1 ASSOCIATION OF XENOBIOTIC METABOLIZING ENZYMES GENE POLYMORPHISM WITH HEPATOCELLULAR CARCINOMA IN EGYPTIAN PATIENTS Manar Obada1, Ashraf El-Fert1,… [602257]

1 ASSOCIATION OF XENOBIOTIC METABOLIZING ENZYMES
GENE POLYMORPHISM WITH HEPATOCELLULAR
CARCINOMA IN EGYPTIAN PATIENTS
Manar Obada1, Ashraf El-Fert1, Asmaa Gomaa2, Mohamed Hashim2, ,
Mohamed Kohla2 ,Wael Abdelrazek2, Om kolsoum Elhadad2 Hala El-Said1
Clinical Biochemistry1, Hepatology2 Departments,
National Liver Institute, Menoufia University

ABSTRACT
Background and objective: Xenobiotics are metabolized by a large
number of metabolizing enzymes, Genetic polymorphism of their
genes were suggested as modifier s of cancer risk. The present study
aimed to investigate the association between xenobiotic metabolizing
enzymes [cytochrome P450 (CYP), N-acetyl transferase 2 (NAT2)
and UDP-glucuronosyl transferase (UGT)] gene polymorphism with
the risk of HCC in patients with chronic HCV-induced cirrhosis.
Methods: this study was performed on 354 subjects, divided into 3
groups, (group I: 150 hepatocellular carcinoma (HCC) patients, group
II: 104 patients with HCV-related chronic liver disease (CLD) and
group III: 100 apparently healthy c ontrol). The studied genes were
genotyped using polymerase chain reac tion-restriction fragment length
polymorphism and allelic discrimination assays.
Results: Genetic polymorphic patterns of NAT2 (M1 and M3), CYP2D
6*6, CYP2D 6*4 and CYP2D 6*3 showed a significant difference in
HCC group compared to other gr oups. NAT2 M2 slow acetylator,
CYP2D6*6 and CYP2D6*3 poor meta bolizers and CYP2D 6*4 rapid
metabolizer were associated with increased HCC risk (OR: 1.23, 4.0,
3.32 and 2.3 respectively).
Conclusion: Increased risk for hepatocellular carcinoma in Egyptian
patients infected with HCV may be associated with the genotypes:
NAT2 (M2), CYP2D 6*6, CYP2D 6*4 and CYP2D6*3 and thus
could help in tailoring individuali zed therapy and serve as potential
target sites for chemotherapy.
Key words: polymorphism, xenobiotic, metabolizing enzymes, genes,
hepatocellular carcinoma.
Corresponding author: [anonimizat]

2 INTRODUCTION
Hepatocellular carcinoma (HCC) is the second leading cause
of cancer-related death worldwide, and its incidence is increasing
(WHO, 2014). Chronic hepatitis C (CHC) vi rus infection is the most
frequent cause of progressive liver disease and liver cancer in Egypt
(El-Zayadi et al., 2005). A decrease in carcinogen metabolism and an
increase in procarcinogen activation have also been documented as
HCC risk factors (Fontham et al., 2009).
Detoxification and elimination of drugs, environmentally
relevant chemicals and endogenous metabolites is a major function of
the liver and important in maintaining the metabolic homeostasis of
the organism. Xenobiotics are metabolized by a large number of
xenobiotic metabolizing enzymes which fall into three broad
categories: phase I, phase II and phase III (Francis et al., 2003).
The major enzymes of phase I metabolism are heme thiolate
proteins of the cytochrome P450 superfamily (CYPs). Phase I
enzymes generate functional groups that may subsequently serve as a
site for conjugation cataly zed by phase II enzymes UDP-
glucuronosyltransferases (UGT), Sulfotransferases (SULT),
Glutathione S transferases (GST), and N-acetyl transferases (NAT)
(Xu et al., 2005) .
CYP2D6 is the most extens ively studied polymorphically
expressed drug metabolizing enzyme in humans As a result of the presence of more than 70 allelic variants of CYP2D6 Gene (Scordo et
al., 2004), metabolism and excretion rates of drugs vary between
individuals, from extrem ely slow to ultra-fast. (Ingelman-Sundberg,
2005)
N acetyl transferase-2 (NAT-2) is a polymorphic enzyme
involved in the activation and deactivation of aromatic and
heterocyclic amines. Polymorphisms of the NAT2 gene result in slow
and rapid acetylators of potentially toxic substances (Hirvonen,
1999).
Human UDP-glucuronosyltransferases (UGTs) can catalyze
the conjugation of hydrophobic com pounds of divergent chemical
classes to form water soluble -D -glucopyranosiduronic acids. These
metabolites then undergo renal or biliary elimination from the body
(Fang et al., 2002).

