Download Azza Ahmed Ibrahim Abo senna_GST paper

Association between glutathione S-transferase genotypes
and risk of acute leukemia
By
Azza Abo Senna, Howayda Kamal , Magda Zedan and *Dalal El- Gezery
Departement of clinical pathology , Banha and Alexandria* faculties of medicine
Abstract :
Glutathione S-transferases (GSTs) are enzymes involved in the detoxification of several
environmental mutagens, carcinogens and anticancer drugs. GST polymorphisms resulting in
decreased enzymatic activity have been associated with several types of tumors. Using PCR,
GSTT1 and GSTM1 genotypes were determined in 40 adults with acute leukemia and 20 age
and sex matched controls. In acute leukemia combined, a slightly higher proportion of cases
displayed the GSTT1 and GSTM1 null genotypes than controls , but not statistically significant .
The null genotype of GSTM1 occurred in 25(62.5%) and 9(45%) of their corresponding
controls (odds ratio (OR) 2.037, 95% confidence interval (CI) 0.686-6.052). 12(30%) of cases
carried GSTT1 null genotype and 4(20%) in controls (OR 1.714, 95% CI 0. 47-6.21). AML
showed the strongest association with GSTM1 null genotype, 15(75%) of AML cases were null
compared with 9(45%) in controls (OR 3.66, 95% CI 1.01-14.03), a slightly higher proportion
of AML cases (25%) displayed GSTT1 null genotype compared with controls (20%), although
the difference was not statistically significant (OR 1.33, 95% CI 0.3-5.926). In ALL 10(50%)
were null in GSTM1 yielding (OR 1.22, 95% CI 0.353-4.235) and 7(35%) of cases were null in
GSTT1 yielding (OR 2.154, 95% CI 0.516-9.00) , so no significant association was found
between either GSTM1 or GSTT1 null genotype and ALL. The interaction between both
genotypes was also studied , in AML increased risk estimates were observed when either allele
was null , it was found a strong association between AML and (GSTM1 null/ GSTT1 present)
genotype (OR 18, 95% CI 1.9-171.9) and also between AML and (GSTT1 null/GSTM1 present)
genotype (OR 18, 95% CI 1.25-260.9) p < 0.05. In ALL , there was no association between
ALL and both genotypes ( GSTM1 null/GSTT1 present) (OR 1.5, 95% CI 0.34-6.53) and
(GSTT1 null/GSTM1 present) (OR 3.0, 95% CI 0.41-21.89). A significant association was
found between smoking and genotype (GSTT1 null/GSTM1 present) (OR 18, 95% CI 1.3255.8)
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Introduction
DNA damage in the hematopoietic precursor cell is the essential prerequisite for
the development of acute leukemia. Such damage may result from the interaction
of reactive species generated by environmental or endogenous metabolites (D’Alo
et al., 2004).
Humans vary in their ability to metabolise such reactive intermediates, this
explains differences in leukemia risk as a result of interplay of genetic
susceptibility and exogenous exposure (Rollinson et al., 2000).
Environmental carcinogens are metabolized in vivo by enzymatic reactions
that are classically divided into two categories: (a) the Phase I enzymes, mediating
oxidation and activation ( ie .Cytochrome oxidase P450 enzymes) and (b) the
Phase II enzymes, mediating glucorinidation, acetylation, or conjugation with
glutathione-S and most often creating a more water-soluble conjugate, which may
be less toxic and more readily excretable (Hohaus et al ., 2003) .
The gene family of glutathione S -transferases (GSTs) , including (GSTM1),
(GSTT1) and (GSTP1), function in the detoxification
of electrophilic
intermediates and in the excretion of reactive species by the addition of
glutathione (Rollinson et al., 2000). they are involved in conjugation of several
environmental pollutants such as, polychromatic hydrocarbons and anticancer
drugs including
alkylating agents ,
anthracyclins ,
and cyclophosphamide
(Salinas and Wang , 1999 and D’Alo et al ., 2004) .
Polymorphisms in several genes of these enzymatic pathways are believed to
be key factors in determining cancer susceptibility to toxic or environmental
chemicals (Hohaus et al., 2003).
Enzymatic deficiencies of GST enzymes have complex metabolic
consequences and the kind of exposure is suspected to be important to detect
whether the GST genotype confers decreased or increased risk of cancer (Hohaus
et al., 2003). Homozygous deletions cause loss of enzymatic functions and have
been shown to be important risk factors for solid tumors (Rebbeck,1997). The role
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of GST genotypes in the pathogenesis of hematological neoplasms, particularly
acute leukemia, has been addressed (Rollinson et al ., 2000).
Aim of the work:
The aim of the present work is to find if there is an association between
Glutathione S- transferase genotypes and risk of acute leukemia.
