Research Journal of Recent Sciences ________________________________________________ ISSN 2277-2502
Vol. 1 (ISC-2011), 219-223 (2012)
Res.J.Recent Sci.

Glutathione S-Transferase gene polymorphisms (GSTT1, GSTM1, GSTP1) as
increased risk factors for asthma and COPD among Isocyanate exposed
population of Bhopal, India
Bose P and Bathri R.
Department of Research, Bhopal Memorial Hospital and Research Centre, Karond Bypass Road, Bhopal (MP), INDIA-462038

Available online at, www.isca.in
(Received 30th November 2011, revised 9th January 2012, accepted 28th January 2012)

Abstract
The release of methyl isocyanate (MIC) in Bhopal, India, caused the worst industrial accident in history. Isocyanates are the
most common low molecular weight chemicals to cause asthma and Chronic Obstructive Pulmonary Disorder (COPD),
whereas their metabolites may be conjugated with glutathione by glutathione S-transferases (GSTs). Glutathione S-transferases
(GSTs) are enzymes involved in the detoxification of hazardous agents. We examined whether polymorphisms in the GSTM1,
GSTP1 and GSTT1 genes modify allergic responses to isocyanate exposure, thus increasing risks for the development of
isocyanate related- pulmonary disorders in a cohort of Bhopal. We compared clinical data - including gender, age and smoking
habits - between the 2 groups. The study population consisted of 54 methyl isocyanate exposed subjects. Genotyping the
polymorphisms in the GSTT1 and GSTM1 genes was performed using the multiplex PCR. The GSTP1 ILe105Val polymorphism
was determined using PCR-RFLP. GSTP1 genotype was significantly associated with the increased risk of asthma and COPD
(RR =1.66, 95%CI, 0.412-3.29 and RR=1.586 ; 95%CI, 0.75-3.33 respectively). Asthmatic had a higher prevalence of the
GSTP1Ile105val allele than the COPD group (53.48% and 44.1%, respectively; p =0 .002). Also, the presence of the GSTP1
homozygote Val/Val was less common in subjects with asthma (39.53%) than in COPD group (62.79%). Polymorphisms within
genes of the GST superfamily were associated with risk of asthma and COPD in Bhopal population. No differences in genotype
frequencies were perceived in patients stratified by age and gender. GSTM1 and GSTT1 had almost no association. The results
suggest, for the first time, that the polymorphic GSTs, especially GST(P1), play an important role in inception of ill effects
related to exposure to methyl isocyanate. Although about half of the subjects with lower FEV1 had never smoked, smoking was
the main risk factor for the decreased FEV1 in COPD and asthmatic subjects. Our series of studies identified GST variants as a
potential susceptibility locus to asthma and to lower lung function in COPD, asthmatic subjects, which may support the
contention that genetic determinants of lung function influence susceptibility to asthma.
Key words: Isocyanate, asthma, chronic obstructive pulmonary disease, airflow obstruction, Bhopal, smoking.

Introduction
Isocyanates are reactive organic chemicals widely used in the
industrial production of polyurethane polymers, pesticides,
fungicides, and other materials. Methyl isocyanate (MIC), a
precursor in pesticide production, was the major causative
agent of the environmental disaster in Bhopal, India,
responsible for more than 3000 immediate deaths and several
thousand additional casualties in the years after the accident.
Dependent on exposure levels and duration, MIC-exposed
individuals present with airway hyperresponsiveness,
inflammation, reactive airway dysfunction syndrome
(RADS) and airway edema and injuries1. To minimise the
potential for oxidative injury, the human lung has an
integrated system of antioxidant enzymes and expendable
soluble molecules. This system includes several mechanisms
by which ROS are converted to products that are further
detoxified by other enzymes. If the oxidant burden is
sufficiently great, ROS may overwhelm the antioxidant

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system leading to a state of “oxidative stress”, which is
thought to contribute to the pathogenesis of a number of
respiratory diseases. Although antioxidant defences are
available to decrease oxidative stress in the airways,
individuals differ in their ability to deal with an oxidant
burden; such differences are, in part, determined genetically2.
This genetic variability may account for the considerable
between-subject variability seen in both the lung function
and airway inflammatory responses to MIC Humans vary in
their ability to metabolize exogenous and endogenous
compounds, and individuals with a genetically determined
reduced capacity to detoxify hazardous compounds may be at
increased risk of adverse health effects compared with those
with unaltered metabolic capacity. In this respect,
conjugation of electrophilic compounds to glutathione,
mediated by glutathione S transferases (GSTs), may be of
great importance. The glutathione S-transferases M1
(GSTM1), T1 (GSTT1) and (GSTP1) P1 genes are

