N- acetyltransferases are coded by the acetyltransferase gene (NA n
1.2.4 N-acetyltransferase 2
1.2.4.1 NAT2 Phenotype and Genetic Polymorph isms
The
polymorphism
oftheN-acetyltransferase
2(NA T2)
gene cluster arises from SNPs whichindividually
or in combinationsgive
rise to the different NAT2 allelic variants(Dandara
elai., 2003).
There is more than 15point
variation on the NAT2 gene
giving
rise to at least 28 allelic variants.Generally.
when
naming
the NAT2alleles,
the mostfunctionally significant
nucleotidesubstitution is considered. The detailed nomenclature for NA T2 alleles is listed in
Appendix
1. Certain variations were more common in certain ethnic groups forexample
variations at 191 G>A which areassigned
to NA T2*14 were foundin African-American
only (Bell
etai., 1993,
Dandara etaI., 2003).
The variations in NA T2 gene result in amino acid substitutions
(Fretland
et
aI., 2001, Hein, 2002)
that can eithercause reduced enzymeactivity
andexpression (Grant
etal., 2000,
Fretland etal., 2001, Hein, 2002),
reducedprotein expression (Hein, 2002,
Fretland etal., 2001),
or reducedprotein
stability (Fretland
etal., 2001, Ferguson
eta/., 1994).
As anexample,
variationsat 191 G>A and 341 T>C were found to have lower N and O
acetylation
capacity
and less stableintrinsically
ascompared
to the referencewild-type
allele(Ferguson
etal., 1994).
Some variations are however silent(Fretland
etai., 2001,
Grantetal., 1992).
The consequence ofSNP's to enzyme activities can be varied. Some may be
expressed,
others are not. Ifexpressed,
their consequence may furtherdepend
on the substrates the enzyme acts upon. Thus forNAT2,
if thesubstrate is a
carcinogen,
slowacetylators
may be at increased risks for exposure and may be athigher
risks todevelop
cancers(Figure 1.3).
If thesubstrate is a
drug
likeINH,
then slowacetylators
may be at increased risks todevelop
dose-related side effects. On the otherhand,
if the substrate is a procarcinogen
or apro-drug,
theopposite
will beexpected.
A
Prodrugl protoxin
I Drugl
toxinI
bioeottvstton
I
Activel drug/
toxinl
bioinactivation
,r
Inactive
drugl
non-toxicmetabolites
Pharmacological
ortoxicological
effectB
Progenotoxin
I
GenotoxinI
�bioactivation
biOinaetlvati01
Epidemiological 1
relationship
I
GenotoxinI
betweengenetic
I Non-genotoxic
metaboliteI
polymorphism in
l
a
biotransformation enzymeand
tumour formation Genotoxic effect
"
Tumor formation
Figure
1.3 Schematicview of the effects ofgenetic polymorphisms ofbiotransforming
enzymeson metabolism of(pro)drugs and (pro)toxins (A) or(pro)genotoxic compounds(B). Depending on whetherthe metabolic reaction involved is
bioactivating orbioinactivating, genetic polymorphisms might increase or reducedrug
efficacy
or(geno)toxic
effects. Also shown is theoften-investigated
epidemiological relationshipwith cancer risk.The SNPs and their effects on NAT2
activity
are listed inAppendix
2. TheNAT2*5cluster
(all possessing
341T>C)
shows thegreatest
reduction in Nacetylation, O-acetylation
andN, O-acetylation
followedby
the NAT2*14 cluster(all possessing
191G>A)
and thenby
the NA T2*6 cluster(all possessing
590G>A) (Hein, 2002).
The dominant reference allele NA T2*4 expresses a full enzymeactivity.
Combinations of theremaining
allelesproduce
enzymes with diminishedactivity
andimpaired stability (Meisel
etal., 2001).
The
expression
of the NA T2 gene hasclearly
been shown to be involved in theacetylation polymorphism.
Thestereotypical pattern
of metabolism has facilitated theanalysis
andquantification
ofan individual'senzymatic
status foreach
drug metabolizing
enzyme(OME) using probes,
substances which are known to be broken downby
therespective
enzyme(substrates).
The commonprocedure
for NAT2phenolyping
is based on the administration of theprobe drug
INH and the measurements of the MR of INH to its metabolite either in theplasma
orin urinepooled
over a certainperiod
oftime afterdrug
administration.The ratio is known as metabolic ratio
(MR) (Sweeney
andBromilow, 2006).