Subjects and methods:
This study included 40 cases of acute leukemia (20 cases of AML and 20
cases of ALL). The cases were from the oncology center of Damanhour and
university hospital of Alexandria. The cases were 30 males and 10 females,their
ages ranged between 16 and 65 years with a mean of 32.88± 14.72. Peripheral
blood samples were obtained at the time of initial diagnosis or during follow-up.
The diagnosis of the cases based on bone marrow aspiration and
immunophenotyping, morphological classification (Fab) for AML cases was also
done.
Twenty normal age and sex-matched individuals, with no history of any
hematological problems, were included in the present work as controls. The
studied control group included 15 males and 5 females. Their ages ranged between
17 and 63 years with a mean of 35.95 ±15.38.
Both cases and controls are subjected to the following: Past history of any
malignancy, family history of any haematological malignancies, History of
smoking , ultimate contact with any chemical compounds and Residence (rural
or town).
Laboratory investigations: Complete blood picture performed using an
automated cell counter ( Sysmex K100) and genotyping of GSTM1 and GSTT1
genetic polymorphism by multiplex PCR
Seven ml (7 ml) venous blood obtained from each subject, five ml of them in a
sterile vacutainer containing EDTA for DNA extraction and performing PCR
and two ml in a tube containing EDTA for a complete blood picture.
Genotyping:
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DNA was extracted from peripheral blood using DNA extraction GFX Genomic
Blood DNA Purification kit (Amersham, Bioscience, USA) following the
manufacturer ’s instructions
The polymorphic deletions of the GSTM1 and GSTT1 genes were genotyped
using multiplex PCR approach described by Chen et al. (1997). The established
multiplex PCR method was used to simultaneously amplify and analyze
GSTM1, GSTT1, and -globin from both patients and controls. The -globin
gene is a housekeeping gene and acts as an internal control.
The PCR Primers used were as follows:
GSTM1 Primers: Sense primer G5-5'GAA CTC CCT GAA AAG CTA AAG C
Antisense primer G6-5'GTT GGG CTC AAA TAT ACG GTG G
GSTT1 Primers: Sense primer T1-5'TTC CTT ACT GGT CCT CAC ATC TC
Antisense primer T2-5'TCA CCG GAT CAT GGC CAG CA
ß- globin primers: Sense primer GH20-5'GAA GAG CCA AGG ACA GGT AC
Antisense primer PC04-5'CAA CTT CAT CCA CGT TCA CC
DNA amplification: All the reactions were performed in a total volume of 50
µl containing: Five µl (5 µl) of PCR buffer (10 xs)(Qiagen), two µl (2 µl) of
MgCl2, 3.1 µl of deoxynucleotide triphosphates (dNTPs)(Stratagene),0.25 µl of
Taq polymerase(Qiagen), One µl (1µl) of each working primer solution.The
total volume is completed to 50 µl of distilled water. Finally, 5µl of the
extracted genomic DNA of each sample was added to the mixture.
The PCR reaction tubes were closed and placed in the heating block in the
DNA thermal cycler (Progene –Techne).
The computerized thermal cycler was programmed for the following
conditions: Denaturation at 94°C for 35 cycles for 1 min, Annealing at
62°C for 1 min and Extension at 72°C for 1 min.
PCR amplification products were run on 3% agarose gel electrophoresis
,visualization of the DNA bands was done through staining of DNA in agarose
gel by fluorescent dye ethidium bromide.
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DNA from patients with positive GSTM1, GSTT1, and β -globin alleles
yielded 219-bp, 480-bp, and 268-bp products, respectively; the absence of
amplifiable GSTM1 or GSTT1 (in the presence of β-globin PCR product)
indicates the respective null genotype for each.
Statistical methods:
The statistical significance of the differences between groups was calculated by the chi-square
test . Odds ratios (ORs) were calculated and given with the 95% confidence intervals(CI) ,
values of P value < o.o5 were considered significant .
The collective data were coded, tabulated and statistically analyzed using statistical package
of social science (SPSS) (version 9).
Results: The distribution of GST genotypes was studied in cases as well as
controls. As regarding GSTM1 polymorphism, in all cases of acute leukemia, it
was found that 25(62.5%) out of 40 cases were null (absence of the enzyme)
compared to 9(45%) out of 20 controls, yielding OR of 2.037 (95% CI 0.6866.052). In AML, 15(75%) out of 20 cases were null in GSTM1 yielding a
significantly elevated OR of 3.667 (95% CI 1.01-14.03) (p value <0.05). In ALL,
10 (50%) out of 20 of cases were null in GSTM1 yielding OR of 1.22 (95% CI
0.353-4.235) (p value >0.05) (Table 1).
