Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona 85721
Received January 14, 2002; accepted March 4, 2003
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ABSTRACT |
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Key Words: N-acetyltransferase; 4-aminobiphenyl; aromatic amines; DNA adducts.
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INTRODUCTION |
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Phase II enzymes, such as the arylamine N-acetyltransferases (NAT) catalyze activation or detoxification reactions for endogenous and exogenous arylamines and hydrazines. The proteins designated NAT catalyze three types of acetylation reactions: N-acetylation of arylamines or hydrazines, O-acetylation of N-hydroxylamines, and the intramolecular acyl transfer with hydroxyarylacetamides. There are two members in this enzyme family, NAT1 and NAT2 (Hein et al., 2002). The importance of these isoforms in the biotransformation of environmental chemicals and therapeutic drugs is well recognized (Hein et al., 2002
). Polymorphisms in NAT1 and particularly NAT2 are linked to susceptibility to therapeutic and adverse chemical effects. Less is known about the physiologic significance of these enzymes; human NAT1 catalyzes the acetylation of a breakdown product of folic acid, p-aminobenzylglutamate, leading to the suggestion that NAT1 may play an as yet undefined role in folic acid metabolism (Minchin, 1995
; Payton et al., 1999
; Smelt et al., 2000
; Upton et al., 2000
; Ward et al., 1995
).
Human NAT1 is present in preimplantation embryos (Smelt et al., 2000) and fetal liver samples acetylate procainamide (Meisel et al., 1986
). Studies in infants and children demonstrate the capacity to acetylate several NAT substrates, providing evidence of the expression of both NAT isoforms (Pariente-Khayat et al., 1991
, 1997
; Szorady et al., 1987
; Vest and Salzberg, 1965
; Zielinska et al., 1999
). Although these studies are consistent with neonatal expression of NAT1 and NAT2, simultaneous investigation of both isoforms during the postnatal period has not been reported.
The present study was initiated to assess postnatal developmental expression of NAT1 and NAT2 in a mouse model and to evaluate the toxicologic consequences of exposure to the aromatic amine carcinogen 4-aminobiphenyl (4ABP) during this period. There are two murine NATs. Based on substrate specificity (Table 1) and amino acid or nucleotide sequence, murine NAT2 is thought to be the functional equivalent of human NAT1, whereas murine NAT1 resembles human NAT2 (Estrada-Rodgers et al., 1998b
; Hein et al., 1988
, 2002
; Martell et al., 1992
; Payton et al, 1999
), although homology has been reported in the noncoding exons of murine NAT2 and human NAT2 (Fakis et al., 2000
; Upton et al., 2001
). In mice, NAT2 has been detected in embryonic stem cells (Payton et al., 1999
), with prenatal expression of NAT1 and NAT2 at the messenger RNA (mRNA) and protein levels (McQueen et al., 2003
; Mitchell et al., 1999
; Stanley et al., 1998
). C57Bl/6 mice ranging in age from neonatal day (ND) 1 to adults were used for the current study. The hypothesis tested was that lower neonatal NAT activity decreases susceptibility to 4ABP genotoxicity.
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MATERIALS AND METHODS |
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Quantitative RT-PCR.
Hepatic RNA was isolated with the Absolutely RNA RT-PCR Miniprep Kit. Replicate assays were performed on a minimum of three livers per age group. The conditions for amplification using a Cephied Systems Thermal Cycler and primer sequences for NAT1, NAT2, and histone 3.3 (HIS3.3) have been described (Futscher et al., 1982, McQueen et al., in press). Briefly, first-strand cDNA was generated with 2-µg RNA using the PE-Biosystems Kit, then amplified using 5-µl cDNA with SYBER-PCR Master Mix and 1 mM of each of the appropriate amplification primers. The reaction started with a denaturing step (95°C, 10 m), followed by 45 amplification cycles: denaturation at 95°C for 4 s, annealing at 56°C for 30 s for NAT2 and HIS and 54°C for NAT1, extension at 72°C for 30 s, and heating the product at 0.2°C/s to 94°C. Double-strand DNA was detected with SYBER Green at each annealing cycle and fluorescence (y axis) plotted against cycle number (x axis) to generate the amplification curve. The rate of change of the slope of the amplification curve (2nd derivative) was used to calculate the threshold cycle (Ct). The amount of RNA was determined from standard curves constructed with known concentrations of RNA, then standardized to HIS3.3. Statistical significance was determined by an analysis of variance (ANOVA), followed by the Student-Newman-Keuls Q test.