Although
manydrugs
are substrates for more than one enzymesystem,
somedrugs
are metabolizedprimarily by
asingle
enzyme and can therefore be used to measure theactivity
of the enzyme. NAT2 substrates includeINH, dapsone, procainamide, sulfamethazine, hydralazine, aminoglutethemide, nitrazepam,
caffeine and
phenelzine (Grant
etal., 1997,
Gaikovitch etal., 2003). However,
caffeine are also a substrate for CYP1A2
(Sweeney
andBromilow, 2006).
Although phenotyping
shouldideally
measure enzymeactivity
that issubsequently explained by
relevantvariation on the gene ofinterest,
itis notalways
exact. Concordance betweengenotype
andphenotype
is sometimes not seen because,apart
fromgenetic variations, phenotypic expression
can be influencedby
both intrinsic and extrinsic factors. Zielinska et al.(1999)
founddiscordance between
acetylator genotype
andphenotype
for NAT2 in children and had difficultiesphenotyping
infants less than 20 weeks. NAT2 is not known to be inducible in humans(O'Neil
eta/., 1997). Although
NAT2activity
is notalso influenced
by gender
or the menstrualcycle phase (Kashuba
etal., 1998),
itwas found to be
significantly
reduced inearly pregnancies (Tsutsumi
eta/., 2001).
NAT2activity
may be alteredby progression
of HIV and AIDS. O'Neil el al.(1997)
found that therewere discordance betweengenotype
andphenotype
of NAT2 in such
patients.
Astudy by
Kaufmann el al.(1996)
however showed concordance betweengenotypes
andphenotypes
of NAT2 inpatients
with HIVinfections and no increase in
prevalence
of slowacetylation
inpatients
withadvanced
stages
of the disease was observed as would beexpected
if the HIVstatus were to
impair activity.
Thediscrepancy
between the studies may be the result of differences in co-medications of thepatients
studied. Such a difference may however beimportant given
thefrequent co-morbidity
of HIV and TB.1.2.4.2 NA T2 and Other Diseases
As NAT2 is involved in the metabolism of many
xenobiotics,
and as mentioned above where NAT2 may be involved in thepathophysiology
ofcancers, NAT2 may be involved in other
environmentally
induced humandiseases.
Thus,
NAT2 has also been associated with diseases such rheumatoid arthritis where it was found that the risk fordeveloping
rheumatoid arthritis wasalmost 5-fold
greater
in slowacetylators compared
to fastacetylators (Pawlik
etal., 2002).
Slowacetylators
were also believed to be more prone todevelop hydralazine-
orprocainamide-induced lupus syndrome
andhaemolytic
anaemiadue to certain sulfonamides. SLE was observed to occur
predominantly
in slowacetylator
where slow metabolism of one or more unknowndietary
orenvironmental substances over many years was believed to
provoke
the disease.1.2.5 Isoniazid
(INH)
INH was first
synthesized
in 1912by Meyer
andMolly
but its antituberculous
properties
were notfound until 40 years laterwhen Robitzek et al.(1952)
tested INH in 92patients
whom he referred to as"mortally
illpatients"
with extensive
pulmonary
TB(Evans, 1989). They
obtainedtherapeutic
benefitsbeyond anything they
had ever seen with anychemotherapeutic agents
everutilized
by
them.INH is a
primary drug
used forfirst line treatment ofTS, usually
incombination with other anti-TB
drugs.
It is also used alone forprophylaxis
of TS.It is available for oral and
parenteral
administration. Thecommonly
useddaily
dose for INH is 5
mglkg
to a maximum of 300 mg(Petri, 2001)
. It ishighly
selective for
mycobacteria
where the minimal tuberculostatic concentration is 0.025to 0.05!-Ig/ml compared
to concentrations in excess of 500!-Ig/ml required
to inhibit the
growth
ofotherorganisms (Petri, 2001).
It is bacteriostatic for"resting"
bacilli but is bactericidal forrapidly dividing microorganisms
and exertsits effect
by inhibiting biosynthesis
ofmycolic acids,
animportant
constituent ofmycobacterial
cell wall.When taken either
orally
orparenterally,
INH wasrapidly
andcompletely
absorbed with
peak plasma
levels attained at 1 to 2 hours afteringestion
andwith a half life of4 hours
(Petri, 2001,
Weber andHein, 1979). Absorption
occurs
mainly
in the small intestine. Itsabsorption
is reducedby
antacids. It is notprotein
bound and metabolized in the liverby
NAT2 enzyme. It iswidely
distributed both
intra-cellularly
andextra-cellularly
wheresignificant
amounts ofthe
drug
were detectable incerebrospinal fluid,
caseousmaterial, pleural (Petri, 2001,
Weber andHein, 1979),
asceticfluids, meninges,
saliva and pus(Ellard
and
Gammon, 1977).