As regarding GSTT1 polymorphism, in all cases of acute leukemia, it was
found that 12(30%) out of 40 cases were null compared to 4(20%) out of 20 of
controls yielding OR of 1.714 (95% CI 0.473- 6.212) .In AML, 5(25%) out of 20
cases were null in GSTT1 yielding OR of 1.33 (95% CI 0.3-5.926). In ALL, 7
(35%) of 20 cases were null in GSTT1 yielding OR of 2.154 (95% CI 0.516-9.00)
(Table 1).
The interaction between GSTM1 and GSTT1 genotypes was studied and the
possible effect of combined genes (GSTM1 and GSTT1) on acute leukemia was
also interpretated. This is by studying the relation between different genotypes of
both genes and their prevalence in acute leukemia. In AML, increased risk
estimates were observed when either allele was null. It was found a strong
association between AML and (GSTM1 null and GSTT1 present) (GSTM1 ¯/
GSTT1+) genotype (OR 18, 95% CI 1.9-171.9) (p = 0.003) and also between
AML and (GSTT1 null and GSTM1 present) (GSTT1¯/GSTM1+) genotype (OR
18, 95% CI 2.15-260.9) (p = 0.018) (Table 2). In ALL, we found no association
between ALL and both genotypes (GSTM1¯/GSTT1 +) (OR 1.5, 95% CI 4..06.53) (p =0.588) and (GSTT1¯/GSTT1+) (OR 3.0, 95% CI 0.41-21.89) (p =0.269)
(Table 3).
The present study was concerned with the history of smoking in acute
leukemic cases. It was found that 52% of acute leukemic cases, with smoking
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habit, were null in GSTM1 yielding OR of 1.238 (95% CI 0.343-4.464) and
66.7% of acute leukemic cases, with smoking habit, were null in GSTT1 yielding
OR of 2.667 (95% CI 0.648-10.97) . No association between smoking and
genotype (GSTM1 null, GSTT1 present) was found (OR 6.6, 95% CI 0.67364.77) (p = 0.078). But, a significant association between smoking and genotype
(GSTT1 null, GSTM1 present) was found which means that smoking could be
considered as a risk factor of acute leukemia if it is combined with (GSTT1 null,
GSTM1 present) genotype (OR 18, 95% CI 2.3-255.8)(p =0.019) .
No correlation between age and sex in relation to GST genotypes was
found. Also, no correlation was found between residence, either rural or town and
GST genotypes .
Table (1): Comparison between cases with acute leukemia, AML, ALL cases and controls
regarding GSTM1and GSTT1 gene polymorphism
Acute leukemia cases n=40
Parameter
Cases
Controls
n(%)
n(%)
Odds ratio
95%CI
2
p-value
GSTM1¯
25
(62.5%)
9
(45%)
2.037
0.686-6.052
1.663
0.197
GSTT1¯
12
(30%)
4
(20%)
1.714
0.473-6.212
0.682
0.409
AML cases n=20
GSTM1¯
15
(75%)
9
(45%)
3.667
1.01-14.03
3.86**
0.049**
GSTT1¯
5
(25%)
4
(20%)
1.33
0.3-5.926
0.143
0.705
ALL cases n=20
GSTM1¯
10
(50%)
9
(45%)
1.22
0.353-4.235
0.1
0.752
GSTT1¯
7
(35%)
4
(20%)
2.154
0.516-9.00
0.129
0.288
* 95% CI : 95% confidence interval
** (2): significance >3.84
***P- Value: significant <0.05
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Table (2): Relation between (GSTM1 null, GSTT1 present) genotype and (GSTT1 null, GSTM1
present) genotype in AML.
Parameter
AML
cases
control
Odds
ratio
95%CI*
GSTM1¯ /GSTT1+
14
(70%)
7
(35%)
18.0
1.9-171.9
GSTM1+ /GSTT1+
1
(5%)
9
(45%)
0
(20%)
1
(10%)
18.0
2.15 -260.9
2
(5%)
9
(45%)
GSTT1- /GSTM1+
GSTT1 +/GSTM1+
2
P- value
8.710**
5.605**
0.003***
0.018***
Table (3): Relation between (GSTM1 null, GSTT1 present) genotype and(GSTT1 null, GSTM1
present) genotype in ALL
ALL cases
Control
Odds
ratio
95%CI*
2
P- value
GSTM1¯ /GSTT1+
7
(35%)
7
(35%)
1.5
4..0-6.53
4.19.
0.588
GSTM1+ /GSTT1+
6
(30%)
9
(45%)
4
(20%)
1
(10%)
3.0
0.41-21.89
2.111
4.169
6
(30%)
9
(45%)
Parameter
GSTT1- /GSTM1+
GSTT1 +/GSTM1+
7
M1
L1
L2
L3
L4
L5
L6
L7
480 bp
268 bp
219 bp
Figure 1: Agarose gel electrophoresis (3%) showing the GSTM1 and GSTT1 polymorphism. The
PCR products amplified from the GSTM1 and GSTT1 loci are 219 bp and 480 bp in size,
respectively. A 268 bp fragment from the β-globin locus was coamplified as an internal control.
