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Vol. 290, Issue 1, 182-187, July 1999
Departments of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville Kentucky (M.A.L., P.N.E., M.A.D., A.J.F., U.-S.D., D.W.H.); and University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota (T.D.R., P.N.E., D.W.H.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic amine identified in the human diet and in cigarette smoke that produces prostate tumors in the rat. PhIP is bioactivated by cytochrome P-450 enzymes to N-hydroxylated metabolites that undergo further activation by conjugation enzymes, including the N-acetyltransferases, NAT1 and NAT2. To investigate the role of prostate-specific expression of human N-acetyltransferase 2 (NAT2) on PhIP-induced prostate cancer, we constructed a transgenic mouse model that targeted expression of human NAT2 to the prostate. Following construction, prostate, liver, lung, colon, small intestine, urinary bladder, and kidney cytosols were tested for human NAT1- and NAT2-specific N-acetyltransferase activities. Human NAT2-specific N-acetyltransferase activities were 15-fold higher in prostate of transgenic mice versus control mice, but were equivalent between transgenic mice and control mice in all other tissues tested. Human NAT1-specific N-acetyltransferase activities did not differ between transgenic and control mice in any tissue tested. Prostate cytosols from transgenic and control mice did not differ in their capacity to catalyze the N-acetylation of 2-aminofluorene, the O-acetylation of N-hydroxy-2-aminofluorene and N-hydroxy-PhIP or the N,O-acetylation of N-hydroxy-2-acetylaminofluorene. Transgenic and control mice administered PhIP did not differ in PhIP-DNA adduct levels in the prostate. This study is the first to report transgenic expression of human NAT2 in the mouse. The results do not support a critical role for bioactivation of heterocyclic amine carcinogens by human N-acetyltransferase-2 in the prostate. However, the lack of an effect may relate to the level of overexpression achieved and the presence of endogenous mouse acetyltransferases and/or sulfotransferases.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Recent
studies have shown that the dietary carcinogen,
2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces tumors in rat prostate (Shirai et al., 1997
). Heterocyclic amine carcinogens such as PhIP may undergo bioactivation by cytochrome P-450 (CYP) 1A2 in
the liver (Butler et al., 1989; Turesky et al., 1998) or by CYP1A1
and/or CYP1B1 in extrahepatic tissues (Crofts et al., 1998). The
N-hydroxy metabolite(s) formed can be further bioactivated by N-acetyltransferases (Hein et al., 1993, 1995; Turesky et
al., 1991; Minchin et al., 1992; Lin et al., 1995) and
sulfotransferases (Chou et al., 1995; Lin et al., 1995) to yield
electrophilic arylnitrenium ions that bind covalently to DNA to form
adducts that can initiate carcinogenesis.
Our laboratory has focused on the role of acetyltransferases in the
bioactivation of heterocyclic amine carcinogens. Two major N-acetyltransferase isozymes [EC 2.3.1.5], NAT1 and NAT2,
are expressed in human and other mammalian species (Vatsis et al., 1995; Grant et al., 1997; Hein et al., 1997). Drugs such as
sulfamethazine (SMZ) are selectively metabolized by human NAT2, whereas
drugs such as p-aminobenzoic acid (PABA) are selectively
metabolized by human NAT1 (Grant et al., 1991; Hein et al., 1993).
Genetic polymorphisms exist for both human NAT1 and
NAT2 (Vatsis et al., 1995; Grant et al., 1997; Hein et al.,
1997). The NAT2 acetylation polymorphism is more completely
characterized than NAT1. NAT2*4 is the most common allele
associated with rapid acetylator phenotype (Vatsis et al., 1995) and
recombinant human NAT2 4 acetyltransferase bioactivates
N-hydroxy-PhIP at higher rates than recombinant human NAT2
proteins encoded by NAT2 alleles from human slow acetylators (Hein et al., 1998). Furthermore, recombinant human NAT2 4 has a higher
capacity to bioactivate N-hydroxy-PhIP than recombinant human NAT1 (Minchin et al., 1992; Hein et al., 1994).
