Introduction

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The N-acetyltransferases (Nats)¹ are evolutionarily well conserved enzymes that catalyze the transfer of an acetyl group from endogenous acetyl-coenzyme A (CoA) to environmental acceptor amines, including arylamine carcinogens, aromatic amines, and hydrazine drugs (Weber, 1987). The study of arylamine Nats has been focused on genetic variations or polymorphisms, which may alter the N-acetylation of therapeutic and carcinogenic compounds. The clinical and toxicological consequences of the acetylation polymorphism in humans include associations between the isoniazid slow acetylator phenotype and various drug toxicities and bladder cancer and between the rapid isoniazid acetylator phenotype and therapeutic drug failure and colorectal cancer (Evans et al., 1960; Iselius and Evans, 1983; Lang et al., 1986; Illet et al., 1987). More recently, a report has linked slow acetylator phenotype and smoking with a higher incidence of breast cancer (Ambrosone et al., 1996). These reports showing statistically significant associations between acetylator phenotype and cancer, however, are still controversial findings that deserve further investigation (Grant et al., 1997). Animal models, including rabbits (Weber et al., 1976), hamsters (Hein et al., 1982), rats (Juberg et al., 1991), and inbred and congenic mouse strains (Mattano et al., 1988), have added useful information regarding the acetylation polymorphism. Outbred mouse strains, however, have not been used, although they may provide a unique model that resembles a genetically heterogeneous population.

In addition to genotype, other factors, including age, gender, and tissue type, may alter Nat activity in mice (Levy and Weber, 1992; Smolen et al., 1993), Syrian hamsters (Menendez-Pelaez et al., 1989), and humans (Evans et al., 1960; Iselius and Evans, 1983; Paulsen and Nilsson, 1985). Smolen et al. (1993) showed a clear developmental influence on the activity of kidney Nat in male, but not female, mice (slow as well as rapid acetylator inbred strains). The gender-related differences in enzyme activity were apparent by weaning age (25 days of age), which coincides with mouse sexual maturation. In addition, the study by Smolen et al. was the first to show that the developmental difference in mouse kidney Nat activity is due to an increase in testosterone. This evidence is of particular interest in light of the differential susceptibility between male and female mice to arylamine-induced carcinogenesis (Brusick et al., 1976). For example, it has been shown that compared with female mice and hamsters, males are more susceptible to 2-acetylaminofluorene-induced bladder tumors (Miller et al., 1964). The Nats Nat1² and Nat2 metabolize 2-aminofluorene in mice. A more recent study reports a differential expression of the Nat genes in the mouse embryo and suggests a potential role for the Nats in development (Mitchell et al., 1999).

Testosterone, produced in large amounts by the testes, plays a major role in the growth and differentiation of reproductive and nonreproductive organs (Bardin and Catterall, 1981). Despite the wide variety of target tissues influenced by androgens, the initial steps of androgen action are common to many tissues. Sex hormones act by binding to members of the zinc finger-containing superfamily of nuclear hormone receptors. The hormone/receptor complex then binds directly to specific DNA recognition sequences known as hormone response elements (HREs) in the promoter region of target genes, resulting in the modulation of transcription. Recently, we reported the identification and characterization of an HRE in the promoter region of mouse Nat2* , which may be a candidate for the in vivo androgenic regulation of this gene in mouse kidney (Estrada-Rodgers et al., 1998). The present work characterizes the Nat2 acetylation polymorphism in the outbred mouse strain CD-1 and explores the developmental-, tissue- and gender-specific expression of Nat2*. In this study, we: 1) determined the correlation of Nat2 genotype and phenotype in a small population of CD-1 adult male mice and 2) examined the kidney and liver Nat2 catalytic activity, immunoreactive protein, and mRNA steady-state levels of male and female mice during development (from birth to 80 days of age).