Lane 1 shows an individual with a homozygous deletion of both GSTM1 and GSTT1. Lane
2 shows an individual in which GSTT1 can be detected but GSTM1 is homozygously deleted.
Lanes 4 and 5 show individuals in which GSTM1 can be detected but GSTT1 is homozygously
deleted. Lanes 3 and 7 show individuals with presence of both GSTM1and GSTT1 genotypes
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Discussion
Acute leukemia is a frequent malignancy affecting both adults and
children. The etiology of acute leukemia is unknown, although many
conditions may influence its development. Like many other cancers, acute
leukemia is considered to be a complex disease, which is determined by
combination of genetic and environmental factors (Arruda et al., 2001).
DNA damage in the hemopoietic precursor cell is the essential prerequisite
for the development of leukemia and the body has developed a series of
mechanisms aimed at preventing such damage. Cytogenetic analysis in acute
leukemia has revealed a great number of non-random chromosome
abnormalities. In many instances, molecular studies of these abnormalities
identified specific genes implicated in the process of leukemogenesis
(Mrozek et al., 2004)
The environmental causes of acute leukemia, which have increased in
the last centuries, have been established. Pollution and occupational hazards
most probably are accepted causes of acute leukemia. There is an increasing
evidence that predisposition to acute leukemia is associated with exposure to
chemicals such as benzene and chemotherapeutic agents (Glass et al., 2003).
Human health is determined by the interplay between genetic factors and the
environment. Many of the genes in human genome influence the impact of
environmental agents on human being (Rosival and Tranovac, 1999).
Humans are polymorphic in their ability to detoxify the products of
environmental pollutants. The enzymes involved in the metabolism of these
carcinogens have received a reasonable level of attention. They are
metabolized in vivo by enzymatic reactions that involves the phase I and the
phase II enzymes, (Hohaus et al., 2003). The equilibrium between phase I
enzymes and phase II enzymes is critical in host response to xenobiotices
9
(drugs and carcinogens).Metabolizing genes could be thus relevant as
genetic factors in acute leukemia following exposure to various chemicals
(Sinnett et al., 2000).
Glutathione S -transferases (GSTs) including (GSTM1), (GSTT1) and
(GSTP1) represent the most important phase II enzymes function in the
detoxification of electrophilic intermediates and in the excretion of reactive
species by the addition of glutathione (Rollinson et al., 2000). They are
involved in the conjugation of several environmental pollutants (D’Alo et al
., 2004).Polymorphisms in several genes of these enzymatic pathways are
believed to be key factors in determining cancer susceptibility to toxic or
environmental chemicals. GST polymorphism have been considered as
possible risk factors of acute leukemia (Zheng and Song, 2005)
GSTM1 or GSTT1 genotypes can be categorized into two classes,
homozygous deletion genotype (denoted null genotype) and genotypes with
one or two undeleted alleles (denoted non-null genotype). The GSTM1 null
and GSTT1 null alleles represent deletion in GSTM1 and GSTT1 genes and
result in a loss of enzymatic activity (Hatagima et al., 2000).
Polymorphism of GSTM1 and GSTT1 exists in all populations. The
GSTM1 class is absent from more than 50% of some populations (with a
42% to 62% range for individual studies).This deficiency appears to be
caused by the deletion of the GSTM1 gene. GSTT1 null genotype in humans
occurs in (10–38) % of various ethnic groups. About 20% of Caucasians are
homozygous for a GSTT1 null allele (Garte et al., 2001). Polymorphism of
GSTM1 and GSTT1 is responsible for many cancers such as, hepatocellular
carcinoma, bladder cancer, colorectal cancer ….. etc. The possible cause is
mostly due to absence of protective GST enzymes, due to deletion of
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GSTM1 or GSTT1 genes, and failure of the body to detoxify the products of
environmental pollutants which have a proved role in precipitating these
cancers (Andonova et al., 2004).
This study aimed to find the association between GSTM1 and GSTT1
genotypes and the risk of acute leukemia in its both types, acute myeloid and
acute lymphoblastic leukemias. Forty patients were diagnosed as acute
leukemia, twenty of them were diagnosed as AML and the other twenty
were diagnosed as ALL. Basic investigations for diagnosis of acute leukemia
were performed including complete blood picture, bone marrow aspiration
and immunophenotyping for detection of different subtypes of both
leukemias. Multiplex PCR method was done to detect GSTM1 and GSTT1
polymorphism.
