Cancer Epidemiology Biomarkers & Prevention Vol. 9, 29-42, January 2000
© 2000 American Association for Cancer Research
Molecular Genetics and Epidemiology of the NAT1 and NAT2 Acetylation Polymorphisms1
David W. Hein2,
Mark A. Doll,
Adrian J. Fretland,
Matthew A. Leff,
Stephanie J. Webb,
Gong H. Xiao,
Udaya-Sankar Devanaboyina,
Norma A. Nangju and
Yi Feng
Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky 40292
 |
Abstract
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The focus of this review is the molecular genetics, including consensus
NAT1 and NAT2 nomenclature, and cancer
epidemiology of the NAT1 and NAT2
acetylation polymorphisms. Two N-acetyltransferase
isozymes, NAT1 and NAT2, are polymorphic and catalyze both
N-acetylation (usually deactivation) and
O-acetylation (usually activation) of aromatic and
heterocyclic amine carcinogens. Epidemiological studies suggest that
the NAT1 and NAT2 acetylation
polymorphisms modify risk of developing urinary bladder,
colorectal, breast, head and neck, lung, and possibly prostate cancers.
Associations between slow NAT2 acetylator genotypes and
urinary bladder cancer and between rapid NAT2 acetylator
genotypes and colorectal cancer are the most consistently reported. The
individual risks associated with NAT1 and/or
NAT2 acetylator genotypes are small, but they increase
when considered in conjunction with other susceptibility genes and/or
aromatic and heterocyclic amine carcinogen exposures. Because of the
relatively high frequency of some NAT1 and
NAT2 genotypes in the population, the attributable
cancer risk may be high. The effect of NAT1 and
NAT2 genotype on cancer risk varies with organ site,
probably reflecting tissue-specific expression of NAT1 and NAT2. Ethnic
differences exist in NAT1 and NAT2
genotype frequencies that may be a factor in cancer incidence.
Large-scale molecular epidemiological studies that investigate the role
of NAT1 and NAT2 genotypes and/or
phenotypes together with other genetic susceptibility gene
polymorphisms and biomarkers of carcinogen exposure are necessary to
expand our current understanding of the role of NAT1 and
NAT2 acetylation polymorphisms in cancer risk.
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Introduction
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Comprehensive reviews of the acetylation polymorphism (NAT2) have
been published previously (1, 2, 3, 4)
. Several shorter reviews
including information on the NAT1 and NAT2
acetylation polymorphisms have been published within the past few years
(e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14
). This review focuses on the
molecular genetics and cancer epidemiology of the NAT1 and
NAT2 acetylation polymorphisms.
The acetylation polymorphism (NAT2) was discovered over 40 years ago
following differences observed in tuberculosis patients to isoniazid
toxicity (15)
. Subsequently, the differences in isoniazid
toxicity were attributed to genetic variability in
N-acetyltransferase (EC 2.3.1.5), a cytosolic phase II
conjugation enzyme primarily responsible for deactivation of isoniazid
(16
, 17)
. Indeed, the polymorphism was termed the
"isoniazid acetylation polymorphism" for many years until the
importance of the polymorphism in the metabolism and disposition of
other drugs and chemical carcinogens was fully appreciated (1
, 2)
.
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N-Acetyltransferase Isozymes
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An early paradox was the observation that the
N-acetylation of isoniazid and other drugs such as
sulfamethazine divided human populations into rapid, intermediate, and
slow acetylator phenotypes, whereas the N-acetylation of
other drugs such as p-aminosalicylic acid yielded an
apparently normal (monomorphic) distribution (18)
. Based
on this observation, drugs such as isoniazid and sulfamethazine were
termed "polymorphic" substrates for N-acetylation,
whereas drugs such as p-aminosalicylic acid were termed
"monomorphic." The biochemical basis for this observation relates
to substrate specificity and molecular genetics of two distinct
N-acetyltransferase isozymes, NAT1 and NAT2. The concept was
suggested by Jenne (18)
, confirmed initially in the Syrian
hamster (19, 20, 21)
, and subsequently confirmed in man
(22)
. The classical isoniazid slow acetylator phenotype(s)
is due, at least in part, to reduction(s) in NAT2 protein (23
, 24)
with a frequency that is approximately 50–60% in Caucasian
populations but shows striking ethnic differences (reviewed in Ref.
2
). For example, slow acetylator phenotype is much more
frequent in Egyptians but is much less frequent in Asians
(2)
. The NAT2 acetylation polymorphism is very important
in clinical pharmacology and toxicology because of its primary role in
the activation and/or deactivation of a large and diverse number of
aromatic amine and hydrazine drugs used in clinical medicine (for
reviews, see Refs. 1
, 2,
and 4
). The NAT1
isozyme was initially described as monomorphic because of its
selectivity for p-aminosalicylic acid and other substrates
yielding "monomorphic" distributions in human populations (1
, 2)
. However, subsequent studies showed that NAT1 is
not monomorphic in human populations but rather is subject to a
separate polymorphism (21
, 22)
. Endogenous substrates for
human NAT1 and NAT2 are not known, although the folic acid catabolite,
p-aminobenzoylglutamate is N-acetylated by NAT1
(27
, 28)
, suggesting a role for NAT1 in folate metabolism.