This makes INH animportant
TBdrug
since it caneasily penetrate
the caseous materials and other cells to exert its effect in TB infection that may not be reachedby
other anti-TBdrugs.
INH also has some otheradvantages.
Itsactivity
is not affectedby
variations inpH
overthe range of 5.0 to8.0,
itreadily
diffuses intomacrophages
and it is effectiveagainst
intracellularas well as extra cellular bacilli
(Ellard
andGammon, 1977).
INH is excreted via thekidneys
either as freedrugs
or as metabolites in an excretion process thatwas
independent
of renal function(Weber
andHein, 1979).
In combination
therapies
with anti-TBdrugs during
the first 2days
oftreatment termed as
early
bactericidalactivity,
INH has thegreatest killing
rateamong all the anti-TB
drugs including rifampicin
and itsactivity
is not affectedby
other
drugs given concurrently (Jindani
etal., 2003).
The metabolism of INH is among the first to be described as
being polymorphic.
Aclearly
bimodal distribution ofplasma
elimination half livesdistinguished
individuals asphenotypically "rapid"
and "slow" inactivator of thedrug (Evans
etal., 1960)
andlater,
Parkin et al.(1997) reported
that INHelimination distribution was
actually
trimodal .Due to the
polymorphic
nature of itsmetabolism,
INH 'dose-related' adverse effects vary among individuals. The list of adverse effects isgiven
inAppendix
3. Elevated liver enzymes arefrequently reported,
however overtclinical
hepatitis
withsymptoms
such asgastrointestinal distress,
nausea,vomiting
andjaundice
occurs in less than 5% ofpatients.
Alcoholconsumption,
advanced age,
acetylator
status andexisting
chronic liver disease have beenreported
to increase risk of anti-TBdrug hepatitis (Huang
etal., 2003).
Itsoccurrence may also differ in
frequencies
in differentpopulations,
a differencethatwas
thought
to be contributedby
thegenetic polymorphisms
of themetabolizing
enzyme(Huang
etal..2002).
INH may also cause
neurotoxicity,
characterizedby generalized seizures.
coma and metabolic
acidosis,
that isusually
associated with doses in excess of100mg/kg
ofbody weight.
Dvorsek et al.(2000)
howeverreported
acute INHneurotoxicity during preventive therapy
where there was no evidence of INH overdose.Therapeutic
doses of INH may alsooccasionally precipitate
convulsions in
patients
with knownepilepsy
orinpatients
with sub-clinicalpyridoxine deficiency,
as itoccurs in pregnancy, cancer,uremia, alcoholism,
chronic liver disease and in advanced age(Martinjak-Dvorsek
etal., 2000).
Pyridoxine (15
to 50mg/day)
should be administered with INH to minimize adversedrug
reactions in malnourishedpatients
and thosepredisposed
toneuropathy
such as inelderly, pregnant
women, HIV infectedindividuals, diabetics,
alcoholics and uremicpatients (Petri, 2001).
INH is also known to cause asensory-dominant peripheral neuropathy.
Yamamoto et al.(1996)
demonstrated that
patients
whodeveloped
INHneuropathy
were all slowacetylator genotype.
NAT2
activity
is not known to be inducible or inhibited. INH on the otherhand may inhibit the
activity
ofCYP2C19, CYP206,
CYP3A4 and CYP2E1(Desta
etaI., 2001).
Some studiessuggested
thatmonoacetylhydrazine
whichis a metabolite of INH may
antagonize
the anti-TBactivity
of INH but othermetabolites such as
Act-INH,
isonicotinic acid anddiacetylhydrazine
were notinhibitory (Weber
andHein, 1979).
INHacetylation
may be reduced when there is concomitant treatment with otherdrugs
metabolizedby
NAT2. Such occurred withprocainamide
where INH half-life wasslightly
butclinically insignificantly
prolonged (Weber
andHein, 1979).
Insulin causesinteresting
alterations in INHpharmacokinetics (Weber
andHein, 1979).
Itwas found to enhance theintestinal
uptake
of INH and increased concentrations of INH in thelungs
andthe liver. Maximum concentrations of the
drug
observed in thekidneys
and thebrain was however reduced. There were also