In the present work, the distribution of GSTM1 and GSTT1 null
genotypes in acute leukemia was studied, both AML and ALL, in
comparison to controls. It was found an increase in GSTM1 and GSTT1 null
genotypes, in cases of acute leukemia, compared with controls. 62.5% of
cases of acute leukemia carry GSTM1 null genotype compared with 45% of
controls and 30% of cases carry GSTT1 null genotype compared with 20%
of controls. Although a slightly higher proportion of cases displayed GSTM1
or GSTT1 null genotypes, the difference was not statistically significant
(table 1). This is maybe due to small sample power.
The significant association was observed between acute myeloid
leukemia and GSTM1 null genotype. 75% of AML cases were null in
GSTM1 compared with 45% of controls, which represents a strong
association between AML and GSTM1 null genotype (table 1) . However, it
was found a mild increase of GSTT1 null genotype among AML cases.
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25% of AML cases were null in GSTT1 genotype compared with 20% of
controls. Although this increase is minute, the role of GSTT1 null genotype
in increasing the risk of AML cannot be neglected. Maybe, because of
normal lower frequency of GSTT1 deletion polymorphism (20% in
population), the number of individuals required for statistical analysis are
much greater than those who were available to us.
The interaction between the GSTM1 and GSTT1 genotypes in acute
leukemia was studied. A statistically significant association was found
between acute myeloid leukemia and both genotypes (GSTM1¯, GSTT1+)
and (GSTT1¯, GSTM1+) (table 2). Only one case, from the twenty cases of
AML, carried the protective genotype (GSTM1+, GSTT1+), which means
that risk of AML is increased when either allele is null. We observed that
GSTM1 and GSTT1 genotypes,when analyzed in combination, the risk of
AML was increased (p value < 0.01) suggesting that the additive effects of
these polymorphisms are important. It was difficult to study the relation
between combined GSTM1 and GSTT1 null genotype (GSTM1¯, GSTT1¯)
and acute leukemia, either in AML or ALL, due to small sample power.
Maybe it can give better results if it is studied on large sample of population
Many studies had confirmed the association between GSTM1 and
GSTT1 gene deletion and risk of AML, which agreed with the present
findings. Arruda et al., (2001) confirmed that the risk for AML is increased
in individuals with GSTM1 and GSTT1 gene defects. Also, Rollinson et al.,
(2000) reported the presence of an association between GSTT1 and GSTM1
null genotypes and risk of AML. Another study found an increased risk for
AML in children with GSTM1 null genotype (Davies et al., 2000).
However, there are few studies failed to find this association. Basu et al.,
(1997) found no increased risk of AML associated with GSTM1 and GSTT1
12
null genotypes in British patients with AML and Whoo et al .,(2000) found
the same in American patients with therapy –related AML.
The association between AML and GST polymorphism was explained
by the major role for these enzyme systems in detoxification of
environmental carcinogens. Myelodysplasia and AML are associated with
exposure to chemicals such as benzene and chemotherapeutic alkylating
agents (Crump et al., 2000). Individuals with a homozygous deletion of the
GSTM1 or GSTT1 genes lack enzymatic conjugation of foreign compounds
with glutathione. This results in diminished ability to detoxify a wide range
of environmental carcinogens. The inherited absence of carcinogen
detoxification pathway in patients makes them more susceptible to develop
acute myeloid leukemia (Arruda et al., 2001).
Also, the absence of GSTM1 and GSTT1 genotypes may help in DNA
damage, which may affect the hemopoietic stem cells and precipitates AML.
DNA is at constant risk from damage by both endogenous and exogenous
sources. A large number of highly complex mechanisms have evolved to
protect DNA from damage including DNA repair pathways and systems that
protect against oxidative stress and other damaging agents. These pathways
play a vital role in maintaining genetic integrity (De-Boer, 2002). The
glutathione S-transferases (GSTs) are a multigene family that detoxify
reactive electrophiles via conjugation to glutathione and, hence, prevent
damage of DNA. A deletion polymorphism in GSTM1 resulting in the
absence of functional GSTM1 protein, which detoxifies a variety of
genotoxic agents ,has been shown to be associated with significantly
elevated levels of DNA adducts in WBCs and with higher levels of sisterchromatid exchange compared with individuals with wild-type GSTM1
protein levels (Seedhouse et al.,2004). Also, Absence of GSTT1 activity in
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blood, corresponding to the GSTT1 null genotype, has been associated with
carcinogen-induced and background chromosomal changes in human
lymphocytes (Crump et al., 2000).
The risk of AML was also referred, not only to the polymorphism in
GST enzymes, including GSTM1 and GSTT1, but also to their interrelation
with other genes such as, xenobiotic- metabolism genes or DNA
homologous recombination (HR) repair genes. The interaction between these
genes may precipitate AML through different mechanisms. (D′Alo et al.,
2004).