NAT activity.
Adult or neonatal animals (minimum of three per group) were killed by CO2 asphyxiation. Livers were removed and homogenized, and S-9 fractions were prepared (Mattano and Weber, 1987). Each liver was analyzed separately. NAT activities were determined in triplicate with isoniazid (INH), p-aminobenzoic acid (PABA), 4ABP, and 2-aminofluorene (2AF). These represent isoform-selective substrates of NATs, as well as those that are substrates for both isoforms (Table 1
). Activities were determined under initial velocity conditions using an acetylcoenzyme A recycling system (Mattano and Weber, 1987
). The assay mixture consisted of 15 mM acetylcarnitine, 2 units carnitine acetyltransferase/µl, 2 mM ethylenediamine tetracetate (EDTA), 2 mM dithioerythritol, 50 mM TrisHCl, pH 7.5, 0.5 mg protein tissue S-9, 0.5 mM acetylcoenzyme A, and 44.4 µM substrate. Incubation was for 10 min at 37°C; the reaction was stopped with ice-cold methanol or trichloroacetic acid. Reverse-phase HPLC was used to separate the acetyl product from the parent compound for SMZ, PABA, and 2AF (McQueen, 2001
, McQueen et al., 2003
). Briefly, samples were eluted from a C18 column with 18% acetonitrile in water for SMZ, 13% for PABA, and 40% for 2AF. In each case, the mobile phase contained 0.5% acetic acid. AcABP was separated from 4ABP using a Partisil 10 ODS column and a linear gradient of acetonitrile containing 0.1% acetic acid: water (50:50) to 100% acetonitrile containing 0.1% acetic acid (McQueen et al., 2003
). The amount of acetyl INH was determined by spectrophotometry (Hein et al., 1982
). The NAT reaction was stopped with 10% trichloroacetic acid, the supernatant mixed with 0.8 M potassium borate, pH 8, and the absorbance measured at 303 nm. For each compound, the amount of the acetyl product was calculated from a standard curve, and activity expressed as nmol product/mg protein/min. Comparisons of activity were done by ANOVA, followed by the Student-Newman-Keuls Q test.
CYP1A2.
Livers were isolated from animals ranging in age from ND 4 to adults. Neonatal livers (three to eight) were pooled to provide sufficient material for analysis. Adult samples were analyzed separately. Microsomes were prepared, and the presence of the CYP1A2 protein was determined by immunochemistry (McQueen et al., 2003). Microsomal protein was boiled in loading buffer (2% SDS, 100 mM dithiothreitol, 10% glycerol, 0.01% bromophenyl blue, and 60 mM Tris-HCl, pH 6.8), then separated by SDS-PAGE (5% stacking gel and 10% separating gel). The proteins were transferred to a nitrocellulose membrane, incubated with 5% (w/v) nonfat dry milk, then with the CYP1A2 antibody (1:1000 dilution). Bands were visualized on the washed membranes with ECL-PLUS Chemiluminescent substrate and the densities of the bands determined.
4ABP-DNA adducts.
Adult and neonatal mice (three to four per group) were given an oral dose of corn oil or 80 mg 4ABP/kg in corn oil. An alternate dosing procedure was also employed at ND 4, with mothers receiving 80 mg 4ABP/kg or corn oil, and the pups were allowed to nurse. In both dosing regimens, the animals were asphyxiated with CO2 24 h later. Livers were isolated, and samples were frozen in OCT compound, using isopentane precooled in liquid nitrogen. Frozen tissues were sectioned (45 microns) on a cryostat (Jung Frigocut 2800N) mounted on slides pretreated with silane and post-fixed in cold (-20°C) 70% ethanol for 20 min.
The 4C11 antibody, which has been characterized for specificity (Al-Atrash et al., 1995), was used to detect 4ABP-DNA adducts. The immunohistochemical method of Al-Atrash et al. (1995)
was used, with minor modification (McQueen et al., in press). Briefly, slides were incubated sequentially with RNase (100 µg/ml) and proteinase K. DNA was denatured with acid, then neutralized with Trizma base. Nonspecific binding was blocked with bovine serum albumin and normal goat serum. The slides were incubated with 4C11, the monoclonal antibody specific for 4ABP-DNA adducts (1:35 dilution), followed by goat antimouse IgG Alexa 488 (1:8 dilution). Nuclei were stained with EthD-1 homodimer (1:4 dilution). The immunofluorescence images were acquired with the Leica TCS-4D Laser Scanning Confocal Microscope and Scanware software (Version 5.10b). A 100x oil immersion lens, a 493-nm excitation filter, and a 634-nm bandpass filter were used for EthD 1; for Alexa 488 fluorescence, a 488-nm excitation filter and a 519-nm bandpass filter were used. The Ar-Kr laser was used at the maximum power setting during each session, and photomultiplier settings were kept consistent.