N-Acetyltransferases metabolize a number of aromatic and
heterocyclic amine carcinogens that produce tumors in rodents
(29)
. Site-directed mutagenesis studies have established a
critical cysteine (amino acid 68) within the catalytic site that
participates directly in acetyl transfer between the acetyl-CoA
cofactor and acceptor substrates (30)
. Aromatic amines and
hydrazines (N-acetylation) and N-hydroxy-aromatic
and -heterocyclic amines (O-acetylation) are both examples
of acceptor substrates that, in general, are deactivated
(N-acetylation) or activated (O-acetylation) by
NAT1 and/or NAT2 (reviewed in Ref. 3
). NAT1 and NAT2 also
catalyze the intramolecular N,O-acetyltransfer of
N-hydroxy-N-acetyl-aromatic amines (3
, 31)
. It is often suggested that human NAT2 activity is highest
in the liver and gastrointestinal tract, whereas human NAT1 activity is
expressed in many extrahepatic tissues, but the relative contribution
of hepatic versus extrahepatic activation and/or
deactivation of these carcinogens by NAT1 and NAT2 is not fully
understood. In Syrian hamsters (32
, 33)
and humans
(34)
, N- and O-acetyltransferase
expression in many extrahepatic tissues is comparable to that in the
liver and underscores the potential importance of
N-acetyltransferases in extraheptic activation/deactivation
pathways. Because both NAT1 and NAT2 catalyze the metabolic activation
(via O-acetylation) of aromatic and heterocyclic amine
carcinogens (31
, 35, 36, 37)
, genetic polymorphism in NAT1
and/or NAT2 may modify the cancer risk related to exposures to these
carcinogens. The metabolic activation of many N-hydroxy
heterocyclic amine carcinogens is catalyzed to a greater extent by
human NAT2 than NAT1 (35
, 37) , but tissue-specific
expression is also important for the effect of the NAT1 and
NAT2 acetylation polymorphisms in cancer risk.
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Molecular Genetics
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NAT1 and NAT2 are products of single, intronless
protein-coding exons of 870-bp open reading frames encoding
290 amino acids (38
, 39)
. NAT1,
NAT2, and a pseudogene, NATP, are located on
human chromosome 8p22 (40
, 41)
, a region frequently
displaying loss of heterozygosity in human tumors (42)
. A
third NAT gene (NAT3) has been identified in the
mouse (43
, 44)
. NAT1 and NAT2 share
87% nucleotide homology in the coding region, yielding 55 amino acid
differences. Whereas NAT1 derives its entire transcript from
a single exon, NAT2 mRNA is derived from both the
protein-coding exon and a second noncoding exon of 100 bp
located about 8 kb upstream of the translation start site (40
, 45)
.
Seven missense (G191A, T341C, A434C, G590A, A803G, A845C, and G857A)
and four silent (T111C, C282T, C481T, and C759T) substitutions have
been identified thus far in the NAT2 coding exon.
NAT2*4 is considered the wild-type allele because of its
absence of any of these substitutions. However, NAT2*4 is
not the most common allele in many ethnic groups, including Caucasians
and Africans. NAT2 alleles containing the G191A, T341C,
A434C, G590A, and/or G857A missense substitutions are associated with
slow acetylator phenotype(s). Striking ethnic differences in the
frequencies of these missense substitutions (reviewed in Ref.
6
) are responsible for the corresponding ethnic
differences in frequency of slow acetylator alleles (reviewed in Ref.
11
) and phenotype(s) (reviewed in Ref. 2
).
For example, the G191A substitution common to the NAT2*14
gene cluster is present in African Americans and native Africans, but
it is virtually absent in Caucasian populations. NAT1
allelic frequency has been reviewed recently (6
, 11)
.
NAT1*4 is the most common wild-type allele in all
populations studied to date. Ethnic differences have been observed. For
example, NAT1*10 is more frequent in Mexican and African
Americans (46)
than in Caucasians (47)
and
Asians (48)
.
As of this date (October 1999), 24 different NAT1 (Table 1)
and 26 different NAT2 (Table 2)
alleles have been identified in human populations. A number of
NAT1 and NAT2 alleles have been identified
recently, and there is a critical need to standardize and widely
publicize consensus gene nomenclature (80, 81, 82)
. A
consensus NAT nomenclature was published in 1995
(26)
. An international NAT nomenclature committee was
formed at the First International Workshop on the Arylamine
N-Acetyltransferases (81)
held in Cairns,
Australia in October 1998 to update, possibly revise, and publish an
internet-accessible website for NAT nomenclature updates
(82)
.3
Some of the nucleotide substitutions and corresponding NAT1
and NAT2 alleles are rare and reflect nucleotide diversity
(frequency of 0.0003–0.005) rather than genetic polymorphism (defined
as frequency greater than 0.01). Moreover, some of the nucleotide
substitutions are silent (do not change primary amino acid sequence)
and may therefore be considered insignificant. However, recent studies
have shown that silent single-nucleotide polymorphisms in gene coding
regions yield allele-specific mRNA variants that differ markedly in
structural folds (83)
that may alter splicing, processing,
translational control, and/or regulation of the mRNA.
Potential genotype and deduced phenotype misclassifications occur using
methods designed to detect a small subset (usually described as
WT, M1, M2, M2, M3, and sometimes M4) of the
alleles (Table 3)
. The M1 allele is detected by possession of the C481T
substitution. This silent substitution is found in many but not all of
the NAT2*5 alleles and is found in NAT2*12C,
which is actually a rapid allele. The M2 allele is detected
by possession of the G590A missense substitution, which is found in
NAT2*5, NAT*6, and NAT2*14 allelic
clusters. The M3 allele is detected by possession of the
G857A missense substitution, which thus far has been identified only on
NAT2*7A and NAT2*7B. An M4 allele was
initially discovered in African Americans (69
, 77)
and is
detected by possession of the G191A missense substitution. Thus far,
this substitution has only been identified on the NAT2*14
gene cluster. The WT allele was formerly defined as the
absence of C481T, G590A, G857A, and sometimes the G191A nucleotide
substitutions. Because NAT1, NAT2, and
NATP are highly homologous, PCR primers must be specific for
NAT1 or NAT2. Because most NAT1 and
NAT2 alleles have multiple nucleotide substitutions, it is
important that the correct phase be determined to identify their
location on one or the other of the homologous chromosomes in the
diploid genome (78)
. This and other pitfalls in
NAT2 genotyping have been reviewed recently
(84)
. Several published methods to detect
NAT1*10 do not distinguish it from NAT1*14A and
perhaps other NAT1 alleles. Genotype misclassification in
epidemiology investigations can produce substantial bias, requiring
large expansions in sample sizes, particularly if gene-environmental
interactions are considered (85
, 86)
.
