratogenicity of arylamines and related compounds have utilized the in vitro rat embryos culture assay [2][3][4][5]8] and assessed the role of microsomal cytochrome P-450 in the bioactivation by supplementing the culture media with adult rodent hepatic S9 fraction and NADPH [2-51. A possible contribution of the peroxidative pathways in the bioactivation of teratogenic arylamines was not investigated. In view of this, it was of interest to evaluate whether 2-AF can serve as a substrate for HTPP.…”

Section: Discussionmentioning

confidence: 99%

“…In vitro studies with rat embryos have shown that reactive metabolites of both acetylated and deacetylated 2-AF exhibit developmental toxicity. However, distinction exists in the type of defects produced [2][3][4][5]8]. The active metabolites of acetylated derivatives mainly produce prosencephalic hypoplasia, microcephaly, and limb bud abnormalities, while abnormalities in axial rotation (flexture) are induced by the ultimate toxicants of deacetylated compounds [2-531.…”

Section: Discussionmentioning

confidence: 99%

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2‐aminofluorene bioactivation by human term placental peroxidase

Murthy

Joseph

Kulkarni

1995

Teratog Carcinog Mutagen

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Earlier investigations from our laboratory demonstrated that human term placental peroxidase (HTPP) is capable of metabolism of xenobiotics and endogenous compounds. In this study, purified HTPP was found to bioactivate 2-aminofluorene (2-AF) in the presence of H2O2. 2-AF oxidation was studied spectrophotometrically while radiometry was employed to assess the bioactivation. The rate of oxidation and covalent binding to protein and DNA was dependent upon the pH of the reaction medium and the concentration of 2-AF, the enzyme, and H2O2. To observe maximal enzyme velocity of oxidation, the presence of 16.5 microM H2O2, 100 microM 2-AF, 37 micrograms of the enzyme protein/ml, and pH 7.2 was required. Under optimal assay conditions, the range of specific activity between 130 and 165 nmol of 2-AF oxidized/min/mg HTPP was observed. Using similar assay conditions, the magnitude of covalent binding of [3H]-2-AF to protein (BSA) and calf thymus DNA was found to be about 508 pmol bound/min/mg HTPP/mg BSA and 84 pmol bound/min/mg HTPP/mg DNA, respectively. Potassium cyanide and sodium azide, the known inhibitors of different peroxidases, significantly blocked both the oxidation and covalent binding of 2-AF in a dose dependent manner. These results strongly suggest that peroxidase may be one of the important pathways responsible for the bioactivation of arylamines in human term placenta.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

2‐aminofluorene bioactivation by human term placental peroxidase

Murthy

Joseph

Kulkarni

1995

Teratog Carcinog Mutagen

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“…The aforementioned approaches require that the metabolic pathway for the compound of interest be well characterized and that each metabolite is available for testing. If this is not the case, research on the role of metabolites can still be pursued by culturing embryos in the presence of an added metabolic activation system, as has been successfully demonstrated with several pro-teratogens such as cyclophosphamide (Piersma et al, 1991), azathiprine, mercatopurine, methotrexate, cyclosporin A (Schmid, 1984), b-aminonicotinamide, and 2-acetylaminofluorene (Faustman-Watts et al, 1985). This method works particularly well with compounds metabolized by liver microsomes (S9), but not for metabolites generated by liver cytoplasmic enzymes, such as the alcohol and aldehyde dehydrogenases (Boerman and Napoli, 1996;Cheever et al, 2001).…”

Section: Identification Of Proximate Toxicant: Role Of Metabolites Inmentioning

confidence: 98%

Whole embryo culture: a “New” technique that enabled decades of mechanistic discoveries

Ellis-Hutchings

Carney

2010

Birth Defects Research Pt B

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Denis New's development of the rodent whole embryo culture (WEC) method in the early 1960s was a groundbreaking achievement that gave embryologists and teratologists an unprecedented degree of access to the developing postimplantation rodent embryo. In the five decades since its development, WEC has enabled detailed investigations into the regulation of normal embryo development as well as a plethora of research on mechanisms of teratogenesis as induced by a wide range of agents. In addition, WEC is one of the few techniques that has been validated for use in teratogenicity screening of drugs and chemicals. In this review, we retrace the steps leading to New's development of WEC, and highlight many examples in which WEC played a crucial role leading to important discoveries in teratological research. The impact of WEC on the field of teratology has been enormous, and it is anticipated that WEC will remain a preferred tool for teratologists and embryologists seeking to interrogate embryo development for many years to come.

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“…The morning after copulation was designated as d 0 of gestation. Explantation of conceptuses was as described previously [4,5]. The conceptuses (d 9.5) were removed from ether-anesthetized dams and were transferred into culture medium saturated with a gas mixture of 02:C02:N2 (5:5:90).…”

Section: Culture Conditionsmentioning

confidence: 99%

Effects of QA 208‐199 and its metabolite 209‐668 on embryonic development in vitro after microinjection into the exocoelomic space or into the amniotic cavity of cultured rat conceptuses

Bechter

Terlouw²,

Lee

et al. 1991

Teratog Carcinog Mutagen

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The objective of this study was to determine the direct embryotoxic effects in vitro of N-hydroxy-N-methyl-7-propoxy-2-naphthalene-ethanamine (QA 208-199, QAB) and of one of its metabolites, 7-propoxy-naphthalene-2-ylacetic acid (209-668, QAA), after circumventing the bioconverting conceptual membranes. The compounds were, therefore, microinjected either into the exocoelomic space or into the amniotic cavity of rat conceptuses of 10 d at prenatal age at doses of up to 84.9 ng (QAA) and 180 ng (QAB) per conceptus respectively. The conceptuses were subsequently cultured for 28 h after which their development was assessed. QAB produced marginal effects on embryonic differentiation only after microinjection of the compound into the amniotic cavity. Dysmorphogenic effects, however, occurred in a dose-dependent fashion after either exocoelomic or intraamniotic microinjections of the compound. The frequencies and types of anomalies were similar after either exposure route and consisted predominantly of anomalies associated with axial rotation. QAA also impaired embryonic differentiation at only the high dose level of 84.9 ng per embryo and after intraamniotic injections only. Dysmorphogenic effects were observed in all experimental groups, although the differences were not statistically significant when compared with the concomitant controls. An increased proportion of anomalies observed were in the cephalic region as compared to the defects produced by QAB. These data suggest that QAA most probably is not the QAB metabolite responsible for the embryotoxic action of QAB in vitro. Furthermore, the results tend to confirm the suggested involvement of the visceral yolk sac membrane in mediating QAB embryotoxicity.

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Analysis of metabolites of 2-acetylaminofluorene generated in an embryo culture system

Cited by 37 publications

References 20 publications

2‐aminofluorene bioactivation by human term placental peroxidase

2‐aminofluorene bioactivation by human term placental peroxidase

Whole embryo culture: a “New” technique that enabled decades of mechanistic discoveries

Effects of QA 208‐199 and its metabolite 209‐668 on embryonic development in vitro after microinjection into the exocoelomic space or into the amniotic cavity of cultured rat conceptuses

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