Considering acute lymphoblastic leukemia (ALL), it was found that
50% of ALL cases were null in GSTM1 compared with 45% of controls
and 35% of ALL cases were null in GSTT1 compared with 20% of controls
(table 1) . No significant association was found between either, GSTM1 or
GSTT1 null genotypes, and ALL (table 1). Also, no association was found
between both genotypes, (GSTM1¯, GSTT1+), (GSTT1¯, GSTM1+) and ALL
(p value > 0.05) which suggests that GST polymorphism has no relation
with the pathogenesis of ALL (table 3) .
These findings is supported the studies done by Davies et al., (2002)
who reported that GST genotypes does not affect etiology or outcome of
childhood ALL. Canalle et al., (2004) also reported that no difference was
found in the prevalence of the GSTM1 and GSTT1 null genotypes between
ALL patients and the controls. However, in contrast to the present work, it
was reported that GSTT1 null genotype conferred a 3-fold increased risk of
adult ALL, although there was no association with GSTM1 genotype
(Rollinson et al., 2000). Other study revealed that The GSTM1 null
genotype was significantly increased in children with ALL, while, the
GSTT1 null genotype did not show this effect (Pakakasama et al., 2005).
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On the other hand, some researches reported that the risk of ALL was
increased in pediatric patients carrying non-null alleles of GSTM1 and
GSTT1, Although GSTM1 and GSTT1 are generally thought to be phase Π
detoxifying enzymes responsible for the inactivation of carcinogens.
However, GSTT1 also is known to have phase Ι activity and ability to
activate carcinogens (Barnette et al., 2004). Guengerich et al., (2003)
agreed with this theory. He suggested that conjugation of foreign compounds
with GSH almost always leads to formation of less reactive products that are
readily excreted. In few instances, the glutathione conjugate is more reactive
than the parent compound and consequently, some of GST substrates that
are activated by conjugation with GSH may lead to genotoxic products
which are capable of modifying DNA and in turn causing cancer. For
example, GSTT1 enzyme can form mutagenic metabolites with some
substances such as, solvent dicloromethane. Thus, the presence of functional
enzyme, while generally protective, may increase the mutagenic risk in some
exposures (Wheeler et al., 2001). These findings suggest that GSTM1 and
GSTT1 enzymes should be studied in ALL patients with different exposure
to different chemicals with taking in consideration the variation in their
reaction with these chemicals either by detoxification or activation
The difference between these studies and the present study may be due
to racial heterogeneity of populations. It is possibly due to different causes
of ALL in different countries which are attributed to different environmental
carcinogens which varies from an area to another. Also, how much these
carcinogens can be detoxified or activated or even not affected by GST
enzymes according to their nature. This can possibly explain why GST null
genotypes are precipitating of ALL in some countries and have no relation
15
with ALL in others and how they can play an unusual protective role with
some chemicals in another areas.
This study also concerned with the history of smoking in acute
leukemic cases. It was found that 66.7% of acute leukemic cases with
smoking habit were null in GSTT1 and 52% were null in GSTM1 . We
found no association between smoking and genotype (GSTM1 null, GSTT1
present) (p value > 0.05). But, a significant association between smoking
and genotype (GSTT1 null, GSTM1 present) was found (p value < 0.05)
which means that smoking could be considered as a risk factor of acute
leukemia if it is combined with (GSTT1 null, GSTM1 wild) genotype .
Some researchers studied the relation between smoking and risk of acute
leukemia in relation to GSTT1 and GSTM1 polymorphic status. Rollinson
et al., (2000) reported that no significant interaction was observed between
smoking and the null genotype of either the GSTT1 or GSTM1. This study
disagreed with the present work, however, other studies confirmed its role in
precipitation of acute leukemia especially in patients with GSTT1 gene
deletion. Cigarette smoking was considered as a risk factor to many diseases
such as lung cancer, bladder cancer and acute leukemia, especially AML
(Kasim et al., 2005).It is common source of exposure to benzene, which is
considered as an established leukemogen present in cigarette smoke. This
benzene is detoxified via GSTT1. It was confirmed that individuals with
GSTT1 null genotypes tended to be more susceptible to benzene toxicity
(Wan et al 2002). So that, absence of GST enzyme due to homozygous
deletion of GSTT1 gene, may decrease the detoxification of benzene and
consequently, leukemia develops (Rollinson et al., 2000).