Acetylation polymorphisms also have been characterized in animal models
(reviewed in Ref. 7
). NAT1 (Table 4)
and NAT2 (Table 5)
in nonhuman species are highly homologous to both human
NAT1 and NAT2 (Table 6)
. In fact, substrate specificities for Syrian hamster, mouse, and rat
NAT2 may resemble human NAT1 more than they do human NAT2
(1)
. Several different mechanisms are responsible for NAT2
polymorphisms in nonhuman species. The molecular basis for slow
acetylation is a NAT2 gene deletion in the rabbit
(87)
, a nonsense mutation yielding a truncated NAT2 enzyme
in the Syrian hamster (88, 89, 90)
, and a missense
substitution (91)
yielding an unstable NAT2 enzyme
(44
, 92)
in the mouse. In contrast, no NAT1
polymorphisms have been identified in nonhuman species except for a
single silent T60C mutation in Syrian hamster NAT1
(93)
.
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Genotype/Phenotype Relationships
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The effects of NAT1 and NAT2 nucleotide
substitutions on acetyltransferase catalytic activities have been
investigated primarily in recombinant expression systems (30
, 31
, 50
, 52
, 55
, 69
, 73
, 94, 95, 96, 97)
. Nucleotide substitutions identified
in human NAT1 and NAT2 allelic variants yield
reductions in substrate affinity, catalytic activity, and/or protein
stability of the recombinant N-acetyltransferase allozyme.
Recombinant human NAT2 5, NAT2 6, NAT2 14, and NAT2 17 clusters yield
variable reductions in catalytic activity associated with the slow
acetylator phenotype, whereas recombinant human NAT2 12 and NAT2 13
clusters catalyze N-,O-, and
N,O-acetyltransferase activities at levels comparable to
those of the rapid acetylator NAT2 4 (Fig. 1
). Although one study suggested that NAT2*13 was associated
with slow acetylator phenotype in vivo (67)
,
the observation related to a NAT2 genotyping artifact
(84)
. A recent study (98)
found that
NAT2*13 is associated with rapid acetylator phenotype
in vivo, consistent with recombinant expression systems
(31
, 94)
. Recombinant NAT2 proteins differ in intrinsic
stability because the NAT2 6, NAT2 7, NAT2 14, and NAT2 18 clusters are
less stable than the others (6
, 69
, 73 , 94)
. The NAT2 7B
allozyme has a higher affinity than other NAT2 allozymes for
sulfamethazine and dapsone (96)
but not for
2-aminofluorene (94
, 96)
, suggesting that expression of
acetylator phenotype is dependent on substrate even if it is
selective for NAT2 versus NAT1. Some, but not all, of the
nucleotide substitutions in human NAT2 yield reductions in
the quantity of recombinant NAT2 protein in eukaryotic expression
systems (24
, 38
, 68
, 73 , 99)
. Because most of the
structure-function information has been derived from recombinant
expression systems, more data from human tissues are needed to
investigate tissue-specific factors and other regulatory factors.
View larger version (29K):
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Fig. 1. Relative capacity of recombinant human
N-acetyltransferase allozymes to catalyze the
N-acetylation of 2-aminofluorene (top),
the O-acetylation of
N-hydroxy-2-aminofluorene (center) and
the N,O-acetylation of
N-hydroxy-N-acetyl-2-aminofluorene
(bottom). Levels of acetyltransferase for each allozyme
were N-acetylation >
O-acetylation > N,O-acetylation.
*, significantly lower than NAT2 4 (P < 0.05).
NAT activities are expressed in pmol/min/unit protein; OAT and
N,O-AT activities are expressed in pmol/min/mg DNA/unit
protein. Adapted from Ref. 31
.
|
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Because multiple mechanisms for reductions in
N-acetyltransferase activity are associated with various
nucleotide substitutions present on NAT2 alleles, the
ability to distinguish acetylator phenotypes is complex and is a
function of the sensitivity and specificity of the phenotype method.
Indeed, some studies report phenotypic differences within the slow
acetylator phenotype (67
, 84
, 100)
. Phenotype is
influenced by a number of factors including diet, disease, and drug
therapy. Depending on the probe drug and analytical method used,
acetylation phenotypes are often not clearly separate or distinct but
rather exhibit continuous and overlapping variability due to numerous
genetic and/or environmental factors, including the large number and
diversity of NAT2 genotypes present in human populations.
This finding is not unique to acetylation phenotypes
(101)
. The relative specificity of the substrate for NAT2
versus NAT1 at the concentrations obtained in
vivo will also affect acetylator phenotype. Aromatic and hydrazine
drugs such as isoniazid, dapsone, procainamide, and sulfamethazine have
also been used in patients treated with these drugs (reviewed in Refs.
1
and 2
). Caffeine is most commonly used as a
probe drug for NAT2 phenotype determinations (102)
, and
excellent NAT2 genotype/phenotype correlations have been
reported (6
, 67)
. Although administration of caffeine is a
relatively noninvasive method for determining NAT2 acetylator
phenotype, this method requires the quantitation of secondary
metabolites with key intermediates that are unknown. Thus,
genetic and/or environmental effects on a number of enzyme systems
(e.g., cytochrome P450 or xanthine oxidase) may affect the
levels of the metabolites used in the phenotype determination. For
example, NAT1 phenotype influences the urinary caffeine ratio used as a
measure of NAT2 phenotype (103)
. Other potential artifacts
in the use of caffeine to determine acetylation phenotype have also
been reported (104)
. An ELISA method to measure caffeine
metabolic ratios is presently under development (105)
.
NAT1 phenotype has been determined in vivo using
p-aminosalicylic acid as a probe drug (51)
.
Measurement of the urinary or plasma metabolite ratio after
administration of low doses of p-aminosalicylic acid allowed
for the identification of individuals with marked impairments of NAT1,
but less than 50% of phenotypic variation in vivo appears
to be related to variation in NAT1 activity (51)
.
Measurement of NAT1 activity in blood lysates in vitro has
been used for phenotype determinations (51
, 55)
, but
tissue-specific regulation of NAT1 is unknown. One study in a Caucasian
Australian population reported a bimodal distribution of NAT1 activity
in human peripheral blood mononuclear cells, with approximately 8%
exhibiting a slow NAT1 acetylator phenotype (55)
. Kinetic
studies showed that slow acetylator NAT1 individuals exhibited
Vmax levels approximately 50% of
rapid acetylators, whereas the Km was
similar for the two phenotypes (55)
. Because the
acetylation activity of the variant NAT1 is minimal, these findings
suggest that NAT1 alleles are codominant (55)
.