3 The aim of this work was to inve stigate the association between the
xenobiotic metabolizing enzymes (Cytochrome P450, N-acetyl
transferase 2 (NAT2) and UDP-glu curonosyl transferase (UGT))
genes polymorphism with the risk of HCV related HCC in Egyptian
patients compared to CHC patients and normal subjects.
MATERIAL AND METHODS
This study was conducted at Na tional Liver Institute, Menoufia
University in the period from 2011-2014. 354 subjects were enrolled
in this study and divided into 3 groups. Group I: 150 patients with
HCV-related HCC. Diagnosis of HCC was based on non-invasive
criteria using multi slice triphasic CT or contrast enhanced dynamic
MRI. The presence of typical features of arterial enhancement and
rapid portal or delayed washout on one imaging technique was
diagnostic of HCC for nodules >2cm in diameter in cirrhotic patients.
In cases of uncertainty or atypical radiological findings, diagnosis was
confirmed by a biopsy (Bruix and Sherman, 2005). Group II: 104
patients with chronic hepatitis C in fection (CHC); Diagnosis of CHC
was based on the presence of posit ive anti-HCV antibody and/or HCV
RNA. Diagnosis of liv er cirrhosis was based on ultrasonographical
findings (shrunken liver, coarse echo pattern, attenuated hepatic vein
and nodular surface) and biochemi cal evidence of parenchymal
damage (Tsai et al., 1994). Group III: 100 apparen tly healthy matched
subjects serving as a co ntrol group, with normal liver and renal tests
and negative for HBs antigen and HCV antibody. Written informed
consents were taken from all participants and the protocol was
approved by the Institution’s ethics committee. HCC patients were
followed up during the study period.
Laboratory Investigations
Basic laboratory investigations were done for all participants;
liver tests, CBC, serum HBs Ag an d Anti-HCV antibodies and serum
AFP using fully automated auto-analyzer SYNCHRON CX9ALX
(Beckman Coulter Inc., CA, US A), Sysmex K-21, (Sysmex
Corporation, Kobe, Japan), an d immunoassay (Abbott Laboratories,
Abbott Park, IL, USA), Automated IMMULITE® 1000 immunoassay

4 analyzer (Siemens Medical Solu tions Diagnostics Corporation,
Erlangen, Germany) respectively.
Genotyping
Genomic DNA was extracted from EDTA-treated whole blood
using Gene JET Whole Blood Genomi c DNA Purification Mini Kit,
(Thermo Fisher Scientific, MA USA). All primers used in this study
were synthesized at Metabion intern ational AG, Martinsried, Germany.
I. NAT 2
Genotyping of NAT2*5(Ml a llele) (C481T) rs1799929, NAT2*6
(M2 allele) (G590A) rs1799930, and NAT2*7 (M3 allele) (G857A)
rs1799931 was carried out as described by Bell et al., (1993) , with
modification. NAT2 genotyping wa s performed by PCR-RFLP. PCR
was carried out using PCR primers (N5, 5'-
GGAACAAATTGGACTTGG-3’; N4, 5'-
TCTAGCATGAATCACTCTGC-3’) Dream Taq Green PCR Master
Mix (Thermo Fisher Scientific, MA, USA) was utilized in the
amplification. The cycling conditio ns consist of 94°C for 3 min,
followed by 35 cycles of 94°C for 30 s, 52°C for 40 s and 72°C for 90
s, with a final extension of 72°C for 5 min. Following PCR, the
amplicon was subjected to restricti on digest with Kpnl (Ml allele),
TaqI (M2 allele), BamHI (M3 allele) restriction enzymes from
(Thermo Fisher Scientific, MA, US A). The restriction digests were
electrophoresed on 3.5% agarose gels.
II. UGT1A7
a. UGT1A7 [codon 208 (nucleotide T622C) rs11692021]
Genotyping of UGT1A7 (W208R) was carried out as described by
Tseng et al. (2005) by PCR-RF LP. The primers U7F3 5’-
TGTCCCCAGACTTCTCTTAG- 3’ and U7R3 5’-
GCTACCCAACAATTAAGTGA-3’ were used to amplify the
specific UGT1A7 fragments that cover nucleotide 622. For the
amplification, Dream Taq Green PCR Master Mix (Thermo Fisher
Scientific, MA, USA) was utilized. The cycling conditions consist of
94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 52°C for 30 s
an and 72°C for 60 s, with a fina l extension of 72°C for 5 min. This
PCR yielded a 447bp fragment, which was digested with restriction