	Materials and Methods

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Animals. Pregnant outbred CD-1 mice were purchased from Charles River Laboratory (Wilmington, MA). For the developmental studies, CD-1 mice were bred and housed in The University of Michigan Medical School animal care facility. All mice were housed with littermates and separated at 21 days of age. Mice were maintained at room temperature on a 12-h light/dark cycle. Purina mouse chow and tap water were provided ad libitum.

Drugs. Reagent-grade p-aminobenzoic acid (PABA) potassium salt, phenylmethylsulfonyl fluoride, acetyl-DL-carnitine hydrochloride, acetyl-CoA sodium salt, leupeptin (acetyl-Leu-Leu-Arg-al) hemisulfate salt, trifluoroacetic acid, 3,3-diaminobenzidine tetrahydrochloride, and hydrogen peroxide were purchased from Sigma Chemical Co. (St. Louis, MO). PABA was obtained from Eastman Organic Chemicals (Rochester, NY). Dithiothreitol (electrophoresis grade) was obtained from Schwarz/Mann Biotech (Cleveland, OH). The Bradford protein assay was obtained from Bio-Rad Laboratories (Hercules, CA). The Maxiscript kit was purchased from Ambion (Austin, TX). The goat anti-rabbit IgG-horseradish peroxidase (blot grade)-conjugated secondary antibody was obtained from Life Technologies (Gaithersburg, MD). 5'-[-³²P]dATP (6000 Ci/mmol) and 5'-[-³²P]dUTP (800 Ci/mmol) were obtained from Amersham (Arlington Heights, IL). The oligonucleotide primers used for PCR amplification and sequencing were synthesized by The University of Michigan DNA synthesis facility. DNA modifying enzymes were from Life Technologies (Grand Island, NY), United States Biochemicals (Cleveland, OH), Promega (Madison, WI), or New England Biolabs (Beverly, MA).

Allele-Specific PCR. Genomic DNA from tail was extracted with the Qiagen QIAamp Tissue Kit according to the manufacturer's recommendations (Qiagen, Santa Clarita, CA). Primer pairs were designed as follows: the 5' allele-specific 16-mer primers were designed to be specific to the adenine (TTAACACTCCAGCCAA) or thymine (TTAACACTCCAGCCAT) base at position 296 and were complementary to nucleotides 280 to 296. The 3' primer hybridizes 146 bases downstream of the sense primer (5'-AAGGAGATCTGAGTTA-3'). Amplification reaction mixtures contained 1 µg of genomic DNA; 300 ng of each primer; 250 µM concentration each of dATP, dCTP, dGTP, and dTTP; 1× polymerase chain reaction (PCR) buffer (10 mM Tris · HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 3 mM dithiothreitol); and 5 U of Taq DNA polymerase (Life Technologies). The reaction mixtures were amplified for 30 cycles, with 90 s at 94°C for denaturation, 90 s at 55°C for annealing, and 40 s at 72°C for extension; one final extension step for 10 min at 72°C was also performed. Amplification reactions were carried out in an Ericomp TwinBlock thermal cycler (San Diego, CA). PCR products were visualized on a 3% NuSieve agarose gel FMC (Rockland, ME) in 1× TAE (40 mM Tris-acetate buffer, pH 8.0, 1 mM EDTA) and stained with ethidium bromide.

Nat Preparation. Mice younger than 15 days of age were sacrificed through decapitation, and older mice were sacrificed through cervical dislocation. Immediately after death, the kidneys and liver were removed, minced, and homogenized in lysis buffer containing 3.3 mM Tris · HCl, pH 7.78, 2 mM EDTA, 2 mM dithiothreitol, 20 µM leupeptin, and 100 µM phenylmethylsulfonyl fluoride. The homogenates were centrifuged at 10,000g for 20 min at 4°C. The resultant supernatant was centrifuged at 100,000g for 1 h at 4°C. The cytosolic fraction was stored at 70°C and assayed for NAT activity within 24 h after preparation. Whole blood was obtained from the mouse tail with the addition of heparin to avoid coagulation. Blood was lysed at room temperature for 3 min and diluted 21-fold in lysis buffer.