Individuals with rapid NAT2 acetylator phenotype who ingest
heterocyclic amines in cooked meats exhibited a significantly higher
incidence of colorectal cancer (Lang et al., 1994). Because heterocyclic amines produce prostate cancer in the rat (Shirai et al.,
1997), it is of interest to investigate the role of human NAT2 in the
bioactivation of heterocylic amine carcinogens within the prostate. The
role and relative importance of hepatic and target organ
acetyltransferases in the bioactivation of heterocyclic amines leading
to prostate tumors is not well understood. A significant role for human
NAT2 would suggest that the NAT2 acetylation polymorphism influences susceptibility to heterocyclic amine-related prostate cancer
analogous to its effect on the susceptibility to colorectal cancer
(Lang et al., 1994).
To investigate the role of human NAT2 in bioactivation within
the prostate, we constructed a prostate-specific transgenic mouse model
using a transgene expression system containing regulatory elements of
the rat probasin (PB)-encoding gene (Rennie et al., 1993; Kasper et
al., 1994). The PB gene encodes an androgen- and zinc-regulated protein
that is specifically expressed in prostate epithelial cells (Kasper et
al., 1994). The ability of the prostate-specific rat PB promoter to
target heterologous genes specifically to the prostate in transgenic
mice has been extensively characterized (Greenberg et al., 1994, 1995;
Barrios et al., 1996). Hormonal induction of PB-transgenes yield
maximal expression of targeted protein at sexual maturity (7 weeks of
age). Following successful construction and characterization, we used
the prostate-specific transgenic mouse model to investigate the effect
of prostate-specific expression of rapid acetylator human
NAT2 (NAT2*4) on the bioactivation of PhIP and
2-aminofluorene (AF), an aromatic amine carcinogen.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Construction of PB-NAT2 Transgene.
Rat genomic DNA was
isolated from a Wistar-Kyoto inbred rat (Charles River Laboratories,
Wilmington, MA) heart by standard methods. Briefly, the heart was
frozen in liquid nitrogen, pulverized with a mortar and pestal,
digested in proteinase K, extracted with phenol and chloroform/isoamyl
alcohol (24:1), and precipitated the genomic DNA with ethanol.
The rat PB gene promotor was amplified by polymerase chain reaction
(PCR) using primers (PB1: 5'-TCTGGATCCCTGTAGGTATCTGGACCTCACTGA-3' and
PB2: 5'-TCTGCGGAGCTCGCGGCCGCAAGCTTCCACAAGTGCATTTAGCCT-3'). PCR was
performed with 100 ng DNA in a 100-µl reaction containing 10 mM
Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 mM each dNTP, 1 µg each of the appropriate primers, and 2.5 units of
Taq DNA polymerase (Perkin-Elmer, Norwalk, CT). The PCR
product (
426/+28 base pair (bp) fragment of the rat PB promoter) was
digested with SacI/BamHI and ligated into the
SacI/BamHI sites of the eukaryotic expression
vector pKS/RIP (Valera et al., 1994) to produce the intermediate
construct pKS/PB. Human genomic DNA was isolated using the Instagene
Genomic DNA kit (Bio-Rad, Hercules, CA) from whole blood obtained from
a person possessing the NAT2*4 allele as determined by PCR
restriction fragment length polymorphism genotyping (Doll et al.,
1995). The NAT2*4 allele was amplified by PCR as
described above using primers (NAT2A:
5'TTAGGAATTCATGGACATTGAAGCATATTTTGAAAGAAT-3' and NAT-2B:
5'-TGTGAATTCAAGGGTTTATTTTGTTCCTTATTCTAAAT-3'). The 870-bp
PCR product (NAT2*4) was inserted as an EcoRI
fragment into the EcoRI site of pKS/PB (Fig.
1). The combination PB promoter region
and NAT2 gene was sequenced via a modified double-stranded dideoxy chain termination method (Sanger et al., 1977) using Sequenase (United States Biochemical, Cleveland, OH), to verify proper base insertion and orientation. A 2.9-kb NotI/XhoI
fragment (Fig. 2) was excised by
digestion with 10 U of NotI and XhoI in 100 mM NaCl, 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, and 1 mM dithiothreitol (DTT) for 1 h at 37°C. The 2.9-kb fragment
(Fig. 2) was subjected to gel electrophoresis and purified using
Prep-A-Gene (BioRad, Hercules, CA).
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Production and Screening of PB-NAT2 Transgenic Mice.
Standard procedures were employed for construction of transgenic mice
(Cameron, 1997). The 2.9-kb PB-NAT2 transgene (Fig. 2) was injected
into one pronucleus of each one-cell mouse embryo of the inbred strain
FVB (Harlan Sprague-Dawley, Indianapolis, IN). Microinjected embryos
were implanted into pseudopregnant females and allowed to come to term.