5 enzyme RsaI (Thermo Fisher Scie ntific, MA, USA). The restriction
digests were electrophoresed on 2% agarose gels.
UGT1A7 [codon 129 (nucleotide T387G) rs17868323]
The single nucleotide polymo rphism of UGT1A7 (N129K) was
detected by using TaqMan al lelic discrimination Assay.
Primers and probes used were: N129K-Forward: 5'-
CACCATTGCGAAGTGCATTT-3'; N129K-Reverse 5'-
GGATCGAGAAACACTGCATCAA-3 '; Probe FAM-N129 5'-
CAGGAGTTTGTTTAATGAC-3'; Probe VIC-K129 5'-
CAGGAGTTTGTTTAAGGAC-3'.
The presence of two primer/probe pairs in each reaction allows
genotyping of the two possible va riants at the single-nucleic
polymorphism site in a target template sequence. This allows the
classification of unknown samples as: homozygote (samples having
only allele 1 or allele 2) or hetero zygote (samples having both allele 1
and allele 2). The genotyping re action mix was prepared using
Maxima Probe qPCR Master Mix (T hermo Fisher Scientific, MA,
USA). The cycling conditions consiste d of initial dena turation step at
940C for 4 minutes, followed by 50 cycl es of denaturation at 940C for
30 seconds, annealing/extension /f luorescence acquisition at 600C for
30 seconds; Using LINE GENE 9660 (BIOER Technology,
Hangzhou, China).
III. Cytochrome P450 (CYP) Gene Polymorphisms
The allelic discrimination of the CYP2D6*3, CYP2D6*4,
CYP2D6*6 polymorphisms were asse ssed with the ABI 7500F Real-
Time PCR Instrument (Applied Bios ystems, Inc. (ABI) Foster City,
CA) using TaqMan assay. The TaqMan Assay-on-Demand reagents
available for CYP2D6*3 2549lAdel where deletion of base A
(rs35742686) [assay ID C-32407232- 50], CYP2D6*4 G1846A
(rs3892097) [assay ID C-27102431-D0], CYP2D6*6 1707Tdel where
deletion of base T occurs (rs5030655) [assay ID C-32407243-20].
TaqMan Universal PCR Master Mix was used for analysis. The PCR
profile consisted of an initial dena turation step at 95°C for 10 minutes
and 40 cycles of 92°C for 15 seco nds and 60°C for 1 minute and
fluorescence acquisition.

Statistic a
Data
Inc., Ch i
studied g
Hardy– W
frequenc y
allele.
The m
were ma
were ma l
were ma l
Table 1.
Fig (1
A. N
M1 ( C
muta n
gener a
a sin g
genot y
B. N
M2 ( G
and 4 3
Lane s
show e
al anal ysis
were statist i
icago, IL, U
genes were c
Weinberg e q
y of the wil d
mean age fo r
les, for CH
les and for h
le. Tumor c
) Representa t
NAT2*5(Ml a l
C) wild typ e
nt type, yiel d
ates two ban d
gle band of 1 0
ypes. M3 all e
NAT2*6 (M2)
G) wild type
33bp, where a
s 2 and 4 sh
ed GA genot y
ically analy z
United Stat e
compared w i
quilibrium ( p
d-type allel e
RE
r HCC patie n
C patients;
healthy subj e
characteristi c
tive example s
llele C481T) a
e shows two
ds a single
ds of 811 an d
093 bp. M1 a
ele in lanes 2
allele G590 A
generated ba n
as (A) mutan t
owed undig e
ype while lan e
6 zed using S
es) The ge n
ith predicta b
p2+ 2pq+q 2
e and q is th e
ESULTS
nts was 54. 8
it was 46. 0
ects it was 4
cs of the H C
s of different
and NAT2*7
bands of 6 6
band of 10 9
d 282bp, whe
allele in lane s
(GG), 3(AA)
A:
nds 380, 317
t type, yields
ested sample s
e 5 showed A
SPSS Versi o
notype freq u
ble values c a
2 = 1); w h
e frequency
84 ± 8.90 ye a
02 ± 6.69 y e
43.2 ± 5.72 y
CC group w
NAT2* gen o
(M3 allele G
60 and 433b p
93 bp. M3
reas (A) mut
s 1 (CT), 4 ( C
and 5 (GG) g
, 226, and 1 7
396, 380, an d
s of 1093 bp ,
AA genotype.
on 17.0 (SP S
uencies of t h
alculated fr o
here p is t h
of the vari a
ars and 79.3
ears and 7 5
years and 7 6
were shown
otypes:
857A )
p, whereas (
(G) wild t y
ant type, yie l
CT) and 6 ( T
genotypes.
70bp bands 6
d 317bp band
, lanes 1 an d