Determinations of N-Acetylation Activity. Nat activity for PABA was determined with triplicate reaction mixtures containing liver or kidney cytosolic fraction (3-25 µg of total protein), 0.1 mM substrate, 0.1 mM acetyl-CoA, and 20 µl of acetyl-CoA regenerating system (5 mM acetyl-DL-carnitine, 0.12 U of carnitine acetyltransferase, 1.5 mM EDTA, 1.5 mM dithiothreitol, and 19.2 mM Tris · HCl buffer, pH 7.5 at 37°C) in a total volume of 100 µl (Martell et al., 1992). Reactions were preincubated at 37°C for 3 min and initiated with the addition of substrate. The reactions were carried out for 10 min at 37°C and terminated with 10 µl of 15% perchloric acid. After precipitation of the denatured protein, the supernatant fraction was assayed for N-acetyl-PABA formation by HPLC with a reversed phase C₁₈ column that was eluted at a flow rate of 1 ml/min. For PABA/N-acetyl-PABA, the solvent system was 0.1% trifluoroacetic acid/methanol with detection at 266 nm (DeLeon, 1996). Liver and kidney cytosolic protein amounts were determined according to the Bradford assay method with BSA as standard. The Biuret assay previously described by Watters (1978) with BSA as a standard determined the blood protein concentrations. Statistical analysis was performed with the Tukey-Kramer multiple comparison test with Instat (GraphPAD Software, San Diego, CA).

Immunoblot Analysis of Liver and Kidney Cytosol. Liver (5 µg) and kidney (3 µg) cytosolic proteins were submitted to electrophoresis on 12% polyacrylamide gels in the presence of SDS. After electrophoretic transfer to Immobilon-P membranes, the filters were incubated for 2 h with 5% BSA in 0.15 M NaCl and 20 µM Tris · HCl buffer, pH 7.4 (TBS), and incubated for 18 to 24 h with a 1:5000 dilution of a primary polyclonal antibody raised against mouse Nat2 (DeLeon et al., 1995), which was previously preabsorbed with Escherichia coli lysate overnight at room temperature. After washing with TBS, the filters were incubated for 1 h with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody. Bands were visualized on the addition of developing solution containing 10 mg of 3,3-diaminobenzidine tetrahydrochloride in TBS, 0.06% hydrogen peroxide, and 0.625 µM nickel chloride.

Plasmid Construct. The template for the 3'-UTR Nat2* antisense RNA probe was constructed through PCR as described in the HybSpeed RPA instruction manual (Ambion, Austin, TX) and cloned into TA cloning vector pCR2.1 (InVitrogen, San Diego, CA). The SP6 phage promoter sequence was appended at the 5' end of the antisense amplification primer (5'-GCGCGCCACGTAGTGATTTAGGTGACACTATAGGATATGGATAATGCTGGT-3'). The sense primer was designed as 5'-GCGGCGTACGTGTAAAGTTTTGGTGTCC-3'. Amplification of the target DNA produced a PCR fragment (215 bp) that contains the SP6 promoter incorporated into the amplified 3'-UTR sequence of Nat2*. The antisense PCR primer also contained additional six bases, which generated a DraIII restriction site, and six extra bases for maximal transcription efficiency. Conditions for the amplification of Nat2* by PCR were as described previously (Vatsis et al., 1991). Selection of positive colonies was performed in the presence of 5-bromo-4-chloro-3-indolyl--D-galactoside, isopropyl -D-thiogalactoside, and 50 µg/ml ampicillin. The positive clones were analyzed for proper orientation and for the correct nucleotide sequence (Sequencing DNA core; The University of Michigan, Ann Arbor, MI). The resulting construct was designated as p3'-UTR Nat2*.