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On the other hand, GSTT1 is expressed in erythrocytes and lymphocytes
and hence acts in the hematopoietic system. Cytogenetic tests such as
chromosome aberration (CA) and sister chromatid exchange (SCE) are most
often applied in the monitoring of the genotoxicity of potentially
carcinogenic chemical in peripheral blood of lymphocytes. The importance
of GSTT1 in the protection of hematopoietic cells from environmental
pollutants has been proven in a population exposed to diepoxybutane (DEB),
an epoxide metabolite of 1,3-butadiene. The- in vitro- sister chromatid
exchange in human lymphocytes in the presence of 1,3-butadiene was 16fold higher in cells from individuals lacking the GSTT1 gene expression. So
that ,GSTT1 can be considered as a major protective factor of individual
susceptibility to benzene-induced chromosomal damage in addition to its
role in urinary excretion of benzene metabolites (Dirksen et al .,2004).
These researches support the present findings which prove the role of
(GSTT1 null, GSTM1 wild) genotype in combination with smoking in
precipitating of acute leukemia.
No correlation between age and GSTM1 or GSTT1 null genotypes
was found. This finding was agreed by Rollinson et al., (2000), who
suggested that there is no association between age and GST null genotypes
in AML or ALL. However, some researchers reported higher frequency of
GSTT1 or GSTM1 homozygous deletion and GSTT1/GSTM1 double null
genotypes in patients over 60 years especially in AML (D′Allo et al., 2004).
This is maybe due to prolonged exposition of hematopoietic progenitor cells
to toxic agents in combination with a reduced capability of detoxification
which might contribute to the pathogenesis of AML in the elderly (Voso et
al., 2002).
17
No correlation between sex and GSTM1 or GSTT1 null genotypes
was found . Most of the studies confirmed the absence of any relation
between sex and GST null genotypes, which agreed with the present study.
D′Alo et al., (2004) and Voso et al., (2002) suggested that there is no
association between sex and GST null genotypes in cases with AML. Davies
et al., (2002) and Chen et al., (1997) mentioned the same in cases with ALL.
This study tried to find if there is any relation between residence of the
patients and their genotypes in order to estimate the effect of GSTs in
detoxification of different pollutants and how the deletion of GST genes can
precipitate the disease through accumulation of environmental carcinogens.
Although, different sources of pollution are present in town more than in
rural areas, pesticides, which increase in rural areas, has already an
important role in precipitation of acute leukemia through different
mechanisms. However, it was found no correlation between residence, either
rural or town, and GSTM1 or GSTT1 null genotypes .
Recommendations:
Further studies on large number of patients and considering of GSTM1 and GSTT1 as
important cytogenetic markers is recommended. Also further studies should be done on
the interaction between GST genotypes and ALL with different exposure to chemical
agents. The interaction between GST genes and other genes such as, phase Ι
detoxification genes or DNA repair genes should be also studied to understand the role
of gene – gene interaction in the pathogenesis of acute leukemia.
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and Ganev VS (2004): Balkan endemic nephropathy and genetic variants of glutathione