SS
he
om
he
ant
%
5%
6%
in

(T)
ype
lds
TT)
60
ds.
d 3

7 
Fig (2) Representative examples of UGT1A7 [codon 208 ]
UGT (T) wild type yielded a single band of 447bp, whereas the
(C) mutant type generates tw o bands of 393 and 54bp. Lanes 1,2,4,6,
and 8 showed the TT genotype, while lanes 3, 5 and 7 showed TC
genotype.
The samples were electrophoresed against100bp DNA ladder (M)
(100bp Gene ruler DNA Ladder, Thermo Scientific Inc., USA. The
land mark band of the ladde r is corresponds to 500bp).
There was a significant differenc e between the studied groups
regarding genotypes of NAT2 (M1 and M3) and CYP2D 6*6, CYP2D
6*4 and CYP2D6*3 genes. Meanwhile, No significant difference was
observed regarding genotypes of UGT gene (Table 2) .
No significant difference between various genotypes of the studied
genes regarding pathological criteria and basic laboratory parameters of
HCC patients except for NAT2 M2 AA (slow acetylator) which showed
lower albumin level compared to ot her genotypes (rap id acetylators)
and for CYP2D6*3 Mut/Mut (poor me tabolizer) which had lower PC%
compared to other genotypes (rapi d metabolizer) (data not shown).
Mutant alleles of NAT2 M1, M2 and M3 (slow acetylators) and
NAT2 M2 (AA) genotype showed incr eased risk of HCC, where ORs
were 1.29, 1.67, 1.99 and 1.23 respectively. Meanwhile, NAT2 M1, M2
and M3 homozygous wild genotypes showed significantly decreased
risk of HCC ( Table 3)
CYP2D6*6 and CYP2D6*3 mutant alleles and mutant genotypes
(poor metabolizers) were associated with increased HCC risk with
ORs of 2.9 and 2.73 respectively fo r mutant alleles, 4.0 and 3.32
respectively for poor metabolizers, but wild genotypes showed

8 decreased risk of HCC. For CYP2 D6*4; mutant allele A and poor
metabolizer genotype (AA) showed decreased HCC risk with ORs
0.55 and 0.16 respectively, while wild genotype (GG) showed
increased risk of HCC with OR 2.3. ( Table 4)
HCC patients were followed wher e, 71 patients received medical
treatment, 4 patients underwent tumor resect ion, 1 patient underwent
tumor resection and transcatheter ar terial chemoembolization (TACE),
32 patients received radiofreque ncy ablation (RFA), 38 underwent
TACE, 3 underwent RFA and TACE and one patient had microwave
treatment. The outcome of the patients were as follows; 63 patients were
still alive, 51 patients died and 36 did not comply with the follow up
schedule. The survival rate was ca lculated using the data of 114 HCC
patients as 36 censored and their outcome was unknown, the median
survival of the involved HCC patient s was 20 months. The survival rate
was not associated with the stud ied genotype patterns in HCC group
(data not shown).
Table (1) Tumor characteristics of group I (HCC patients)
Variable Value
Tumor diameter (cm), mean ± SD 4.9 ± 2.8
TTV (cm3), median (IQR) 33.49 (0.52 – 8177.1)
AFP, mean± SD 117.2 ± 105.5
Ascites, n (%)
absent/ Moderate/Tense 83 (55.3), 50(33.3), 17(11.3)
Lymph node invasion, n (%) 8 (5)
Vascular invasion, n (%) 16 (10.6)
Child Pugh Class, n (%)
A/B/C 72 (48)/ 56 (37.3)/ 22 (14.6)
BCLC stage, n (%)
A/B/C/D 90 (60)/ 20 (13.3)/ 19 (12.6)/ 21 (14)
Number of tumor nodules, n (%)
Solitary/ 2 nodules/ ≥3 nodules 116 (77.4)/32 (21.3)/ 2 (1.3)
Affected lobe(s), n (%)
Right lobe/ Left lobe/ Both lobes 94 (62.7)/ 30 (20)/ 26 (17.3)

9 TTV: total tumor volume , AFP: alpha fetoprotein, BCLC: Barcelona Clinic
Liver Cancer, IQR: interquartile range, SD: standard deviation.
Table (2) Polymorphic patterns of the studied genes among the
different groups
Genotypes Group I
n=150 (%) Group II
n=104 (%) Group III
n=100(%) P
NAT (M1) CC 29 (19.3)a 34 (32.7) 24 (24)
0.036* TT 38 (25.3) 27 (26) 17 (17)
CT 83 (55.3) 43 (41.3)b 59 (59)
NAT2 (M2) GG 63 (42) 60 (57.7) 57 (57)
0.052 AA 24 (16) 11 (10.6) 8 (8)
GA 63 (42) 33 (31.7) 35 (35)
NAT2 (M3) GG 96 (64)ab 82 (78.8) 80 (80)
0.032* AA 9 (6) 3 (2.9) 3 (3)
GA 45 (30) 19 (18.3) 17 (17)
UGT1A7
(codon208) TT 42 (28) 37 (35.6) 35 (35)
0.713
CC 23 (15.3) 14 (13.5) 14 (14)
TC 85 (56.7) 53 (51) 51 (51)
UGT1A7
(codon129) TT 44 (29.3) 18 (17.3) 21 (21)
0.054
GG 55 (36.7) 33 (31.7) 39 (39)
TG 51 (34) 53 (51) 40 (40)
CYP2D6*6 Wt/Wt 1 (0.7)b 4 (3.8)b 27 (27)
<0.0001* Mut/Mu t 84 (56)ab 33 (31.7)b 16 (16)
Wt/Mu t 65 (43.3)a 67 (64.4) 57 (57)
CYP2D6*4 GG 121 (80.7)b 74 (71.2) 57 (57)
<0.0001* AA 17 (11.3) 17 (16.3) 11 (11)
GA 12 (8)b 13 (12.5) 32 (32)a