To identify transgenic founder mice, genomic DNA was isolated from 1-cm
tail clips from 3- to 4-week-old mice. DNA was subjected to PCR as
described above using primers specific for mouse NAT1 (M1:
5'-TGGTGTCTCCAGGTTAATCA-3' and M2: 5'-GGTGGAGCCCACTAAACAGT-3'), to
verify intact DNA, and primers specific for PB-NAT2 (1:
5'-TCAGTGAGGTCCAGATACCTACAG-3' and 2: 5'-CTTCTGTCAAGCAGAAAATG-3').
Founder (mice generated from injected mouse embryos that contained the
PB-NAT2 construct) mice were bred with FVB inbred mice and transgenic
offspring were identified by PCR as described above. Transgenic
positive (heterozygotes) and negative littermates were used for
experiments at 7 weeks of age, when maximum transgene expression in the
mouse prostate is observed (Greenberg et al., 1994; Barrios et al.,
1996).
N-Acetyltransferase Assays.
SMZ, PABA, and AF
N-acetyltransferase activities were measured using HPLC to
separate N-acetyl-product from aromatic amine substrate
using modifications of previous methods (Doll et al., 1997). Male mice
(transgenic and control) were sacrificed at 7 weeks of age and organs
were harvested and snap frozen. Tissue samples were homogenized (1 g/3
ml) in 20 mM sodium phosphate buffer (pH 7.4), containing 1 mM EDTA, 1 mM DTT, 100 µM phenylmethylsulfonylfluoride, and 10 mM leupeptin.
Homogenates were centrifuged at 100,000g for 60 min to
prepare cytosols.
). Briefly, undiluted transgenic and control mouse prostate cytosols were incubated at 37°C with 2 mM PhIP and 2 mM [3H]acetyl-coenzyme A.
O-Acetyltransferase Assays.
The activation of
N-hydroxy-PhIP and N-hydroxy-AF (via
O-acetylation) to species that are capable of binding DNA
were evaluated in prostate cytosols from transgenic and control mice as
previously described (Hein et al., 1995). Briefly, prostate tissue
cytosols were incubated at 37°C for 20 min in a reaction that
included [ring-3H]N-hydroxy-PhIP
(100 µM) or [ring-3H]N-hydroxy-AF
(100 µM), 20 mM sodium phosphate buffer (pH 7.4), 2 mM acetyl
coenzyme A, 1 mM DTT, 1 mM EDTA, and 1 mg/ml calf thymus DNA. In
controls, distilled water was substituted for acetyl coenzyme A.
N,O-Acetyltransferase assays.
The
activation (via N,O-acetylation) of
N-hydroxy-N-acetyl-AF to species capable of
binding DNA were evaluated in prostate cytosols from transgenic and
control mice as previously described (Hein et al., 1995). The reactions
were carried out with 100 µM [3H]-N-hydroxy-N-acetyl-AF
as described above except that water was substituted for acetyl
coenzyme A and controls used heat-denatured enzymes.
Measurement of PhIP DNA-Adduct Levels In Vivo.
PhIP (50 mg/kg) was dissolved in 100% dimethyl sulfoxide and administered i.p.
to transgenic and control mice (7 weeks of age). Controls were
administered vehicle. Mice were sacrificed 6 h after injection and
the prostate tissues were collected, snap frozen in liquid nitrogen,
and DNA isolated by proteinase K digestion followed by
phenol/chloroform extraction. DNA was treated with RNase A and T1 and
quantified by spectrophotometric analysis at 260 nm. DNA isolated from
prostate glands (10 µg) or reference DNA (1.48 pmol of PhIP-DNA
adducts/mg DNA) was hydrolyzed to 3'-monophosphates by incubating with
micrococcal nuclease (3.3 µg; Sigma, St. Louis, MO) and spleen
phosphodiesterase (3.3 µg; Sigma) in 10 mM sodium succinate, 5 mM
calcium chloride, pH 6.0, at 37°C for 5 h. DNA adducts were
enriched by n-butanol extraction method (Gupta, 1985). PhIP-DNA adducts were 32P-labeled in the presence
of T4 polynucleotide kinase and [
-32P]ATP
(50 µCi/sample, specific activity 7000 Ci/mmol; ICN, Costa Mesa, CA).