This is analogous to NAT2 alleles in the Syrian hamster,
which exhibit a striking Mendelian intermediary inheritance, yielding
doubled expression of both N- and O-acetylation
in the presence of two functional (i.e., homozygous rapid)
alleles (Refs. 19
and 106, 107, 108
; Table 7
). Preliminary studies suggest that NAT1 is subject to regulatory
control (55
, 109
, 110)
. Recently, a hormone response
element was identified in the NAT2 promoter region that
mediates androgenic regulation of N-acetyltransferase
activity in mouse kidney (111)
.
Several NAT1 alleles (Table 1)
have been associated
with reduced function in vivo or via recombinant expression
in bacteria (51
, 55 , 56)
. Recombinant expression of
NAT1*21, NAT1*24, and NAT1*25 in
bacterial systems resulted in allozymes with
N-acetyltransferase activities 2–3-fold higher than NAT1 4
(56)
. Some studies suggest that NAT1*10 may be
a rapid acetylator allele because it has been associated with slight
increases in N-acetyltransferase activity in bladder and
colon (112)
, liver (113)
, and erythrocytes
(52)
and increased carcinogen-DNA adduct
binding in the urinary bladder (114)
. Each of these
studies reported that individuals possessing the NAT1*10
allele had slightly higher levels of N-acetyltransferase
activity. However, in each study, there was considerable overlap, and
the increase was either nonsignificant or of marginal significance. The
instability of human NAT1 (22
, 36)
is also a problem for
investigations in human tissues. NAT1*10 has no nucleotide
substitutions in the coding region, but the substitution (T1088A) in
the 3'-untranslated region alters a polyadenylation signal
(TAATAA
TAAAAA) that may enhance mRNA
stability (112
, 115)
. However, other studies did not find
that NAT1 10 was a high activity allozyme (51
, 55 , 116)
.
Thus, the relationship between NAT1 genotype and phenotype
remains poorly understood.
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Molecular Epidemiology
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Human epidemiological studies have investigated the role of
NAT1 and/or NAT2 polymorphisms in a number of
cancers. Aromatic amines such as 4-aminobiphenyl and heterocyclic
amines such as PhIP4
are present in cigarette smoke (117
, 118)
. Heterocyclic
amines are also present in the diet as protein pyrolysis products
formed when meat is cooked well done (29)
. Aromatic and
heterocyclic amine carcinogens produce tumors at a number of sites in
rodents but require metabolic activation to mutate DNA and initiate
carcinogenesis. After N-oxidation, the
N-hydroxy-aromatic and N-hydroxy-heterocyclic
amines are further activated (via O-acetylation) by
N-acetyltransferases to acetoxy intermediates, which react
spontaneously with DNA to form DNA adducts (3
, 31, 32, 33, 34, 35, 36, 37)
.
Thus, biological plausibility for relationships between the
NAT1 and/or NAT2 acetylation polymorphisms are
strongest for cancers related to aromatic or heterocyclic amine
exposures.
The role of NAT1 versus NAT2 and/or the role of rapid
versus slow acetylator genotype in cancer predisposition
differs between organ sites, as might be expected with tissue-specific
expression of the NAT1 and NAT2 enzymes. Earlier studies primarily
investigated relationships between acetylator phenotype (NAT2) and
cancer incidence. Since the identification and cloning of human
NAT1 and NAT2 about 10 years ago, most studies
have investigated NAT2 genotype and cancer incidence, either
alone or in combination with phenotype. Recent studies have
investigated NAT1 genotype, usually in combination with
NAT2 genotype. Because multiple carcinogen metabolism
enzymes are involved in the activation and deactivation of chemical
carcinogens, many studies test interactions among multiple carcinogen
metabolizing genes. A discussion of these gene-gene interactions is
beyond the scope of this review. Similarly, many studies attempt to
estimate exposure to chemical carcinogens and test for
gene-environmental interactions. Investigations of gene-gene and/or
gene-environmental interactions require larger sample sizes to ensure
sufficient statistical power. New genotype technologies
(e.g., Refs. 78
and 119, 120, 121, 122
) are
facilitating these studies, and many large epidemiological studies are
ongoing. As such, this review represents a status of the field, which
is expected to expand exponentially in the near future. Present
information on the role of NAT1 and/or NAT2
acetylation polymorphism(s) in predisposition to urinary bladder,
colorectal, breast, lung, head and neck, and prostate cancers is
summarized below.
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Urinary Bladder Cancer
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The first association between slow acetylator phenotype and
urinary bladder cancer was reported 20 years ago (123)
.
The mechanism for this association suggests that slow NAT2 acetylation
of aromatic amine carcinogens competes poorly with metabolic activation
via cytochrome P450(s) and/or prostaglandin H-synthases, thus
accounting for higher risk in the slow NAT2 phenotype(s). In a
subsequent study (100)
, English chemical dye workers with
documented exposure to aromatic amine carcinogens showed a striking
association (odds ratio = 16.7; P = 0.00005)
between urinary bladder cancer and slow acetylator phenotypes. The
association was strongest in the slowest acetylator phenotypes. As
previously reviewed (3)
, a number of studies confirmed
these observations, and, as expected, the associations between slow
acetylator phenotype(s) and urinary bladder cancer are strongest in
studies in which there are documented exposures to aromatic amine
carcinogens. One prominent exception is a study of a cohort of workers
in China who had been employed in benzidine production and use. A
case-control study of surviving bladder cancer cases from this cohort
showed that neither slow NAT2 acetylator phenotype nor genotype was
associated with increased risk of urinary bladder cancer
(124)
. However, these results are explained by the
observation that benzidine is a much better substrate for NAT1 than
NAT2 (113)
. Recent NAT2 genotype studies
(125, 126, 127, 128)
show associations with urinary bladder cancer
that are highest for particular NAT2 alleles associated with
slow acetylator phenotype(s), consistent with the earlier phenotypic
studies (100)
. Among smokers, NAT2 slow acetylators have
higher levels of 4-aminobiphenyl hemoglobin adducts than rapid
acetylators (129
, 130)
.