10 CYP2D6*3 Wt/Wt 3 (2)b 7 (6.7) 17 (17)
<0.0001* Mut/Mu t 99 (66)ab 41 (39.4) 34 (34)
Wt/Mu t 48 (32)ab 56 (53.8) 49 (49)
Data are n (%). aP<0.05 compared to group II, bP<0.05 compared to group
III, *p<0.05: significant, p>0.05: non-significant.
Table (3) frequency of NAT2 alleles and genotypes in group I (HCC
patients) and non-HCC controls (group II & III) and associated HCC risk
Frequency Group I
(no=150)
No (%) Controls
(no=204)
No (%) p-value OR (95% CI)
NAT2 M1
T mutant allele C wild allele 159 (53)
141 (47) 190 (46.6)
218 (53.4) 0.045* 1.29 (0.95-1.74)
NAT2 M1
Dominant model
CC CT + TT 29 (19.3)
121(80.7) 58 (28.4)
146 (71.6) 0.024* 0.06(0.36-0.99)
NAT2 M1
Recessive model
TT
CT + CC 38 (25.3) 11
(74.7) 44 (21.6)
160 (78.4) 0.21 1.23 (0.74-2.03)
NAT2 M2
A mutant allele
G wild allele 111 (37)
189 (63) 106 (26)
302 (74) 0.0008* 1.67 (1.21-2.3)
NAT2 M2
Dominant model
GG GA + AA 63 (42)
87 (58) 117 (57.4)
87 (42.6) 0.0002* 0.54 (0.35-0.83)
NAT2 M2
Recessive model
AA
GA + GG 24 (16)
126 (84) 19 (9.3)
185 (90.6) 0.03* 1.85 (0.97-3.56)
NAT2 M3
A mutant allele
G wild allele 63 (21)
237 (79) 48 (11.8)
360 (88.2)
0.0004*
1.99 (1.32-3.01)

11 NAT2 M3
Dominant model
GG GA + AA 96 (64)
54 (36) 162 (79.4)
42 (20.6) 0.0007* 0.46 (0.28-0.74)
NAT2 M3
Recessive model
AA
GA + GG 9 (6)
141 (94) 6 (2.9)
198 (97.1) 0.86 2.1 (0.73-6.05)
* p<0.05: significant, p>0.05: non-significant
Table (4) frequency of CYP6* alleles and genotypes in group I (HCC
patients) and non-HCC controls (group II & III) and associated HCC risk
Frequency Group I
(no=150)
No (%) Controls
(no=204)
No (%) p-value OR (95% CI)
CYP2D6*6
Mutant allele Wild allele
233(77.7) 67(22.3)
222 (54.4) 186 (45.6) <0.00001* 2.9 (2.09-4.1)
CYP2D6*6
Dominant Model
W/W
W/M + M/M
1 (0.7)
149 (99.3)
31(15.2)
173 (84.8)
<0.00001* 0.037 (0.002-
0.2)
CYP2D6*6
Recessive Model
M/M
W/M + W/W
84 (56%)
66 (44)
49 (24)
155 (76)
<0.00001* 4.0 (2.5 6.4)
CYP2D6*4
A mutant allele G wild allele
46 (15.3) 254 (84.7)
101 (24.8) 307 (75.2)
<0.001* 0.55 (0.37-0.8)
CYP2D6*4
Dominant Model
GG
GA + AA
121 (80.7)
29 (19.3)
131(64.2)
73(35.8) 0.0003* 2.3 (1.42-3.85)
CYP2D6*4
Recessive Model
AA
GA + GG
17 (11.3)
133 (88.7)
88 (43.1)
116 (56.9)
<0.00001*
0.16 (0.09- 0.29)
CYP2D6*3
Mutant allele Wild allele
246 (82) 54 (18)
255(62.5) 153(37.5)
<0.00001* 2.73 (1.9 3.9)

