Prenatal expression of N-acetyltransferases in C57Bl/6 mice

Abstract

Exposure to carcinogens such as 4-aminobiphenyl (4ABP), found in tobacco smoke and other combustion products, results in the formation of detectable levels of 4ABP–hemoglobin adducts in cord blood and 4ABP–DNA adducts in conceptal tissue. The presence of these adducts requires that the parent compound undergo biotransformation. When exposure occurs in utero, the maternal, placental and conceptal tissues are all possible sites for the formation of DNA-reactive products. One step in the activation of 4ABP is catalyzed by N-acetyltransferases (NAT). The expression of NAT was evaluated in gestational day (GD) 10–18 conceptal tissues from C57Bl/6 mice. There was a quantitative increase in NAT1 and NAT2 mRNAs with increasing gestational age that was also reflected in age-related changes in functional protein measured as 4ABP–NAT activity. The ability to acetylate 4ABP increased from GD10 to 18 and was lower in conceptal tissue than in adult liver. The potential toxicologic significance of prenatal NAT expression was assessed by formation of 4ABP–DNA adducts. At GD 15 and 18, 4ABP–DNA adducts were detected by immunohistochemistry 24 h following a single oral dose of 120 mg 4ABP/kg. Based on nuclear fluorescence, conceptual 4ABP–DNA adducts were present at similar levels at GD15 and 18. Levels of 4ABP–DNA adducts were significantly higher in maternal liver compared with the conceptus. Results from this study show that both NAT genes were expressed prenatally and that functional enzymes were present. These data support the possible in situ generation of reactive products by the conceptus. The relative contributions of maternal activation of 4ABP and that by the conceptus remain to be determined.

Introduction

Developmental changes in the expression of biotransformation enzymes affect the susceptibility of fetuses, infants and children to the therapeutic and toxic effects of chemicals [1], [2], [3]. It is essential to know the ontogeny of protein expression during gestation and the balance between the isoforms involved in activation and detoxification of xenobiotics in order to develop effective therapies and prevent adverse reactions.

The arylamine N-acetyltransferase (NAT) family of enzymes metabolizes aromatic amines and hydrazines and has been investigated for their metabolic role in the fetus. Two genes, NAT1 and NAT2, have been identified in humans with allelic variants present for both genes [4], [5], [6]. It has been suggested that NAT1 plays a role in regulating folate levels [7]. The breakdown product of folic acid, p-aminobenzylglutamate, is a specific substrate for NAT1 [7], [8], [9], [10], [11]. Expression in pre-implantation embryos and developing neural tissue is consistent with a critical developmental role for NAT1 although it has not yet been defined [11], [12]. In contrast the biotransformation of xenobiotics by NAT1 and NAT2 is well-characterized [5], [6].

4-Aminobiphenyl (4ABP) is a human and animal carcinogen [13]. This compound is genotoxic requiring the generation of electrophilic products with NAT playing a key role [14], [15], [16]. NAT1 and NAT2 each catalyze three types of acetylation: the N-acetylation of arylamines, the O-acetylation of N-hydroxylamines and the N,O-acetyltransfer of arylhydroxamic acids [17], [18], [19], [20], [21]. The major route of hepatic bioactivation of 4ABP begins with the N-hydroxylation of the arylamine catalyzed by hepatic CYP1A2 [22], [23]. The N-hydroxylamine undergoes O-acetylation [18] resulting in an unstable N-acetoxy ester that can dissociate to the reactive arylnitrenium ion [24], forming a DNA adduct at the C-8 position of guanine [18]. Additionally, the N-hydroxylamine can serve as a substrate for UDP-glucuronosyltransferase yielding a glucuronide conjugate that is transported to the urinary bladder, a site for human tumors induced by 4ABP [24], [25]. There are a number of reactions that compete with this activation scheme. For example, N-acetylation of the arylamine is considered to be a detoxification step since the resulting arylacetamide is a poor substrate for CYP 1A2.

In adults, variation in NAT genotypes and phenotypes is associated with the development of toxicity with differences in the ability to acetylate xenobiotics affecting susceptibility to adverse chemical effects. Therefore, it is possible that variation in fetal expression may play a role in developmental toxicity. Enzymes involved in the activation of aromatic amines are present before birth, although these activities are often lower than those of adults [26], [27], [28], [29], [30]. In mice, qualitative studies show that NAT1 and NAT2 are expressed during development. Murine NAT2 m-RNA is detected in embryonic stem cells [7] and NAT1 and NAT2 mRNAs are present from GD10 through 18 [31]. The NAT2 protein is present in neuronal tissue from GD9.5 through 13.5 [12]. Although there is evidence that one or both of these genes are expressed before birth, the presence of functional NAT1 and NAT2 proteins has not been demonstrated nor has the level of expression been quantitated. In the present study, the hypothesis that conceptal tissue catalyzes the acetylation of 4ABP was tested in C57Bl/6 mice. This strain carries the NAT2*8 allele which results in greater activity with several substrates of NATs including 4ABP. Since many biotransformation enzymes are present at lower levels before birth, the higher activity of the C57/Bl6 mice facilitates this study. Expression of NAT1 and NAT2 was assessed by real time reverse transcriptase-PCR. N-Acetylation of 4ABP was measured to determine the presence of functional enzyme and 4ABP–DNA adduct formation was used to monitor the generation of reactive products.