An association between urinary bladder cancer and smokers possessing
the NAT1*10 allele was reported in two studies (131
, 132)
but not in two others (116
, 127)
. In the two
former studies (131
, 132) , the association was highest
among smokers who possessed both the NAT1*10 allele and were
slow NAT2 acetylators, suggesting that higher levels of
NAT1-catalyzed activation (O-acetylation) within the urinary
bladder increase risk. Immunochemical detection of NAT1 has been
reported in human urinary bladder, whereas NAT2 was below the level of
detection with the antibody used (133)
. However, both
NAT1 and NAT2 mRNA are readily detectable
(134)
. High levels of metabolic activation of
N-hydroxy-aromatic amines have been reported in human
urinary bladder cytosol (135)
and human uroepithelial
cells (136)
. Levels of activation were slightly higher in
individuals possessing the NAT1*10 allele
(112)
. Administration of aromatic amines to mice
(137)
and rats (138)
, but not to congenic
Syrian hamsters (139
, 140)
, resulted in higher DNA adducts
in the urinary bladder of slow acetylators than rapid acetylators. NAT2
activity expressed in urinary bladder is comparable to that observed in
other extrahepatic tissues in Syrian hamster (141)
, rat
(142)
, and mouse (143)
. In mouse and human,
immunohistochemistry has revealed that N-acetyltransferase
expression is limited to the transitional epithelium and the linings of
minor blood vessels (133
, 144)
.
N-Acetyltransferase expression in the kidney has been
detected in the cuboidal epithelium of the proximal convoluted tubules,
with more intense staining in the cortex than in the medulla
(133)
. Expression in the proximal convoluted tubules is
consistent with a role for N-acetyltransferase(s) in the
activation and/or deactivation of carcinogens excreted into the urinary
bladder. Moreover, a very recent study reported an association between
slow acetylator NAT2 genotype and renal cell carcinoma
(145)
.
|
Colorectal Cancer
|
|---|
Heterocyclic amines are associated with colorectal cancer in
rodents (29)
but are poor substrates for
N-acetylation (33
, 34
, 36
, 146 , 147)
. Thus, a
biologically plausible mechanistic hypothesis suggests that rapid
NAT1 and/or NAT2 acetylators should more readily
activate N-hydroxy-heterocyclic amine carcinogens within the
colon to their ultimate carcinogenic forms, thereby predisposing them
to colorectal cancer. Human colon cytosols activate
N-hydroxy heterocyclic amine carcinogens to DNA adducts
catalyzed by N-acetyltransferases (34
, 112
, 148)
. Because human populations are genetically heterogeneous,
and exposures to heterocyclic amines are difficult to estimate, it is
not surprising that the results are inconsistent. Several studies
(149, 150, 151)
found an association between rapid
NAT2 acetylator phenotype and colorectal cancer, whereas
other studies (152, 153, 154, 155, 156, 157)
did not. Five studies
(158, 159, 160, 161, 162)
reported an association between rapid
NAT2 acetylator phenotype and colorectal cancer for
individuals consuming well-done meat and, presumably, higher levels of
heterocyclic amine carcinogens (163)
. Consistent with this
hypothesis, rapid NAT2 acetylators who consumed pan-fried
meats had higher levels of urinary mutagenicity than slow acetylators
(164)
. One study found the association to colorectal
cancer was limited to homozygous rapid
(NAT2*4/*4) acetylators (151)
, a
finding that was also observed for lung (165)
and
laryngeal (166)
cancers.
Bell et al. (115)
found an association between
the NAT1*10 allele and colorectal cancer, and the risk was
highest among NAT2 rapid acetylators. Another study also
showed a higher risk for colorectal cancer in individuals who consumed
well-done meat and possessed both the NAT1*10 allele and
rapid acetylator NAT2 genotype (161)
.
NAT1*10 and NAT2*4 are in linkage disequilibrium
(166
, 167) , which may be a factor in the association of
the NAT1*10 allele with colorectal cancer. Two studies
reported a lack of association between the NAT1*10 allele
and colorectal cancer (48
, 56)
. The disparity in results
may relate to misclassification of NAT1*10 alleles because a
common test to detect the NAT1*10 allele does not
distinguish between NAT1*10 and NAT1*14A
(55)
or other NAT1 alleles.
The role of the NAT2 acetylator polymorphism in
colorectal cancer has been investigated in animal models. These models
are particularly useful in that age, carcinogen exposure, and diet can
be carefully matched, and genetic variability in genes other than
NAT2 is virtually eliminated in the congenic models.
Furthermore, the presence of a single mechanism for slow acetylator
genotype in these models, resulting from homozygosity for a single slow
acetylator NAT2 allele (Table 5)
, results in clear and
unambiguous genotype/phenotype correlations, as illustrated by the
levels of N-acetyltransferase activity expressed in the
colon (Table 7)
. This is particularly true when the NAT1 and NAT2
isozymes in colon mucosa are isolated, separated from each other, and
tested separately (Table 7)
. NAT1 and NAT2 mRNA are widely distributed
in humans (168)
, but comprehensive data on NAT1 and NAT2
activities are lacking for human tissues, reflecting the availability
of fresh human tissue and the instability of human NAT1. NAT1 and NAT2
are distributed differently in rabbit, Syrian hamster, mouse, and rat
small intestine (169
, 170)
. N-Acetyltransferase
expression at the cellular level was restricted to epithelial cells
exposed to the lumen of both the small intestine and colon.
Immunohistochemical staining of mouse tissues with anti-NAT2 antibody
demonstrated intense staining in the columnar epithelial cells of the
villus tip of small intestine, with stain intensity decreasing along
the crypt/villus axis, and little to no staining in the crypts of
Lieberkuhn (144)
. Similarly, in rat small intestine,
N-acetyltransferase expression was greatest in epithelial
cells isolated from mid-villus and villus tip (171)
.
