Teratogenicity in vitro of 2‐acetylaminofluorene: Role of biotransformation in the rat

Abstract

2-Acetylaminofluorene (AAF), a highly effective mutagen and carcinogen, was tested as a direct-acting teratogen in an embryo culture system. Concentrations of up to 75 kg/ml (336 kM) of AAF produced no detectable malformations and only minimal decreases in viability and growth of rat embryos explanted on day 10 (11th day of gestation; the day after fertilization was designated as day 0). Inclusion of a microsomal monooxygenase system (prepared from rat liver) in the culture medium, however, resulted in marked changes in viability and growth. All explanted embryos exposed to concentrations of AAF above 60 Fg/ml (269 (LM) plus the microsomal monooxygenase system exhibited readily observable malformations as well as significant decreases in size and macromolecular content. Two metabolites, N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-acetoxy-2-acetylaminofluorene (AAAF), each produced malformations in the absence of a monooxygenase system. At concentrations of 25 Fgl ml (105 and 88 FM) approximately 50% of the embryos exposed to these metabolites were malformed. The predominant malformations associated with the metabolites were ventrolateral protrusions and hypoplasia of the prosencephalic region, whereas AAF plus the monooxygenase system produced an interruption of neural tube closure. This suggested the possibility that other metabolites may participate in the AAF-induced malformation syndrome. The addition of the monooxygenase system to cultures containing N-OH-AAF or AAAF increased the frequency of malformations without increasing lethality or decreasing growth.2-Acetylaminofluorene (AAF) is an extremely effective mutagenic and carcinogenic aromatic amine. It is utilized extensively as a

2‐aminofluorene bioactivation by human term placental peroxidase

“…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.…”

“…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.…”

The potential role of redox cycling as a mechanism for chemical teratogenesis.

“…Several metabolites of 2-acetylaminofluorene (AAF) have been investigated as embryotoxins in whole embryo cultures (6,(31)(32)(33). A number of metabolites of AAF have the potential to behave as redox cyclers although this mechanism has not been seriously considered in terms of its carcinogenicity or mutagenicity.…”

“…Ascertainment of the identities of ultimate chemical dysmorphogenic agents and of regulatory factors governing their generation during organogenesis will undoubtedly assist in elucidating teratogenic mechanisms. Thus far, focus in this research area has been on the cytochrome P-450dependent generation of reactive intermediates by both extraembryonic (3)(4)(5)(6) and embryonic (7-9) enzyme sources. The purpose of this article is to discuss some initial and preliminary findings with respect to cytochrome P-450-independent embryonic systems that appear capable of converting potential prodysmorphogenic agents to their ultimate, biologically active forms.…”

Quantitation and immunohistochemical localization of DNA adducts in rat embryos and associated yolk sac membranes exposed in vitro to N‐acetoxy‐2‐acetylaminofluorene (N‐Ac‐AAF)

“…AAF and some of its metabolites have previously been shown to produce concentration-related effects on growth and development of rat embryos cultured in vitro [ 12,131. The activated forms, N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-acetoxy-2-acetylaminofluorene (N-Ac-AAF) produce prosencephalic hypoplasia, microcephaly, and limb bud abnormalities, while abnormalities in axial rotation (flexure) are associated with exposure to 2-nitrosofluorene and N-OH-AAF [ 131.…”