cRNA Probe and Sense RNA Synthesis. cRNA probes for RNase protection assays were synthesized as described previously (Ballestero et al., 1995; Seasholtz et al., 1995). Briefly, the in vitro transcription reaction was carried out for 1 h at 22°C in a 10-µl reaction mixture containing 1× transcription buffer (Life Technologies); 10 mM dithiothreitol; 10 U of RNasin (Promega); 333 µM concentration each of ATP, CTP, and GTP; 5 µM UTP; 4 µl of [-³²P]UTP (3000 Ci/mmol; Amersham); 1 µg of linearized template; and 1 U of SP6 RNA polymerase (Life Technologies). For sense-strand RNA synthesis, p3'-UTR Nat2* was digested with MseI (New England Biolabs), and RNA was synthesized by using T7 RNA polymerase (Life Technologies). The RNA was quantified by spectrophotometry and verified by formaldehyde/agarose gel electrophoresis followed by ethidium bromide staining. Serial dilutions were made for the sense-strand standards. For the internal control, we used the pTRI-Cyclophilin-Mouse antisense control template (Ambion), which contains a 103-bp cDNA insert of highly conserved region of the mouse cyclophilin gene spanning exons 1 and 2 (Hasel and Sutcliffe, 1990). The riboprobe was synthesized with T7 RNA polymerase according to the manufacturer's instructions (Ambion).

Ribonuclease Protection Assay. Total RNA (100 µg) of tissue samples isolated with the use of the Qiagen (Santa Clarita, CA) Maxi Prep Kit or 100 µg of yeast RNA (Life Technologies) were hybridized with 5 × 10⁵ cpm of 3'UTR Nat2* probe and 1 × 10⁵ cpm of the pTRI-Cyclophilin-Mouse antisense control template in 40 µl of RNase protection assay hybridization buffer [10 mM Tris · HCl, pH 7.5, 0.4 M NaCl, 1 mM EDTA, and 75% (v/v) formamide] at 55°C for 19 h. The indicated number of picograms of sense-strand RNA were hybridized with 5 × 10⁵ cpm of 3'-UTR Nat2* probe. The RNase protection assay procedure was performed as described by Seasholtz et al. (1995). Briefly, samples were heated at 95°C for 10 min and hybridized overnight at 55°C. The next day, the reactions were digested with 200 U of RNase T₁ at 30°C for 30 min. Proteinase K (0.1 mg) and 10 µl of Sarkosyl (10%) were added, and the reaction mixtures were incubated at 37°C for 30 min. The RNA was then purified in the presence of guanidinium isothiocyanate/tRNA and precipitated with isopropanol. After centrifugation, the pellets were washed with 70% ethanol, dried in a Speedvac, and resuspended in sequencing loading buffer. Finally, the reactions were boiled for 5 min and submitted to electrophoresis through a 6% polyacrylamide denaturing gel. The dried gel was exposed to Kodak X-OMAT autoradiographic film at 70°C for 5 days with an intensifying screen. For quantification, the protected fragments were analyzed with a Bio-Rad densitometer (Hercules, CA).