12 CYP2D6*3
Dominant Model
W/W
W/M + M/M
3 (2)
147 (98)
24 (11.8)
180 (88.2)
0.0003*
0.15(0.036-0.47)
CYP2D6*3
Recessive Model
M/M
W/M + W/W
99 (66)
51 (34)
75 (36.7)
129 (63.2)
<0.00001*
3.32 (2.1-5.2)
* p<0.05: significant,
DISCUSSION
One of the many risk factors of HCC is the patient’s capability to
metabolize xenobiotics, because so me xenobiotics play a role in
inducing cancer as procarcinogen or carcinogen (Farker et al., 2003).
N-acetyltransferase (NAT2) is involved in the metabolic
activation and detoxification of arom atic amines, which are potential
hepatocarcinogens in humans. Gene tic polymorphism is associated
with slow acetylation which predis poses to HCC development because
of decreased deactivation of aromatic amines (Chang-Claude et al.,
2002) .
The current study augmented these findings as there were
increased frequencies of NAT2 M1, M2 and M3 mutant alleles (slow
acetylators) and NAT 2 M2 slow acet ylator (AA) in HCC compared to
other groups, also they were associated with increased risk of HCC
compared to rapid acetylators alle les and genotypes. These findings
are consistent with those of Agundez et al. (1995), and Khalaf et al.,
(2012). They suggested that the NAT2 enzyme behaves as an
inactivating enzyme of carcinogens and plays a protective role in
averting the development of HCC.
However, other studies have shown that rapid acetylators are at
a higher risk of developing HCC (Huang et al., 2003) based on the
hypothesis that the risk of cancer fo r rapid acetylators is due to the
activation of procarcinogens such as heterocyclic amines.
This discrepancy over the role of NAT2 polymorphism in
HCC may be explained by the fact that NAT2 is involved in different
metabolic pathways of various aromatic amines (Gelatti et al., 2005).

13 Also, exposure to different types of carcinogens varies from one
region to another.
CYP2D6 is perhaps the most extensively studied
polymorphically expressed drug metabolizing enzyme in humans and
its polymorphism has a high clini cal importance. There are four
polymorphism-related phenotypes; poor (PM), intermediate (IM),
extensive (EM), or ultrarapid metabolizers (UM) (Ingelman-
Sundberg, 2005).
The current study detected that the frequency of mutant
genotypes (poor metabolizers) of CYP2D 6*6 and of CYP2D6*3
showed significant elevation in HCC group compared to other groups,
and were significantly associated with increased risk of HCC.
These findings were in agreement with the study of Silvestri et
al. (2003) . The authors reported that CYP2D6 genotypes have an
effect on liver disease progression as shown by the distribution of
different genotypes according to th e severity of liver lesions.
G to A (G1934A) transition (CYP2D6*4 allele) at the
intron3/exon4 boundary of the CYP2D 6*4 gene leads to incorrect
splicing of mRNA resulting in a frame shift and premature termination
(Surekha et al ., 2010). This mutation result in decreased or lack of
CYP2D6 isoenzyme activity, lead ing to poor metabolizer (PM)
phenotype (Van Der Weide and Steijns, 1999).
In the present study, the fre quency of poor metabolizer
genotype of CYP2D6*4 (A/A) s howed no significant difference
between HCC patients, CHC patients and controls. The extensive
metabolizer (G/G) genotype was sign ificantly increased in HCC group
compared to the healthy subjects only (80.75 vs, 57%), and was
significantly associated with increas ed risk of HCC. However, the
poor metabolizer A/A genotype was associated with significantly
decreased HCC risk. These observa tions were in consistence with
other studies; Kimura et al . (2005), Mochizuki et al . (2005) and
Sayed and Imam, (2012).
Several studies have shown th at the extensive metabolizer
phenotype is associated with increased risk of various cancers
(Raunio et al., 1995), and reported that; th e CYP2D6*4 may mediate

14 the activation of procar cinogenic agents present in environment and
the CYP2D6*4 gene might be in an association dise quilibrium with
the contributing genes.
UGT1A7 plays a critical role in the detoxification of
carcinogens. Specifically, it wa s shown previously that
polymorphisms in codons 129, 131, or 208 markedly decreased the
carcinogen detoxification activity of UGT1A7 (Strassburg et al .,
2002).
Our results demonstrated that no significant difference was
detected between HCC, CLD patients and controls regarding the
studied genotypes of UGT, also polymorphic pattern of UGT did not
associate with susceptibility to HCC. These results agreed with study
of Stücker et al., (2007).
On the contrary , Wang et al ., (2004) found that UGT1A7
alleles with lower activity were associated with HCC risk; UGT1A7
gene plays a critical role in the de toxification of hepatocarcinogens at
the epithelia of the lung and gastrointestinal tract, which are thought to
be entry sites for mutagens. Alte rnatively, there may be an unknown
gene that is directly associated w ith HCC and that acts together with
the UGT1A7 polymorphisms, or perhaps an unknown function of
UGT1A7 affects the liver through actions at a di fferent site.
CONCLUSIONS
Increased risk for liver cancer in Egyptian patients infected
with HCV may be associated with the genotypes: NAT2 (M2), CYP2D 6*6, CYP2D 6*4 and CYP2D 6*3 and thus could help in
tailoring individualized th erapy and serve as pote ntial target sites for
chemotherapy.
ACKNOWLEDGEMENT and FI NANCIAL DISCLOSURE
The research was fully funded by a National Grant from Science
and Technology development Fund (STDF), Egypt.
None of the contributing authors have any conflicts of interest.