PhIP-DNA adducts were resolved by polyethyleneimine-cellulose thin-layer chromatography using slight modifications of previously published methods (Peluso et al., 1991). Briefly, DNA was separated using thin-layer chromatography on PEI Cellulose F (Alltech, Deerfield, IL) in D1 (top to bottom; 1 M sodium phosphate, pH 5.7), followed by D3
(bottom to top; 4 M lithium formate/8.5 M urea, pH 3.5), followed by D4
(left to right; 0.8 M LiCl, 0.5 M Tris-HCl, and 7 M urea, pH 8.0).
PhIP-DNA adducts were quantified by Instant Imager electronic
autoradiography (Packard Instruments, Chicago, IL) and compared with
the intensity of the PhIP-DNA adduct standard; N-(deoxyguanosin-8-yl)-PhIP (Lin et al., 1992).
Data Analysis. Differences in enzyme activity and PhIP-DNA adduct levels measured in vivo between transgenic and control mice were analyzed for significance by Student's t test.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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To investigate whether the PB promoter fragment could regulate the
expression of human NAT2*4, transgenic mice were constructed by microinjection of a 2.9-kb transgene carrying the 426/+28 PB
promoter fragment upstream of the 870-bp intronless NAT2*4 (Fig. 2). As shown in Fig. 3, founder
transgenic mice were identified by PCR amplification of the PB-NAT2
transgene using genomic DNA isolated from tail clips. Intact DNA was
verified from tail clips by amplification of mouse NAT1. The
PB-NAT2 construct was used as a control to verify proper
amplification of the PB-NAT2 fragment from mouse genomic
DNA. Two founder mice were identified (Fig. 3, lane 7 [DWH2.1] and
lane 11 [DWH2.2]) that possessed the PB-NAT2 transgene. Both founder
transgenic mice transmitted the transgene to approximately 50% of
their offspring.
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To determine whether PB-NAT2 transgene expression yielded functional
NAT2 enzyme activity restricted to the prostate, male transgenic and
control mice were sacrificed at 7 weeks of age. In comparisons between
transgenic and control mice, significant increases in SMZ (human
NAT2-specific) N-acetyltransferase activity were observed in
the prostate but not in any other tissue. As shown in Fig.
4A, transgenic line DWH2.1 demonstrated a
15-fold (p < .001) increase in SMZ
N-acetyltransferase activity compared with control mice. In
contrast, no significant increase in SMZ N-acetyltransferase
activity was observed in any other tissue tested (Fig. 4A). Transgenic
line DWH2.2 did not express an increase in SMZ N-acetyltransferase
activity in any tissue (data not shown).
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Transgenic mice did not demonstrate significantly higher levels of PABA (human NAT1-specific) N-acetyltransferase activity in the prostate compared with control mice (Fig. 4B). Moreover, no significant differences were observed between transgenic and control mice for PABA N-acetyltransferase activity in other tissues analyzed (Fig. 4B). These data suggest that the elevated SMZ N-acetyltransferase activities observed in the prostate of the transgenic mice were due to specific overexpression of human NAT2 in the prostate.
As shown in Table 1, transgenic and control prostate cytosols did not differ significantly in their ability to catalyze the O-acetylation of N-hydroxy-PhIP or N-hydroxy-AF. Moreover, there were no significant differences observed in the N-acetylation of AF and the N,O-acetylation of N-hydroxy-N-acetyl-AF between transgenic and control prostate cytosols. PhIP N-acetyltransferase activities were below the limit of detection in both transgenic and control prostate cytosols.
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PhIP-DNA adducts were detected in the prostate of both transgenic and
control mice treated with PhIP, but not in those treated with vehicle
(Fig. 5). The primary DNA adduct found in
prostate was identified as N-(deoxyguanosin-8-yl)-PhIP
adduct by chromatographic comparisons with the synthetic standard. The
PhIP-DNA adduct levels did not differ significantly between transgenic
and control prostate DNA (Fig. 6).