	Results

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Amplification and Direct Sequencing of Nat2* from CD-1 Mouse Strain. To determine whether the CD-1 Nat2* nucleotide sequence was similar to the previously reported sequence for mouse Nat2*, a 1.162-kb fragment was amplified from liver genomic DNA isolated from the CD-1 mouse outbred strain. The sequence autoradiograms showed that CD-1 Nat2* is identical to the previously reported nucleotide sequence for slow acetylator A/J mouse strain, allele Nat2*9 (Martell et al., 1991). This finding was unexpected because we had previously phenotyped several mice, and the data had classified CD-1 as a rapid acetylator strain. A small population of adult male CD-1 mice (nine animals, mice 26-34) was phenotyped through allele-specific PCR, an expeditious and accurate genotyping tool, and phenotyped with PABA, a mouse Nat2-selective substrate (Fig. 1, A and B). Two of the nine mice are genotyped as slow acetylator (mice 27 and 34), whereas the remaining seven are of rapid homozygous genotype (mice 26, 28, 29, 31, 32, and 33; mouse 30 is not shown). Mice 28 and 29 could be classified as heterozygous, but the presence of both alleles would have to be confirmed by direct sequencing. For each mouse, we determined the rate of PABA acetylation in whole blood (nmol N-acetyl-PABA/min/50 µl of whole blood) as described in Materials and Methods (Fig. 1B). The concentration of total protein in 50 µl of blood did not vary significantly among the mice studied. The B6.D2 congenic rapid acetylator strain shows 38-fold higher formation of N-acetylated PABA by Nat2 than the slow congenic strain, B6.A. CD-1 mice 27 and 34 exhibit rates of acetylation comparable with those of the slow acetylator congenic strain (B6.A), which is in complete concordance with the genotype determined by allele-specific PCR. The other seven animals show 6- to 18-fold higher acetylating activity than slow acetylator mice, a phenotype that is associated with heterozygous or homozygous rapid acetylator genotype. The variation in acetylation of PABA observed among the rapid acetylators may be due to the outbred nature of the strain and the role played by other genes.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Determination of the Nat2* genotype and phenotype in CD-1 mice. A, genomic DNA was isolated from the tail of seven CD-1 mice and submitted to allele-specific PCR as described in Materials and Methods. Amplified DNA was submitted to gel electrophoresis on a 3% NuSieve agarose gel and visualized with ethidium bromide. Arrows, expected amplified fragment (146 bp). S, slow acetylator allele; R, rapid acetylator allele. B, whole blood from the indicated mice was assayed for acetylating activity with PABA in the presence of 0.1 mM acetyl-CoA. N-Acetylated product was separated and quantified by HPLC. The activities (mean ± S.E. of triplicate determinations per animal) are expressed in nmol of N-acetylated PABA/min/50 µl of whole blood. The amount of total protein in whole blood measured by the Biuret assay did not vary significantly between samples.

Developmental Pattern of N-Acetylation Activities in Kidney and Liver of CD-1 Mice. We determined the kidney and liver N-acetyltransferase-acetylating activities with PABA in CD-1 mice of both sexes from birth to 80 days of age. In kidney, statistically significant differences were shown between Nat2-specific PABA N-acetylation activity from male and female mice (P < .01 to .001) as demonstrated in Fig. 2. (Throughout this report, the Nat2 activities are compared with those measured on postnatal day 1.) Female Nat2-specific activity exhibits only a 2.5-fold increase by 80 days of age. On the other hand, PABA-Nat2 catalytic activity determined for males increased by 4.3-fold at 25 days of age and by 5.8-fold at 80 days of age. Rates of N-acetylation were also determined with liver cytosol from male and female CD-1 mice, and the results are depicted in Fig. 3. Liver PABA-Nat2 catalytic activity showed no gender-related difference in the age range studied. The liver Nat2 activities, however, were 3.7- and 3.8-fold higher in males and females, respectively, by 80 days of age (Fig. 3). These results concur with those reported for the developmental pattern of Nat activity in kidney and liver of A/J and C57BL/6J mice (Smolen et al., 1993), and as proposed by those authors, these data suggest androgenic control of kidney Nat activity in mice.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Developmental pattern of kidney acetyl-CoA-dependent PABA-Nat2 activities in CD-1 mice. Kidney cytosol from female and male mice were assayed for catalytic activity from 1 to 80 days of age (n = 3-10/group) with 0.1 mM PABA in the presence of 0.1 mM acetyl-CoA. N-Acetylated products were separated and quantified by HPLC. Specific activities (mean ± S.E. of triplicate determinations per animal) are expressed in nmol/min/mg of total protein. **P < .01, ***P < .001.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Developmental pattern of liver acetyl-CoA-dependent PABA-Nat2 activities in CD-1 mice. Liver cytosol from female and male mice were assayed for catalytic activity from 1 to 80 days of age (n = 3-10/group) with 0.1 mM PABA in the presence of 0.1 mM acetyl-CoA. N-Acetylated products were separated and quantified by HPLC. Specific activities (mean ± S.E. of triplicate determinations per animal) are expressed in nmol/min/mg of total protein.