N-Acetyltransferase expression in humans has been observed
in epithelial cells of all types in the small intestine villus, but not
in the crypts of Lieberkuhn (172)
. Staining was restricted
to epithelial cells at the luminal surface, with no staining in the
crypts. Under controlled exposure and genotype, a higher frequency of
aberrant crypt foci (a preneoplastic lesion for colorectal cancer) is
found in rapid acetylators than in slow acetylators using two different
congenic hamster models and an inbred rat model (Table 8)
. Recently, higher levels of PhIP-DNA adducts were detected in the
colons of rapid versus slow acetylator rats given PhIP
(176)
. These studies support the role of the
NAT2 acetylation polymorphism in genetic predisposition to
colorectal cancer, suggesting that homozygous rapid acetylators exposed
to high levels of heterocyclic amines through consumption of
consistently well-done meat are at a higher risk.
View this table:
[in this window]
[in a new window]
|
Table 8 Frequency of aberrant crypt foci in homozgous rapid and slow acetylator
congenic hamsters and inbred rats administered
3,2-dimethyl-4-aminobiphenyla
|
|
|
Breast Cancer
|
|---|
Several studies have investigated an association between NAT2
acetylator phenotype or genotype and breast cancer, but the findings
have been very inconsistent. NAT2 acetylator phenotype was not
associated with breast cancer in three (177, 178, 179)
,
studies. However, rapid acetylator phenotype was associated with breast
cancer risk (180
, 181)
or advanced disease at first
presentation (182)
in three other studies. Recently, the
association between NAT2 acetylator genotype and breast
cancer has been investigated in relation to smoking and diet. Two
studies found that red meat consumption and NAT2 genotype
were not associated with breast cancer risk (183
, 184)
.
However, in a preliminary report, rapid/intermediate NAT2
genotypes were associated with breast cancer risk in women who
consistently consume very well-done meat (185)
. The
relationship between NAT2 genotype and breast cancer among
smoking women has varied. Two studies reported a higher risk among slow
acetylators (186
, 187)
, one found no difference between
rapid and slow acetylators (188)
and one reported a higher
risk among rapid acetylators (189)
. NAT1 and
NAT2 mRNA has been detected in human mammary cells
(190)
. Human mammary cells from rapid acetylators
activated heterocyclic amines to DNA adducts to a greater extent than
cells derived from slow acetylators (191)
. However, higher
levels of DNA adducts were reported in mammary tissue from slow
acetylators than in mammary tissue from rapid acetylators
(192)
.
Recent investigations have explored the relationship between
NAT1 acetylation polymorphism and breast cancer risk. NAT1,
but not NAT2, enzymatic activity was detected in human mammary
epithelial cells (190)
. One study (189)
observed little modification of smoking effects for breast cancer
according to NAT1 genotype, except among postmenopausal
women with the NAT1*10 allele. A more recent study reported
an elevated risk of breast cancer in smokers who consistently consumed
well-done red meat and possessed the NAT1*11 allele
(47)
. The acetylator phenotype of individuals possessing
the NAT1*11 allele is not yet fully understood. The G445A;
Val129Ile substitution present in
NAT1*11 (50)
was not included in previous
descriptions of this allele (26)
. A subsequent study found
that this missense substitution yielded recombinant NAT1 protein that
catalyzed the metabolic activation of N-hydroxy-aromatic
amines at rates up to 2-fold higher than that of wild-type
recombinant NAT1 (50)
. However, another study
(51)
reported that recombinant expression of
NAT1*11 did not produce a higher activity allozyme.
|
Lung Cancer
|
|---|
Early studies investigating the role of the NAT2 phenotype in
susceptibility to lung cancer were either negative (193)
or showed a slight overrepresentation of rapid acetylators (194
, 195)
. A subsequent NAT2 genotype study
(165)
basically confirmed the latter finding but showed
more clearly that the highest risk was found in smokers with the
homozygous rapid acetylator (NAT2*4/*4) genotype. Similarly,
Nyberg et al. (196)
reported an increased risk
for lung cancer for rapid acetylators who were smokers. In contrast,
two studies (70
, 197)
did not find an association between
NAT2 acetylator genotype and lung cancer. Increased risks of
asbestos-associated malignant mesothelioma were observed in slow
acetylators in Finnish studies, especially when exposed to high levels
of asbestos (198
, 199)
. The role of NAT1
genotype in lung cancer risk has also been investigated. One study
(197)
reported an association between low activity
NAT1 alleles (NAT1*14 and
NAT1*15) with lung cancer. A second study
(46)
did not find an association between NAT1
genotype and lung cancer, but the genotype assay did not distinguish
the NAT1*14 or NAT1*15 alleles.
|
Head and Neck Cancer
|
|---|
Head and neck cancers are strongly associated with smoking, and
several studies have explored the role of NAT1 and
NAT2 polymorphisms in the incidence of head and neck cancer
in smokers. The slow NAT2 acetylator phenotype was associated with the
development of head and neck cancer in Caucasians (200
, 201)
and with the development of esophageal (202)
and laryngeal (203)
cancers in Japan. The homozygous slow
acetylator NAT2 genotype was associated with an increased risk of
oral/pharyngeal cancer, but not laryngeal cancer (204)
.
The homozygous rapid acetylator (NAT2*4/NAT2*4) genotype was
strongly associated with laryngeal cancer in a German study
(167)
. NAT1*10 allele, but not NAT2 phenotype,
was associated with oral squamous cell carcinoma in a Japanese study
(205)
. NAT1*10 was associated with head and
neck cancers in one study (206)
but with neither
oral/pharyngeal nor laryngeal cancer in another study
(204)
.
|
Prostate Cancer
|
|---|
Three studies have explored associations between acetylator
genotypes and prostate cancer. No relationship between NAT2
genotype and prostate cancer was observed in two studies (207
, 208)
, and aromatic amine N-acetyltransferase activity
levels in human prostates were independent of NAT2 genotype
(207)
. An association between NAT1*10 and
prostate cancer was reported recently (209)
. Higher levels
of DNA adducts in the prostate were reported in slow versus
rapid acetylator rats given 3,2'dimethyl-4-aminobiphenyl
(138)
, but the opposite was observed in rapid and slow
acetylator rats given PhIP (176)
. Transgenic mice in which
human NAT2 was overexpressed specifically in the prostate did not
increase levels of DNA adducts in the prostate of mice given PhIP
(210)
.