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Vol. 1(ISC-2011), 219-223 (2012)
Res.J.Recent.Sci
polymorphic in humans, and a phenotypic absence of
enzyme activity results from the homozygous deletion of the
respective genes (i.e., the null genotypes). The GSTM1 and
GSTT1 genes exhibit null (i.e., deletion) polymorphisms; in
specific individuals, homozygous deletion (i.e., both copies
lost) of these genes can be detected3.
The current authors investigated the relationships between
common polymorphisms in these three genes involved in
response to oxidative stress, and decline in respiratory
symptoms and lung function in response to isocyanate
among asthmatics and Chronic Obstructive Pulmonary
Disorder (COPD). The full spectrum of exposures and
susceptibility genes involved in the pathogenesis of asthma
and wheezing have yet to be established. Tobacco smoke is
an exposure of interest, especially among groups with high
prevalence of asthma and increased sensitivity to air
pollutants. An extensive body of evidence indicates that
involuntary tobacco smoke exposure increases the prevalence
of wheezing, cough, and phlegm, and that household ETS
(Environmental
tobacco
smoke)
exposures
cause
exacerbations in asthma and COPD4.
Pulmonary function is among the most important health
indicators, being a strong predictor of long-term morbidity
and mortality. Impaired pulmonary function is a strong risk
factor for the development of asthma and COPD and a
marker of disease severity. Chronic stimulation of the innate
immune system by microbes, inhaled allergens or
components of tobacco smoke is involved in the
inflammation and remodeling of airways that underlies
asthma and COPD, and certain genetic variants may play a
role in the common pathogenesis of both asthma and COPD 5.
Our group has recently identified the GST gene as an asthma
susceptibility gene among the methyl isocyanate exposed
cohort in Bhopal. Since it has been suggested that genetic
determinants of lung function influence susceptibility to
asthma as well as COPD, our finding may indicate a role of
GST in the development of airflow obstruction that originates
from oxidative stress.

Material and Methods
Study Subjects: This was a cross-sectional analysis of 54
Indian men and women resident of Bhopal city since past 3
decades aged 35 to 67 years who visited the Bhopal
Memorial Hospital and Research Centre for annual medical
checkup since past years. Detailed information on respiratory
health, lifestyle, and exposure to MIC, other environmental
irritants such as tobacco smoking, allergens, and air
pollutions was collected. Based on a detailed questionnaire
on pulmonary symptoms we carefully excluded the presence
of pulmonary diseases such as chronic bronchitis, old
tuberculosis, and pulmonary fibrosis.
Our diagnosis of asthma basically relied on the physicians
diagnosis. Clinical diagnosis of COPD was defined as
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reporting of any respiratory symptom (exertional
breathlessness, chronic cough, regular sputum, frequent
winter bronchitis, or wheeze) and evidence of airway
obstruction on spirometry (both FEV1/FVC < 0.7 and FEV1
< 80% predicted).
Classification of the subjects: Of the 54 subjects, 20
subjects with a history of asthma, 23 subjects with COPD
participated in the study. Of the total subjects 14 subjects had
FEV1/FVC < 70%.
Pulmonary function test (PFT): Data for FEV1, FVC, and
FEV1/FVC ratio were available for all participants. Lung
function was measured in the standing position using an
electronic spirometer (Autospiro series; Minato Medical
Science Co., Ltd, Osaka, Japan). Participants performed up
to 3 forced expiratory maneuvers to obtain acceptable
maneuvers; FEV1 and FVC values taken to characterize each
participant were the maximum results obtained from
acceptable maneuvers. FEV1/FVC ratio was calculated using
actual values.
Determination of Genetic Polymorphisms: DNA was
isolated from clot with a Qiamp Blood DNA Maxi kit
(Qiagen Inc., Santa Clarita, CA, USA) in accordance with
the manufacturer's instructions and stored at -80°C until use.
Genotyping for the GST(s) polymorphism was carried out
following a previously reported protocol6.
Descriptive statistics were expressed as mean ± standard
deviation (SD). We compared clinical data – gender, age,
body mass index (BMI), smoking habits (never smoker, exsmoker, and current smoker), using a 2-tailed Student’s t-test
or Pearson’s chi-square test as appropriate. Smoking index
was calculated by multiplying smoking dose (cigarettes per
day) and duration (years smoked).
Statistical Methods The odds ratio (OR) and risk ratio (RR)
is an estimate of the relative risk of disease associated with
specific genotypes and is defined as the odds of a case
patient having the at-risk genotype divided by the odds of a
control subject having the same genotype. ORs and 95%
confidence intervals (CIs) were calculated from 2 x 2 tables
with the Fisher's exact model, and Pearson coefficients of
correlation were calculated (Statistical Package for the social
sciences software-SPSS 16 for windows, SPSS Inc.,
Chicago, III, USA).
Ethics This study was undertaken after ethical clearance
from the Institutional Review Board and informed written
consent was taken from each patient after briefing them
regarding the study.