Kidney and Hepatic Nat Protein Levels during Mouse Development. To determine whether the increase observed in Nat2-acetylating activity in male kidney is associated with higher protein levels, Nat protein content was evaluated through immunochemistry in aliquots of kidney and liver cytosolic fraction in CD-1 mice of each gender from 1 to 80 days of age (n = 2). The extent of immunoreactive kidney cytosolic Nat protein represented by the 34-kDa band (Fig. 4, A and B, arrows) increased by 2.2-fold at 80 days of age in males (Fig. 4, A and C). In females, only a 1.4-fold increase in immunoreactive Nat protein was as determined through densitometry (Fig. 4, A and C). At 80 days of age, the difference in Nat protein between males and females was 2-fold (Fig. 4C). In contrast, in liver, the Nat immunoreactive protein (Fig. 5, A and B, arrows) increases in both males and females by 1.7-fold at 80 days of age compared with 1-day-old mice (males, Fig. 5, A and C; females, Fig. 5, B and C). This information indicates that the observed gender-related difference in Nat2 catalytic activity in mouse kidney is associated with a gender-related difference in amounts of Nat protein.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Immunodetection of kidney cytosolic Nat during development of CD-1 mice. Kidney cytosol (5 µg of total protein) from male (A) and female (B) CD-1 mice from 1 to 80 days of age were loaded onto a 12% polyacrylamide gel and submitted to electrophoresis in the presence of SDS. The proteins were transferred to an Immobilon-P membrane and reacted with Nat2 antiserum. The size of the band was determined with appropriate molecular weight markers. Arrows, Nat protein. C, the graph reports densitometric quantification (absorbance × mm2) of the immunoreactive bands detected in kidney samples.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Immunodetection of liver cytosolic Nat during development of CD-1 mice. Liver cytosol (3 µg of total protein) from male (A) and female (B) CD-1 mice from 1 to 80 days of age were loaded onto a 12% polyacrylamide gel and submitted to electrophoresis in the presence of SDS. The proteins were transferred to an Immobilon-P membrane and reacted with Nat2 antiserum. The size of the band was determined with appropriate molecular weight markers. Arrows, the Nat protein. C, the graph reports densitometric quantification (absorbance × mm2) of the immunoreactive bands detected in liver samples.

Nat2 Transcript Levels during Mouse Development. Having established the kidney and hepatic Nat2 catalytic activity and protein levels during mouse development, the next step was to examine the steady-state level of Nat2 transcript. RNase protection assays were performed to quantify the expression of Nat2 in mouse kidney and liver (Fig. 6A; n = 2) as described in Materials and Methods. The Nat2 RNA steady-state levels were calculated by normalizing the Nat2 band intensity determined through densitometry to the intensity of the control probe, cyclophilin. The RNA steady-state levels of Nat2 in mouse kidney of males increased 4.2-, 9.7-, and 7.6-fold by 25, 35, and 80 days of age, respectively, as determined through densitometry (Fig. 6B). In contrast, the Nat2 transcript levels remained unchanged in female mouse kidney (Fig. 6, A and B). Similar gender-associated differences in Nat2 RNA were absent in mouse liver (Fig. 6, A and C). These observations correlate with the Nat2 catalytic activities and protein levels during CD-1 development and suggest that the androgenic modulation of Nat2 is accomplished through an increase in transcript levels that is translated into an increase in Nat2 protein and, subsequently, Nat2-acetylating activity.

View larger version (37K): [in this window] [in a new window]   Fig. 6.   RNase protection analysis of Nat2 mRNA in mouse kidney and liver. A, antisense Nat2 cRNA probe (250 nucleotides) was hybridized to Nat2 sense RNA standards (0-30 pg; arrowhead) or to 100 µg of total RNA isolated from mouse kidney or liver or from yeast. As internal control, antisense mouse cyclophilin cRNA probe (165 nucleotides) was included in the tissue sample reactions. The 220-nucleotide fragment in mouse kidney and liver corresponds to protected Nat2 mRNA transcript (Nat2). The 103-nucleotide fragment in mouse kidney and liver samples corresponds to the protected cyclophilin mRNA (Cyc) transcript. The sizes indicated were determined through comparison with RNA size markers. Autoradiograms were exposed for 5 days at 70°C with intensifying screens. B, densitometric quantification of the protected fragments detected by RNase protection assay performed with kidney samples. The number of pg of Nat2 mRNA/pg of total RNA was calculated by comparison with the sense controls. The values were corrected for RNA loading according to the cyclophilin (103 bp) internal control intensities. C, densitometric quantitation of the protected fragments detected by RNase protection assay performed with liver samples. Calculations were as indicated for kidney samples. This figure is a representation of two independent experiments.