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18
Arruda VR, Lima CS, Grignoli CR, De-Melo MB, Metze LI, Alberto FL, Saad ST and
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Garte S. Gaspari L, Alexandrie AK, Ambrosone C, Autrup H, Aurup JL, Baranova H,
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21
‫الملخص العربي‬
‫يعد مرض سرطان الدم الحاا حداد ح األ ااماراض النارطاي ج يا نح ا حيحاا العاالأل ‪ .‬سا ا ااباابج ب الا الحارض لا ف معركياا كل ا‬
‫يعت ر مرضاا مرب اا ماج معح ماج ماج الع امان الع اج ك ال ع اج ‪ .‬يعاد التما أل ماج ح األ ااسا ار الح ياج لمحارض ماج طريا‬
‫ال ا ك الري ا(‬
‫الا التما‬
‫ن اا الصااال بالصاياا امم الح يااج لمادم ع لاللس يعااد العناأل طر ااا مدياد لح ا‬
‫ما‬
‫ك يتواااكب ال يار يا‬
‫الحاام‬
‫أل مما‬
‫اد‬
‫التصمص مج له الحم ثاب ال ع ج‪.‬‬
‫مائمج ن ج العم ااث ن س‬
‫الثاي الت‬
‫رايناو راا التا‬
‫‪:‬األ (‬
‫ا‬
‫نا( س‬
‫سم ‪2‬ا ع نا( س‬
‫ا ‪2‬ا‬
‫ا‬
‫عاد ماج ا األ سيايحااب ال ا‬
‫يترك ي محم ج ام تران بالعديد مج الحم ثاب ال ع ج ك التصمص م ا ك يُعتقد حن التعد الي م ي بعا‬
‫الحنا اب اايايح ج‬
‫الحوتاح الرئ ن ي‬
‫الع ااب الصاباج ب اله‬
‫حديد ابم ج الور لإلبابج بحرض النرطان يت عج التعرض لم حاكيااب ال ع اج الناامج ‪ .‬بحاا ايا‬
‫د أبد ك اايحاط الع ج الصابج ب لا الع ج ي م يأ حك ام الدم كبااخص سرطان الدم الحا ‪.‬‬
‫رايناو راا كخ ار التعارض لإلباابج بحارض‬
‫له الد اسج دف سل( سيعا العا ج ب ج اايحااط الع اج ايايحااب العم ااث ن س‬
‫سرطان الدم الحا ‪ .‬ك لقد حنريت اله الد اساج مما( ‪ 04‬شصصاا األ يص صا أل بحاامب مصاابج بحارض سارطان الادم الحاا ع ما أل ‪ 14‬األ‬
‫يص ص أل بحامب مصابج بحرض سرطان الصايا المحواكيج الحا ك ‪ 14‬أل يص ص أل بحامب مصابج بحرض سرطان الادم ال صاام الحاا ‪.‬‬
‫ك د أل محن التحال ن ااساسا ج لتياص ص الا الحارض ك ياحن با‬
‫الادم ال امماج عيحاص ل صااا الع اامع ال ارا ال ا رياج الح ام اج لتحدياد‬
‫ااي اا الحصتموج لنرطان الدم الحا ‪ .‬بللس أل استصدام طريقج وامن نمنن ال محر لم ي‬
‫س‬
‫ا( سم ‪2‬ا ك نا(‬
‫ماج اايحااط الع اج نا( س‬
‫ى (‪2‬ا ي( م اب الدم كسظ ا ا ب اس ج ن اا الردان ال ربائ باستصدام حنا كا ن ان ‪ .%.‬ك لقاد ‪:‬اح ت اله الرساالج حي‪:‬اا ‪14‬‬
‫شصصا بح حا بحعح مج ضاب ج‪.‬‬
‫ك د حظ رب له الرسالج ا اطا اما ب ج مرض سرطان الدم ال صام الحا ك ال راا الع ا( ال اا ص لعا ج نا( س‬
‫حي‪:‬ا كك ند ا اطا يا ب ج مرض سرطان الدم ال صام الحا ك ب ج ال راا الع ( ال ا ص لع ج ن( س‬
‫ا‬
‫ا ‪ 2‬ا كاي‪:‬اا ال ااراا الع اا( ال اا ص لعا ج ناا( س‬
‫اابابج بالحرض يا كنا‬
‫خ را ينحح ب‬
‫ا‬
‫سم ‪2‬ع ع ال يط لع ج نا( س‬
‫ا ‪2‬ع ع ال ياط لعا ج ناا( س‬
‫ا‬
‫سم ‪ 2‬ا ك الاال ياادى مما( ايااا خ اار‬
‫ح م اا كلاللس يح اج امت اا يقاد التحاثان الايعا( لمع ا ج نا( س‬
‫ا‬
‫ا ‪ 2‬ا مااما‬
‫سم ‪2‬ا حك نا( س‬
‫ا‬
‫الحرض ‪.‬‬
‫مم( الع ف ع لأل نت‬
‫له الد اسج سيعا ح ا اط ب ج يقاد التحاثان الايعا( لمع ا ج نا( س‬
‫ا‬
‫سم ‪2‬ا حك نا( س‬
‫مرض سرطان الصايا الم حواكيج الحا ع لللس م يعت ر يقد التحاثن الايع( ل ليج الع ج ماما خ را ينحح ب‬
‫حظ رب له الرسالج ا اطا اما ب ج التدخ ج ك ال اراا الع ا( ال اا ص لعا ج نا( س‬
‫لللس يح ج امت ا التدخ ج ماما خ را ينحح ب‬
‫لدخان الت غ حدد م امن الص‬
‫اابابج بالحرض يت عج ل ن‬
‫لأل نت‬
‫ا‬
‫سم ‪2‬اع‬
‫الحارض بيارط حن يصااد‬
‫ا‬
‫الحرض ‪.‬‬
‫ا ‪2‬ع ال ياط لعا ج نا( س‬
‫ا‬
‫لإلبابج بحرض سرطان الدم الحا ك مم( الرغأل مج حن لا التعارض ضاع ا ك ل ا يح اج حن يا‬
‫ال راا الع ( ال ا ص لع ج ن( س‬
‫‪2‬ع ال يط لع ج ن( س‬
‫راينو راا ‪.‬‬
‫‪22‬‬
‫سم ‪ 2‬ا‬
‫الا ال اراا الع ا(‪ .‬ك مما( للاس يوتارض حن التعارض‬
‫له الد اسج سيعا ح ا ااط با ج الناج حك العا ف حك م اان س اماج الحرضا( سا ا يا الريا‬
‫الحصتموج لع ج العم اث ن س‬
‫ا‬
‫ا ‪2‬ا ك‬
‫سلا‬
‫سم ‪ 2‬ا‪.‬‬
‫حك الحدي اج كبا ج اايحااط الع اج‬