|
Summary
|
|---|
Polymorphisms exist for both NAT1 and NAT2. Because they catalyze
the N-acetylation (usually deactivation) and
O-acetylation (usually activation) of aromatic and
heterocyclic amine carcinogens, genetic polymorphisms in NAT1 and/or
NAT2 may modify risk associated with carcinogen exposures. Although our
understanding is still incomplete, particularly for NAT1, information
on the molecular genetics and structure-function of NAT1 and NAT2 has
increased substantially over the past decade, facilitating the
development of molecular epidemiological studies exploring the
relationship between the acetylation polymorphisms and cancer. Animal
models have well-defined molecular mechanisms for the slow NAT2
acetylator phenotype. In contrast, molecular mechanisms for slow NAT2
acetylator phenotypes in humans appear to be much more complex and are
not well understood. Human epidemiological studies suggest that the
NAT1 and NAT2 acetylation polymorphisms modify predisposition to
urinary bladder, colorectal, breast, head and neck, lung, and possibly
prostate cancers, but there is inconsistency in the results. The
inconsistencies may relate, in part, to differences in carcinogen
exposures, genotype and/or phenotype methods, insufficient sample
sizes, and/or other susceptibility genes and factors. Associations
between slow NAT2 acetylator genotypes and urinary bladder
cancer and between rapid NAT2 acetylator genotypes and
colorectal cancer are the most consistently reported associations.
Individual risks associated with NAT1 and/or NAT2
genotypes are small, but they increase when considered in conjunction
with other susceptibility genes and/or aromatic and heterocyclic amine
carcinogen exposures. Because of the relatively high frequency of some
NAT1 and NAT2 genotypes, the attributable risk of
cancer in the population may be high. Because most aromatic and
heterocyclic amine carcinogens are metabolized by NAT1 and NAT2, both
genotypes should be determined using methods that minimize or eliminate
allele misclassification. Data on carcinogen exposure should also be
assessed when possible. The effect of NAT1 and
NAT2 genotype on cancer risk varies with organ site,
probably reflecting tissue-specific expression of NAT1 and NAT2. Ethnic
differences in NAT1 and NAT2 genotype frequencies
may be a factor in cancer incidence. Large-scale molecular
epidemiological studies that investigate the role of NAT1
and NAT2 genotypes and/or phenotypes together with other
genetic susceptibility gene polymorphisms and biomarkers of carcinogen
exposure are necessary to improve our understanding of the role of the
NAT1 and NAT2 acetylation polymorphisms in cancer
risk.
|
Footnotes
|
|---|
1 Supported in part by USPHS Grant CA-34627 from
the National Cancer Institute. 
2 To whom requests for reprints should be
addressed. Phone: (502) 852-5141; Fax: (502) 852-7868; E-mail: d.hein{at}louisville.edu
3 The website is presently located at
www.louisville.edu/medschool/pharmacology/NAT.html.
4 The abbreviation used is: PhIP,
2-amino-1-methyl-6-phenylimidazo[4,5-b]
pyridine.
Received 8/ 5/99;
revised 10/18/99;
accepted 10/20/99.
|
References
|
|---|
-
Weber W. W., Hein D. W. N-Acetylation pharmacogenetics. Pharmacol. Rev., 37: 25-79, 1985.[Medline]
-
Weber W. W. . The Acetylator Genes and Drug Response, Oxford University Press New York 1987.
-
Hein D. W. Acetylator genotype and arylamine-induced carcinogenesis. Biochim. Biophys. Acta, 948: 37-66, 1988.[Medline]
-
Evans D. A. P. N-Acetyltransferase. Pharmacol. Ther., 42: 157-234, 1989.[Medline]
-
Evans D. A. P. . Genetic Factors in Drug Therapy: Clinical and Molecular Pharmacogenetics, Cambridge University Press Cambridge, UK 1993.
-
Grant D. M., Hughes N. C., Janezic S. A., Goodfellow G. H., Chen H. J., Gaedigk A., Yu V. L., Grewal R. Human acetyltransferase polymorphisms. Mutat. Res., 376: 61-70, 1997.[Medline]
-
Hein D. W., Doll M. A., Fretland A. J., Gray K., Deitz A. C., Feng Y., Jiang W., Rustan T. D., Satran S., Wilkie T. R. Rodent models of the human acetylation polymorphism: comparisons of recombinant acetyltransferases. Mutat. Res., 376: 101-106, 1997.[Medline]
-
Lang N. P. Acetylation as an indicator of risk. Environ. Health Perspect., 105: 763-766, 1997.
-
Meyer U. A., Zanger U. M. Molecular mechanisms of genetic polymorphisms of drug metabolism. Annu. Rev. Pharmacol. Toxicol., 37: 269-296, 1997.[Medline]
-
Weber W. W. . Pharmacogenetics, Oxford University Press New York 1997.
-
Cascorbi I., Brockmoller J., Mrozikiewicz P. M., Muller A., Roots I. Arylamine N-acetyltransferase activity in man. Drug Metab. Rev., 31: 489-502, 1999.[Medline]
-
Hulla J. E., Miller M. S., Taylor J. A., Hein D. W., Furlong C. E., Omiecinski C. J., Kunkel T. A. Symposium overview: the role of genetic polymorphisms and repair deficiencies in environmental disease. Toxicol. Sci., 47: 135-143, 1999.[Abstract/Free Full Text]
-
Wormhoudt L. W., Commandeur J. N. M., Vermeulen N. P. E. Genetic polymorphisms of human N-acetyltransferase, cytochrome P450, glutathione-S-transferase and epoxide hydrolase enzymes: relevance to xenobiotic metabolism and toxicity. Crit. Rev. Toxicol., 29: 59-124, 1999.[Medline]
-
Hein D. W. N-Acetyltransferase genetics and their role in predisposition to aromatic and heterocyclic amine-induced carcinogenesis. Toxicol Lett., : in press
-
Hughes H. B., Biehl J. P., Jones A. P., Schmidt L. H. Metabolism of isoniazid in man as related to the occurrence of peripheral neuritis. Am. Rev. Res. Dis., 70: 266-273, 1954.