Nuclear fluorescence of Alexa 488 images were compared in corn oil- and 4ABP-treated mice to assess levels of 4ABP-DNA adducts. Fluorescence intensities (mean gray levels) of 3050 nuclei from at least three regions in each section were determined using ImagePro Plus, Version 4.0 (Media Cybernetics, Silver Spring, MD). For each nucleus, the fluorescence intensity was normalized to account for variations in nuclear sizes. The weighted mean fluorescence intensity equals (nuclear optical density) (nuclear area)/
nuclear area. For a given nucleus, the Alexa 488 and EthD-1 stained images were converted to gray scale and overlayed to determine 4C11 nuclear fluorescence. Little or no fluorescence for 4C11 was seen in corn oil-exposed samples (mean ± SD = 1.7 ± 0.6). Statistical significance was determined by ANOVA, followed by the Student-Newman-Keuls Q test.
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RESULTS |
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DISCUSSION |
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Although age-related changes are seen in mRNA levels and activities measured with several NAT substrates, there is no direct correlation between the two parameters. The formation of acetylated product is based on total hepatic protein, and changes in specific NAT proteins may not be readily apparent. Immunodetection of NAT2 in CD1 mouse liver shows there is a 50% increase in the protein between 1 and 80 days after birth (Estrada et al., 2000). PABA NAT activity increased threefold during the same period (Estrada et al., 2000
).
The pattern of expression of NATs during the neonatal period, although lower than seen in adult, does not show a linear rate of change. By either parameter, increases in expression are greater in the early neonatal period, followed by a plateau. Similar results are seen in CD1 mice and NAT2 developmental changes (Estrada et al., 2000). Because little is known about the control of expression of NAT genes, the basis for the observed pattern remains to be elucidated. NAT1 and NAT2 are both located on murine chromosome 8 and are independently controlled (Cornish et al., 2002
; Mattano et al., 1988
). These genes have separate tissue-specific patterns of expression with both genes expressed in liver (Debeic-Rychter et al., 1996
, Stanley et al., 1997
). Examination of the promoter region of murine NAT2 reveals a hormone-responsive element that is implicated in gender differences in expression of that gene in mouse kidney (Estrada-Rodgers et al., 1998a
,b
). A better understanding of what controls developmental expression of NATs is needed.
The current data support and expand previous studies of developmental expression of NATs in animals. In rabbits, acetylation of INH at 1 week after birth is 10% of that in adults (Cohen et al., 1973). The developmental expression of NAT2 in CD1 mice increases during the neonatal period (Estrada et al., 2000
). NAT2 mRNA and PABA NAT activity are progressively greater during the first 80 days after birth. No gender differences are present in hepatic PABA NAT activity, although renal acetylation of PABA is greater in males than in females. The age-dependent changes in mRNA and protein that have been reported for NAT2 are part of the continuing expression of this gene, beginning before birth. NAT2 is expressed in mouse embryonic stem cells and is detected in neural tissue from gestational day (GD) 9.5 through 18 (Smelt et al., 2000
; Stanley et al., 1998
). Both NAT1 and NAT2 mRNAs and the capacity to acetylate 4ABP are found in conceptual tissue from C57Bl/6 mice at GD 10 through 18 (McQueen et al., 2003
).