REFERENCES
WHO: International Agency for Research on Cancer (2014): PRESS
RELEASE N° 224. Feb 3; Lyon/ London. Available from:

15 URL:http://www.iarc.fr/en/me diacentre/pr/2014/pdfs/pr224_E.pdf.
Accessed December 5, 2015
El-Zayadi AR1, Badran HM, Barakat EM, et al. (2005):
Hepatocellular carcinoma in Egypt : a single center study over a
decade. World J Gastroenterol. Sep 7; 11(33):5193-8.
Fontham ET, Thun MJ, Ward E, et al. (2009): Cancer ACS and the
Environment S. American Cancer Society perspectives on
environmental factors and cancer. CA Cancer J Clin.; 59:343-351.
Francis GA, Fayard E, Picard F and Auwerx J (2003) : Nuclear
receptors and the control of metabolism. Annu Rev Physiol 65:
261–311.
Xu C, Li CY and Kong AN (20050 ): Induction of phase I, II and III
drug metabolism/transport by xe nobiotics. Arch Pharm Res;
28:249-68.
Scordo MA, Caputi AP , D’Arrigo C, et al. (2004): Allele and
genotype frequencies of CYP2C9, CYP2C19 and CYP2D6 in an
Italian population. Pharmacol. Res.; 50:195-200.
Ingelman-Sundberg M. (2005): Genetic polymorphisms of
cytochrome P450 2D6 (CYP2D6) : clinical consequences,
evolutionary aspects and functiona l diversity. Pharmacogenomics
J.; 5:6-13.
Hirvonen A. (1999): Polymorphisms of xenobiotic-metabolizing
enzymes and susceptibility to cance r. Environ Health Perspect. ;
107 Suppl 1:37-47.
Fang JL, Beland FA, Doerge DR, et al. (2002): Characterization of
benzo(α)pyrene-trans-7,8-dihydrodio l glucuronidation by human
tissue microsomes and over-expressed UDP-
glucuronosyltransferase enzy mes. Cancer Res; 62:1978–86.
Bruix, J., and Sherman, M. (2005): Management of hepatocellular
carcinoma, Hepatology; 42 (5): 1208-1236.
Tsai J F, Chang W Y, Jeng J E, et al. (1994): Hepatitis B and C virus
infection as risk factors for liver cirrhosis and cirrhotic
hepatocellular carcinoma: a case- control study, Liver; 14 (2): 98-
9102.

16 Bell DA, Taylor JA, Butler MA, et al. (1993): Genotype/phenotype
discordance for human arylamine N-acetyltransferase (NAT2) reveals a new slow-acetylator allele common in African- Americans. Carcinogenesis; 14:1689–92.

Farker K, Schotte U, Scheele J and Hoffmann A (2003): Impact of
N-acetyltransferase polymorphism (NAT2 in hepatocellular
carcinoma (HCC) – an investigation in a department of surgical
medicine. Experimental and Toxi cologic Pathology; 54 (5–6): 387–
391.
Chang-Claude J, Kropp S, Jager B, et al. (2002): Differential effect
of NAT2 on the association be tween active and passive smoke
exposure and breast cancer risk. Cancer Epidemiol Biomarkers
Prev; 11: 698–704.
Agundez JA, Ledesma MC, Benitez J, et al. (1995): CYP2D6 genes
and risk of liver cancer. Lancet; 345:830–1.
Khalaf F , Basuni A, El Daly M, et al. (2012): Genetic polymorphism
of N acetyltransferase 2 and its a ssociation with tumor markers and
cigarette smoking in hepatocellu lar carcinoma. The Egyptian
Journal of Biochemistry and Mo lecular Biology; 31(2): 119-142.
Huang YS1, Chern HD, Wu JC, et al. (2003): Polymorphism of the
N acetyltransferase 2 gene, red m eat intake, and the susceptibility
of hepatocellular carcinoma. Am J Gastroenterol. Jun; 98(6):
14117-22.
Gelatti U, Covolo L, Talamini R, et al. (2005): N-Acetyltransferase-2,
glutathione S-transferase M1 and T1genetic polymorphisms,
cigarette smoking and hepatocellular carcinoma: a case-control
study. Int J Cancer; 115: 301–6.
Silvestri L, Sonzogni L, De Silvestri A, et al. (2003): CYP enzyme
polymorphisms and susceptibility to HCV-related chronic liver
disease and liver cancer. In t J Cancer; 104: 310-317.
Surekha D, Sailaja K, Rao DN, et al. (2010): Association of a CYP17
gene polymorphism with developmen t of breast cancer in India.
Asian Pac J Cancer Prev.; 11(6):1653-7.
Van Der Weide J and Steijns L. (1999): Cytochrome P450 enzyme
system: genetic pharmacology. Ann. Clin. Biochem. 36:722-9.