-
Evans D. A. P., White T. A. Human acetylation polymorphism. J. Lab. Clin. Med., 63: 394-403, 1964.
-
Weber W. W., Hein D. W. Clinical pharmacokinetics of isoniazid. Clin. Pharmacokinet., 4: 401-422, 1979.[Medline]
-
Jenne J. W. Partial purification and properties of the isoniazid transacetylase in human liver: its relationship to the acetylation of p-aminosalicylic acid. J. Clin. Investig., 44: 1992-2002, 1965.
-
Hein D. W., Kirlin W. G., Ferguson R. J., Weber W. W. Biochemical investigation of the basis for the genetic N-acetylation polymorphism in the inbred hamster. J. Pharmacol. Exp. Ther., 234: 358-364, 1985.[Abstract/Free Full Text]
-
Smith T. J., Hanna P. E. N-Acetyltransferase multiplicity and bioactivation of N-arylhydroxamic acids by hamster hepatic and intestinal enzymes. Carcinogenesis (Lond.), 7: 697-702, 1986.[Abstract/Free Full Text]
-
Ozawa S., Abu-Zeid M., Kawakubo Y., Toyama S., Yamazoe Y., Kato R. Monomorphic and polymorphic isozymes of arylamine N-acetyltransferases in hamster liver: purification of the isozymes and genetic basis of N-acetylation polymorphism. Carcinogenesis (Lond.), 11: 2137-2144, 1990.[Abstract/Free Full Text]
-
Grant D. M., Beer M., Blum M., Meyer U. A. Monomorphic and polymorphic human arylamine N-acetyltransferases: a comparison of liver isozymes and expressed products of two cloned genes. Mol. Pharmacol., 39: 184-191, 1991.[Abstract]
-
Grant D. M., Morike K., Eichelbaum M., Meyer U. A. Acetylation pharmacogenetics: the slow acetylator phenotype is caused by decreased or absent arylamine N-acetyltransferase in human liver. J. Clin. Investig., 85: 968-972, 1990.
-
Deguchi T. Sequences and expression of alleles of polymorphic arylamine N-acetyltransferase of human liver. J. Biol. Chem., 267: 18140-18147, 1992.[Abstract/Free Full Text]
-
Vatsis K. P., Weber W. W. Structural heterogeneity of Caucasian N-acetyltransferase at the NAT1 gene locus. Arch. Biochem. Biophys., 301: 71-76, 1993.[Medline]
-
Vatsis K. P., Weber W. W., Bell D. A., Dupret J-M., Price-Evans D. A., Grant D. M., Hein D. W., Lin H. J., Meyer U. A., Relling M. V., Sim E., Suzuki T., Yamazoe Y. Nomenclature for N-acetyltransferases. Pharmacogenetics, 5: 1-17, 1995.[Medline]
-
Minchin R. F. Acetylation of p-aminobenzoylglutamate, a folic acid catabolite, by recombinant human arylamine N-acetyltransferase and U937 cells. Biochem. J., 307: 1-3, 1995.
-
Ward A., Summer M. J., Sim E. Purification of recombinant human N-acetyltransferase type 1 (NAT1) expressed in E. coli and characterization of its potential role in folate metabolism. Biochem. Pharmacol., 49: 1759-1767, 1995.
-
Layton D. W., Bogen K. T., Knize M. G., Hatch F. T., Johnson V. M., Felton J. S. Cancer risk of heterocyclic amines in cooked foods: an analysis and implications for research. Carcinogenesis (Lond.), 16: 39-52, 1995.[Abstract/Free Full Text]
-
Dupret J-M., Grant D. M. Site-directed mutagenesis of recombinant human arylamine N-acetyltransferase expressed in E. coli: evidence for direct involvement of Cys68 in the catalytic mechanism of polymorphic human NAT2. J. Biol. Chem., 267: 1992/DATE>7381-7385, [Abstract/Free Full Text]
-
Hein D. W., Doll M. A., Rustan T. D., Ferguson R. J. Metabolic activation of N-hydroxyarylamines and N-hydroxyarylamides by 16 recombinant human NAT2 allozymes: effects of 7 specific NAT2 nucleic acid substitutions. Cancer Res., 55: 3531-3536, 1995.[Abstract/Free Full Text]
-
Hein D. W., Flammang T. J., Kirlin W. G., Trinidad A., Ogolla F. Acetylator genotype dependent metabolic activation of carcinogenic N-hydroxy-arylamines by S-acetyl coenzyme A-dependent enzymes of inbred hamster tissue cytosols: relationship to arylamine N-acetyltransferase. Carcinogenesis (Lond.), 8: 1767-1774, 1987.[Abstract/Free Full Text]
-
Ogolla F., Ferguson R. J., Kirlin W. G., Trinidad A., Andrews A. F., Mpezo M., Hein D. W. Acetylator genotype-dependent expression of arylamine N-acetyltransferase and N-hydroxy-arylamine O-acetyltransferase in Syrian inbred hamster intestine and colon: identity with the hepatic acetylation polymorphism. Drug Metab. Dispos., 18: 680-685, 1990.[Abstract]
-
Turesky R. J., Lang N. P., Butler M. A., Teitel C. H., Kadlubar F. F. Metabolic activation of carcinogenic heterocyclic aromatic amines by human liver and colon. Carcinogenesis (Lond.), 12: 1839-1845, 1991.[Abstract/Free Full Text]
-
Minchin R. F., Reeves P. T., Teitel C. H., McManus M. E., Mojarrabi B., Ilett K. F., Kadlubar F. F. N- and O-acetylation of aromatic and heterocyclic amine carcinogens by human monomorphic and polymorphic acetyltransferases expressed in COS-1 cells. Biochem. Biophys. Res. Commun., 185: 839-844, 1992.[Medline]
-
Hein D. W., Doll M. A., Rustan T. D., Gray K., Feng Y., Ferguson R. J., Grant D. M. Metabolic activation and deactivation of arylamine carcinogens by recombinant human NAT1 and polymorphic NAT2 acetyltransferases. Carcinogenesis (Lond.), 14: 1633-1638, 1993.
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Abstract
Introduction