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Discussion |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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N-acetyltransferases bioactivate heterocyclic amines
via O-acetylation to ultimate carcinogens and individuals
segregate into rapid, intermediate, or slow acetylator phenotypes
(Grant et al., 1997; Hein et al., 1997). Thus, rates of heterocyclic
amine O-acetylation may be increased in rapid acetylators
(Hein et al., 1995). Molecular models that increase carcinogen
biotransformation, as it relates to genetic polymorphisms in activating
enzymes such as the N-acetyltransferases, may aid in the
understanding of complex pathways that either propagate elimination or
enhance genotoxicity of carcinogens. The prostate is a target organ for
the carcinogenic effects of PhIP in rats (Shirai et al., 1997).
However, the underlying mechanisms that initiate heterocyclic
amine-induced prostate tumor formation are unclear.
A working hypothesis includes PhIP bioactivation in the liver to form N-hydroxy-PhIP, and transport to extrahepatic organs where further bioactivation, via O-acetylation or O-sulfation, forms electrophilic species that bind DNA and initiates neoplasia. To study the role of extrahepatic O-acetylation in the bioactivation of N-hydroxy-PhIP within the prostate, we constructed a transgenic mouse model in which human NAT2 was specifically overexpressed in the prostate.
Our results are consistent with a previous study that found that mice
express relatively high levels of PABA N-acetyltransferase activity and very low levels of SMZ N-acetyltransferase
activity (Glowinski and Weber, 1982). Another study found that
recombinant mouse NAT1 and NAT2 proteins were unable to catalyze SMZ
N-acetylation at detectable levels (Martell et al., 1992).
Because SMZ is a very selective substrate for human NAT2 (Grant et al.,
1991; Hein et al., 1993), we used SMZ N-acetyltransferase
activity to assess the functional expression of the human
NAT2*4 transgene. The specific expression of this transgene
was clearly shown by the 15-fold increase observed in prostate cytosols
of transgenic mice, whereas significant increases were not observed in
other tissues. These results provide further evidence of the
specificity of the PB promoter in targeting prostate-specific transgene expression.
Previous studies found that transgene copy number does not correlate
with expression of two different transgenes using the same PB promoter
employed in our study (Greenberg et al., 1994, 1995). Consequently,
copy number was not determined. However, the Mendelian inheritance
observed in our study suggests a single insertion site for the transgene.
Previous studies have shown relatively high levels of PABA
N-acetyltransferase activity in rat (Hein et al., 1991) and
hamster (Hein et al., 1992) prostate that was significantly higher in rapid versus slow acetylator animals. Other studies in rodents have
shown that genetic differences in N-acetyltransferase
activity can affect DNA adduct levels. For example, higher levels of
aromatic amine DNA adduct levels were observed in urinary bladder of
rapid acetylator versus slow acetylator congenic mice (Levy and Weber, 1989) and congenic hamsters (Feng et al., 1996) following
administration of AF. Assuming that bioactivation of PhIP to
N-hydroxy-PhIP occurs in the liver with further
bioactivation in the prostate, we tested the hypothesis that
overexpression of rapid acetylator human N-acetyltransferase in mouse prostate would also result in higher levels of DNA adducts both in vitro and in vivo.
The results of this study did not support this hypothesis. Although we
successfully overexpressed human NAT2*4 in mouse prostate, we did not observe higher levels of PhIP DNA adducts in transgenic versus control mice. Similarly, higher levels of N-,
O-, or N,O-acetyltransferase activity were
not observed in vitro. The significant increase in SMZ
N-acetyltransferase activity in the transgenic mouse
prostate is most likely due to the specificity of human NAT2 to
N-acetylate SMZ (Grant et al., 1991; Hein et al., 1993), and
the very low ability of endogenous mouse
N-acetyltransferases to N-acetylate SMZ
(Glowinski and Weber, 1982; Martell et al. 1992). Furthermore, we did
not observe a significant increase in O-acetylation in the
prostates of transgenic mice. One explanation is that
N-hydroxy amine derivatives, such as N-hydroxy-AF
and N-hydroxy-PhIP, are not specific substrates for human
NAT2 and these compounds may be activated by endogenous mouse
acetyltransferases or sulfotransferases. The relative importance of
acetylation versus sulfation in this bioactivation pathway varies with
species (Lin et al., 1995). Compared with mice and rats, humans have
high O-acetyltransferase activity, but the lowest
O-sulfotransferase activity for the bioactivation of
N-hydroxy-PhIP (Lin et al., 1995). Mice, in contrast, have the lowest O-acetyltransferase activity, but the highest
O-sulfotransferase activity toward the bioactivation of
N-hydroxy-PhIP (Lin et al., 1995). Previous studies have
suggested that O-sulfotransferase predominates over
O-acetyltransferase in the bioactivation of N-hydroxy heterocyclic amine carcinogens in the mouse
(Buonarati et al., 1990; Lin et al., 1995).