Although there are species differences in the developmental expression of biotransformation enzymes, these animal studies support the more limited data available from humans. NAT1 but not NAT2 transcripts are present in human preimplantation embryos at the blastocyte stage (Smelt et al., 2000). Fetal liver samples acetylate procainamide, a selective substrate for human NAT2 (Meisel et al., 1986
). Several studies in infants and children demonstrate they have the capacity to acetylate NAT substrates. Following dosing of infants intravenously with PABA, a human NAT1-selective substrate (Table 1
), the acetyl product is detectable in the blood and urine (Vest and Salzberg, 1965
). Determination of NAT2 phenotype in children using sulphadimide, INH, or caffeine shows that the percentage of children designated as slow acetylators is greater than that seen in the corresponding population of adults (Pariente-Khayat et al., 1991
, 1997
; Szorady et al., 1987
). This trend is most apparent in the younger children. The urinary caffeine metabolite profile characteristic of the NAT2 slow phenotype occurs in all the children younger than 83 days, whereas both phenotypes are present in the group of older children (Pariente-Khayat et al., 1991
,). Similar results are seen with INH (Pariente-Khayat et al., 1997
). Sequential testing of 44 children one to five times during the course of their treatment for tuberculosis shows NAT2-catalyzed acetylation of INH increasing from birth to 4 years (Pariente-Khayat et al., 1997
). It should be noted that these studies use the metabolic ratios representing rapid and slow acetylator phenotypes determined in adults. The differences in the population frequencies of the phenotypes in adults and children, as well as the change in phenotype with age, suggest that children have a lower expression of NAT2 than do adults, resulting in a lower urinary excretion of acetylated products. Consequently, the use of ratios established from studies in adults results in inaccuracies and possible phenotypic misclassification. This is supported by an investigation of phenotype and genotype in children ages 1 month to 17 years (Zielinska et al., 1999
), showing that urinary caffeine metabolite ratios are unreliable for determining NAT2 phenotype in children less than 1 year old. These authors also note that children have a decreased rate of urine flow, which may also contribute to age-related differences in caffeine metabolic ratios.
NATs are only one of the biotransformation enzymes exhibiting developmental patterns of expression in humans (Hines and McCarver, 2002; McCarver and Hines, 2002
). Arylamines are substrates for CYP1A2, which is undetectable in human liver before birth. Expression increases after birth and is approximately 50% of the adult level by age 1 year (Sonnier and Cresteil, 1998
). In C57Bl/6 mice, there is a similar pattern of increase in the CYP1A2 protein (Fig. 4
). Thus, both species exhibit developmental changes in enzymes that biotransform arylamines.
In the current study, the potential for alterations in response to a genotoxic carcinogen due to lower expression of biotransformation enzymes was evaluated. The data show age-related changes in 4ABP-DNA adducts, with lower levels in neonates than in adults (Table 4). The formation of DNA-damaging products involves multiple reactions that can include NAT. In liver, the initial activation step is a CYP1A2-catalyzed N-hydroxylation, followed by a NAT-mediated O-acetylation of the hydroxylamine, yielding an unstable N-acetoxy ester that can dissociate to the reactive arylnitrenium ion (Butler et al., 1989
; Flammang and Kadlubar, 1986
; Fretland et al., 1997
; Hammons et al., 1997
) and form a DNA adduct at the C-8 position of guanine (Flammang and Kadlubar, 1986
). N-acetylation of the arylamine is viewed as a detoxification step because the resulting arylacetamide is a poor substrate for CYP1A2. Examination of neonatal livers shows that at least two of the enzymes involved in the activation of arylamines are lower in neonates, compared with adults. Consequently, the age-related increase in both 4ABP-NAT activity and CYP1A2 in neonatal liver is associated with an age-related increase in the formation of 4ABP-DNA adducts. The changes observed in 4ABP-DNA adduct levels are not of the same magnitude as those seen in NAT expression. Other CYP isoforms and phase II enzymes can contribute to the biotransformation of 4ABP (Kimura et al., 1999
; Lin et al., 1995
). It should also be noted that detection of fewer 4ABP-DNA adducts does not necessarily mean that fewer tumors will be seen. The neoplastic potential of the compound is also influenced by the ability of the neonate to detoxify xenobiotics and the capacity to repair damage to DNA. 4ABP has been shown to induce hepatic tumors following neonatal exposure (Dooley et al., 1992
). Further studies are needed to better characterize carcinogenic potency of arylamines in neonates.
In summary, the current study demonstrates in a mouse model that the expression of the NAT1 and NAT2 genes increases during the period from birth to sexual maturity. Compared with adult, neonatal mouse liver has a significantly lower ability to acetylate aromatic amines. The decreased capacity for this and other enzymatic reactions needed to form reactive products contributes to less 4ABP genotoxicity in neonates than in adults. Direct extrapolation of this finding to humans is premature and requires a better understanding of developmental expression in both species. However, the human studies showing a lower capacity for acetylation of NAT substrates in children are consistent with the suggestion that children and adults will respond differently to exposure to arylamines. Depending on whether acetylation of the xenobiotic is an activation or detoxification reaction, changes in therapeutic doses and exposure limits may be warranted. Further studies are required to investigate this issue fully.
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ACKNOWLEDGMENTS |
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NOTES |
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