	Discussion

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In mice, the N-acetylation conjugation reaction is modulated in kidney by testosterone. Smolen et al. (1993) showed greater kidney Nat activity determined with PABA in male (C57BL/6J and A/J inbred strains) than in female mice. This sexual dimorphism was evident by 30 days after birth and persisted into adulthood (>200 days of age). Furthermore, castration decreased male kidney PABA-Nat activity to female levels, and the administration of testosterone to either castrated or ovariectomized mice increased kidney PABA-Nat activity to levels equivalent to that of intact males (Smolen et al., 1993). Interestingly, the androgenic modulation of mouse Nat activity was not observed in liver. The authors proposed that testosterone is a key element in the regulation of acetylation of arylamine compounds by the kidney, which could result in differential susceptibility between male and female mice to arylamine-induced carcinogenesis. However, the study of Smolen et al. (1993) only looked at the PABA-Nat catalytic activities of inbred mice 10 days old or older. In addition, Smolen et al. did not evaluate the molecular events that lead to the androgenic regulation of kidney Nat2 during mouse development.

Acetylator inbred and congenic models have been developed and characterized, but no study of the acetylation polymorphism in outbred mice has been reported. The large litter size of CD-1 compared with that of the C57BL/6J or A/J strains offered another great advantage to study mouse development. Using CD-1 mice as a representation of an outbred strain, we determined the phenotype and genotype of the Nat2* gene in a population of nine adult male mice. Two of the nine mice were of the slow genotype, whereas the remaining seven were either heterozygous or rapid homozygous genotypes. Nat2 phenotypes were determined in whole blood with PABA, and the results demonstrated complete concordance of genotype and phenotype. Therefore, the outbred CD-1 mouse strain could be a substitute for the C57BL/6J and A/J strains as another useful model of the acetylation polymorphism in a random population.

To establish the site of Nat androgen regulation, in the present study we investigated not only the N-acetylation of amine substrates but also the associated steady-state RNA and protein levels from early development to maturity (1-80 days) in mouse kidney and liver in the outbred mouse strain, CD-1. Several observations show that age, gender, and tissue type each influences the acetylating capacity of mice. First, we observed a clear developmental increase in kidney (Fig. 1) and liver (Fig. 2) Nat2 catalytic activity in CD-1 mice in males and females. Males have higher Nat2 catalytic activity than females. Variations in Nat2 activity during development have been shown previously in other species, such as guinea pigs (Sonawane, 1982), rabbits (Cohen et al., 1973), and humans (Vest, 1965; Pacifici et al., 1986). Recently, prenatal (gestational days 10, 15, and 18) mRNA expression of Nat1 and Nat2 has been detected in hepatic and extrahepatic tissues (Mitchell et al., 1999). Nat1 expression, however, was not detected in liver. This pattern of Nat1 expression continued after birth (neonatal day 3). The authors proposed that the expression of Nat1 and Nat2 during mouse development might allow the embryo to activate or detoxify endogenous (p-aminobenzoyl-L-glutamate) and exogenous aromatic amines (Mitchell et al., 1999).