17 Kimura Y, Selmi C, Leung PS, et al. (2005): Genetic polymorphisms
influencing xenobiotic metabolism a nd transport in patients with
primary biliary cirrhosis. Hepatology ; 41(1):55-63.
Mochizuki J, Murakami S, Sanjo A, et al. (2005): Genetic
polymorphisms of cytochrome P450 in patients with hepatitis C
virus-associated hepatocellular carcinoma. J Gastroenterol Hepatol.
; 20(8):1191-1197.
Sayed S and Imam H (2012): Study of the Associ ation of CYP2D6*4
Polymorphism with the Susceptibility of HCV- Related Liver
Cirrhosis and Liver Cancer. Life Science Journal; 9(3): 1571.
Raunio H, Husgafvel-Pursiainen K, Anttila S, et al.(1995):
Diagnosis of polymorphisms in carcinogen-activating and
inactivating enzymes and cancer su sceptibility–A Review. Gene.
14; 159(1):113-21.
Strassburg CP, Vogel A, Kn eip S, et al. (2002): Polymorphisms of
the human UDP-glucuronosyltran sferase (UGT) 1A7 gene in
colorectal cancer. Gut; 50:851–6.
Stücker I, Loriot MA, N'koutchou G et al. (2007): UDP-
glucuronosyltransferase UGT1A7 ge neticpolymorphisms in hepato
cellular carcinoma: a differential impact according to seropositivity of HBV or HCV markers? BMC cancer. Nv 19; 7:214.
Wang Y, Kato N, Hoshida Y, et al. (2004): UDP-
glucuronosyltransferase 1A7 genetic polymorphisms are associated
with hepatocellular carcinoma in Ja panese patients with hepatitis C
virus infection. Clin Cancer Res; 10:2441– 6.

18  بين العالقة الجينية لجيناتالصور CYP, NAT and UDP االصابة خطر و
فى الكبد مرضىالبسرطان المصابين المصرين سىباإللتھ الفيروسى الكبدى اب
*عبادة منار*, الفرت أشرف**, جمعة أسماء**, ھاشم محمد**, عبدالرازق وائل**, كحلة محمد
** الحداد كلثوم أم*, السعيد ھالة
*االكلينيكية الحيوية الكيمياء قسم** – الكبد امراض قسم. القومى الكبد معھد – المنوفية جامعة

لمشتقات الغذائى التمثيل يتم xenobiotics يؤثر وقد األيضية، اإلنزيمات من كبير بعدد
الجيني األشكال تعدد لجيناتة االنزيمات ھذه االصابةعلى معدالت سرطانب الكبدية الخالية.
ھدفت ھذه الدراسة العالقة ايجاد الى لجينات الجينى التعدد بين P450 CYP،2
) NAT2 (و glucuronosyl transferase UDP- راالصابةوخط ب في الكبد سرطان
يعانون الذين المزمنالمرضى سى بفيروس االصابة من .
الطرق : على الدراسة ھذه أجريت 354 وشخص تقسيمھم، تم الى3 مجموعات،
و)األولى المجموعة: 150 الكبد سرطان) HCC (, الثانية المجموعة: 104 مرضى
المزمن سى الكبدى بااللتھاب( CHC) الثا لثةوالمجموعة: 100 من اصحاء كمجموعة
ضابطة . دراسةتم المتسلسل البلمرة تفاعل باستخدام الجينات القاطعة اإلنزيمات بإستخدام ثم
ان الدراسة اظھرت وقد من الوراثية األشكال NAT2 M1 وM3 ، CYP2D 6 * 6 ،
CYP2D 6 * 4 و CYP2D 6 * 3 مجموعة في كبيرا فرقا أظھرت HCC مع بالمقارنة
األخرى المجموعات . وأن NAT2 M2 ، CYP2D6 * 6 و CYP2D6 * 3
و CYP2D6*4 ارتبطت مع خطر بزيادة الكبداالصابة سرطان .
الخالصة : المصابين المصريين المرضى في الكبد سرطان خطر ببااللتھازيادة الكبدى
المزمن سى الفيروسى الوراثية األنماط مع: NAT2 (M2) ، CYP2D 6*6 ، CYP6 *
4 و CYP2D6 * 3 باعتبارھا العالج في تساعد أن يمكن وبالتالي من المستھدفة المواقع
الكيميائي للعالج .