The results of this study suggest that bioactivation via
O-acetylation within the mouse prostate is of limited
importance in the generation of PhIP-DNA adducts, and presumably
tumors. These results are consistent with a recent report by Agundez et al. (1998) that found very low levels of SMZ
N-acetyltransferase activity in human prostate that was
independent of the NAT2 genotype. However, our results need
to be interpreted with caution. As described above, mice express very
high levels of sulfotransferase activity, and it is possible that the
high levels of sulfotransferase overwhelmed the increase in
acetyltransferase activity achieved in the transgenic mice. In
addition, mice have endogenous N-acetyltransferase
activities that also contribute to the bioactivation (Fretland et al.,
1997; Hein et al., 1997). These endogenous
N-acetyltransferases may also have overwhelmed the modest
increase (15-fold) in human NAT2 4 that we achieved in this transgenic model.
In summary, to our knowledge, this is the first study to report construction of a transgenic mouse that overexpresses human N-acetyltransferase. The overexpression was successfully targeted to the prostate. Differences were not observed between transgenic and control mice in bioactivation of heterocyclic amine carcinogens within the prostate. Although these results do not support a role for bioactivation of heterocyclic amine carcinogens by human NAT2 within the prostate, the lack of an effect may relate to endogenous mouse N-acetyltransferases or sulfotransferases in the prostate and the modest level of human NAT2 over-expression observed.
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Acknowledgments |
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We thank Dr. Fred Kadlubar, National Center for Toxicological Research, Jefferson, Arkansas, for his generous donation of N-hydroxy arylamine and arylamide substrates and the PhIP-DNA adduct standard. We also thank Dr. Fatima Bosch, University of Barcelona, Bellaterra, Spain, for kind donation of the pKS/RIP expression vector.
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Footnotes |
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Accepted for publication March 23, 1999.
Received for publication December 29, 1998.
1 This work was partially supported by United States Public Health Service Grant CA34627 from the National Cancer Institute. A preliminary report of this work was presented at the 1998 annual meeting of the Society of Toxicology (Leff et al., 1998; Toxicol Sci 42:318).
2 This work constitutes partial fulfillment by Matthew Leff for the Ph.D. in Pharmacology and Toxicology at the University of Louisville.
3 Present address: Toxicology and Pathology Services, Inc., 10424 Middle Mount Vernon Rd., Mt. Vernon, IN 47620.
Send reprint requests to: David W. Hein, Ph.D., Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40292. E-mail: d.hein{at}louisville.edu
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Abbreviations |
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PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; NAT2, N-acetyltransferase 2; NAT1, N-acetyltransferase 1; SMZ, sulfamethazine; PABA, p-aminobenzoic acid; PB, probasin; PCR, polymerase chain reaction; bp, base pair; AF, 2-aminofluorene; DTT, dithiothreitol.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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 C. P. Nelson, L. C. R. Kidd, J. Sauvageot, W. B. Isaacs, A. M. De Marzo, J. D. Groopman, W. G. Nelson, and T. W. Kensler Protection against 2-Hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine Cytotoxicity and DNA Adduct Formation in Human Prostate by Glutathione S-Transferase P1 Cancer Res., January 1, 2001; 61(1): 103 - 109. [Abstract] [Full Text]
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D. W. Hein, M. A. Doll, A. J. Fretland, M. A. Leff, S. J. Webb, G. H. Xiao, U.-S. Devanaboyina, N. A. Nangju, and Y. Feng Molecular Genetics and Epidemiology of the NAT1 and NAT2 Acetylation Polymorphisms Cancer Epidemiol. Biomarkers Prev., January 1, 2000; 9(1): 29 - 42. [Abstract] [Full Text]
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 M. A. Leff, A. J. Fretland, M. A. Doll, and D. W. Hein Novel Human N-Acetyltransferase 2 Alleles That Differ in Mechanism for Slow Acetylator Phenotype J. Biol. Chem., December 3, 1999; 274(49): 34519 - 34522. [Abstract] [Full Text] [PDF]
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-globin
gene that includes the last exon (open sections) and the human
NAT2*4 open reading frame (slashed section).