In addition to the increase in catalytic activity during mouse development, the Nat2 activity found in CD-1 mouse kidney differs according to gender. The effect of gender on Nat2-acetylating activity is first apparent at 20 days of age and becomes statistically significant at 25 days of age. These gender-related differences are sustained through adulthood. Smolen et al. (1993) reported a similar pattern of results in both slow (A/J) and rapid acetylator (C57BL/6J) inbred mouse strains. In view of our findings on CD-1 mice, the gender-related difference in Nat2 activity is not a mouse strain-specific occurrence. Gender also plays a role in the extent of N-acetylating activity in the Harderian gland in Syrian hamsters (Payne et al., 1977; Menendez-Pelaez et al., 1989) and in rats (Zidek and Janku, 1981). Gender has also been suggested to influence acetylating capacity in humans (Iselius and Evans, 1983; Paulsen and Nilsson, 1985). We also observed that although the Nat2 activity of CD-1 mouse liver increased with age (Fig. 3), a gender-related difference was not evident. This suggests that the regulatory control of Nat2 activity in mouse liver differs from that of mouse kidney.

Second, we examined the Nat protein levels in kidney and liver during CD-1 development through immunoblot analysis. We found a 2-fold higher kidney Nat immunoreactive protein level in males than in females at 80 days of age (see Figs. 3 and 4). There also was an increase (1.7-fold) at 80 days of age in liver Nat protein level, but there was no difference between males and females. We detected a 5.8-fold increase in male mouse kidney Nat2 activity, whereas the Nat protein steady-state levels increased by only 2.2-fold at 80 days of age. The greater fold increase in activity than protein is most likely explained by the fact that although the polyclonal Nat antibody used for the immunoblot analysis was raised against mouse Nat2, it also recognizes Nat1 (DeLeon et al., 1995) and, possibly, mouse Nat3. Therefore, the 2-fold increase in immunoreactive Nat protein we observed in mouse kidney of males probably corresponds to a greater increase in Nat2-specific protein.

Third, we determined the Nat2 mRNA levels during CD-1 development in kidney and liver by the ribonuclease protection assay. The results suggest that the increase in Nat activity and protein are associated with an increase in transcript levels (Fig. 5). The Nat2 transcript levels in kidney of males increased substantially during the age range tested but were unchanged in female kidney (Fig. 6B). In liver, however, Nat2 mRNA levels remained relatively constant in both male and female mice (Fig. 6C). Altogether, the data reported in this work support the hypothesis that the androgenic modulation of kidney Nat2 during male mouse development is achieved by an increase in Nat2 transcript. Further investigation is needed to determine whether this increase in transcript level is due to enhanced transcription rate or to mRNA stabilization. It should be noted that steroid hormones have been shown to modulate both events (Beato et al., 1996).

It is generally accepted that the steroid hormone/receptor complex promotes transcription by binding directly to specific DNA recognition sequences, termed HREs, in the promoter region of target genes (Yamamoto, 1985; Beato et al., 1996; Meier, 1997). In Nat2* of CD-1, as well as C57BL/6J and A/J mice, we have identified a palindromic HRE in the promoter region as a potential candidate for androgen modulation in mouse kidney (Estrada-Rodgers et al., 1998). Functional analysis has demonstrated that the Nat2*-HRE sequence can confer androgen regulation on the heterologous HSV-tk1 promoter in transiently transfected CV1 cells (Estrada-Rodgers et al., 1998).

Interestingly, the gender-related differences in Nat2 activity, protein, and transcript observed in kidney of mice were absent from liver. This finding strongly suggests tissue-specific regulation of Nat2* gene expression, but the mechanism by which specific genes are expressed in a developmentally or tissue-specific manner is yet not fully understood. Nevertheless, interactions between transcription factors and cis-acting DNA sequences are widely accepted as required for eukaryotic gene regulation (Maniatis et al., 1987; Arnone and Davidson, 1997). Although advances have been made toward the understanding of gene regulatory networks, current challenges include how specific protein/DNA interactions regulate gene expression and how these interactions are linked to the pattern of gene regulation during development. The tissue-, gender-, and developmentally specific regulation of mouse Nat2* gene expression provides another useful model to study the interactions between cis- and trans-acting regulatory systems. It will also be important to study the effects of age, gender, and tissue on the regulation of mouse Nat1* and Nat3*.