Results and Discussion
The association between GST genotype and susceptibility
was studied in 20 unrelated asthmatic and 23 COPD patients
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residing at varying radial distance from Union Carbide India
Limited (UCIL), using a control group of 50 healthy
individuals. Table-1 summarizes the characteristics of
subjects conducted in this study. In total, 34% of asthmatic
children and 21% of COPD patients were active smokers or
subjects with a past smoking history of 2 to 5 years. With an
average age of 54 (ranging between 35 and 67 years). Low
Body mass index (BMI) among COPD patients were obvious
as compared to asthmatics (20.1 and 23.6 respectively). The
odds of Asthma and COPD occurring in the isocyanate
exposed population of Bhopal was 1.8 and 4.43 times greater
than the not exposed population (ORAsthma, 1.8, 95%CI,0.674.81 ; ORCOPD,6.67, 95%CI, 2.06-21.56). The control group
had significantly higher lung function parameters than the
population under study. Smoking was found to be highest
among the gas affected population viz. 32% as compared to
the control population being 16%. Our data exhibited very
significant association of smoking resulting in asthma and
COPD (ODAsthma,1.6829,95%CI, 0.5417 – 5.2288 ; ORCOPD,
6.67, 95%CI, 2.06-21.56 respectively). Table-2 summarizes
the data found regarding the genotype frequencies for the
RFLP in the GSTP1 gene, as well as the homozygous
deletions of the GSTM1 and GSTT1 genes. Genotype
frequencies (GSTP1, GSTM1, and GSTT1) were found that
GSTP1 genotype was significantly associated with increased
risk of asthma (p = .002). Indeed, the GSTM1 null genotype
was present among 30% of the asthmatics and among 8.6%
of the COPD group. On the other hand, GSTT1 null
genotype was present in 40% asthmatics and 17.39% COPD
subjects. As for the GSTP1, the homozygote GSTP1 Val/Val
genotype and heterozygote Ile/Val genotype was
significantly higher and common among the COPD patients
than in the asthmatic group .
Table- 1
Demographic description of studied
cohort from Bhopal
Asthma
COPD Control
Parameters
(n=20)
(n=23)
(n=50)
Age range (years)
39-62
47-67
35-66
Av.Body Mass Index
23.6
20.1
25.8
(BMI)
Gender
76
84
63
( %Male)
Income below Rs.1000/59
96
55
per month (%)
Smoking history (%)
34
21
16
Av. FEV1/FVC ratio
0.65
0.51
0.83
Within 5 kms. radial
distance of residence from
62
88
59
UCIL plant (Kms.)(%)
Note , FEV1, Forced Expiratory volume in one second, FVC,
Forced Vital Capacity , BMI , Body Mass Index
Subjects with the GSTP1Val/GSTP1Val genotype registered
a 1.22 fold (OR) lower risk of asthma and 2.01(OR) in case

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of COPD patients. This was in comparison with the
GSTP1Ile/Ile genotype (95% CI 0.31–4.74 and 95% CI 0.656.19 respectively). Between both study samples, there was a
significant difference in the frequency of the GSTP1 alleles
(p = .02).
The presence of the GSTT1 null polymorphism was
compared in both groups. The difference showed to be
nonsignificant (P> .05) between COPD and asthmatics,
17.39% and 40%, respectively. The characteristics of the
population studied are shown in table-1. We studied lung
function results from a these subjects with pulmonary
disease. This study showed that the group of individuals who
have normal FEV1/FVC ratio and decreased FEV1 mainly
consists of smokers with higher cumulative tobacco
consumption. About 20 subjects who had a reduced FEV1
(<80% of predicted) could not be classified as having COPD
because they had a FEV1/FVC ratio ≥70%.
In cross-sectional analyses, the percentage of males in the
lower FEV1 group was significantly higher than the
percentage of males in the higher FEV1 group. No significant
differences in the following factors were observed between
the 2 groups – lower FEV1 and higher FEV1, age (range),
Among subjects who had a normal FEV1/FVC ratio and a
decreased FEV1% (the lower FEV1 group), 48.8% had never
smoked. Nevertheless, smokers were more common in the
lower FEV1 group than in the higher FEV1 group (P <
0.0001). The smoking index was also significantly higher in
the lower FEV1 group than in the higher FEV1 group (P <
0.0001).
In contrast, there was no association between the genotype
GSTP1 and the FEV1% predicted or the FEV1/FVC
phenotype. We did not observe any significant association
between any one of these SNPs and the decline in FEV 1.
Furthermore, this study provided preliminary evidence that
functional SNPs in the GST gene influence FEV1 and
FEV1/FVC in healthy subjects independently of the impact of
tobacco smoke, emphasizing a distinct role for GST in
airway obstruction that is a potentially important shared risk
factor for asthma and COPD.
Although smoking is a major, modifiable risk factor,
nonsmokers are similarly at risk for lung function decline.
Therefore, our findings indicate that the group of healthy
subjects with lower FEV1 is a mixture of individuals who are
susceptible to 1 or more irritants including microbial
infections, allergens, or tobacco smoking7. As seen in figure1, GSTT1 and GSTM1 genotypes desplayed weak correlation
(0.001 and 0.28 respectively, p,0.05).
On the other hand, GSTP1 showed significant correlation
0.5, p, 0.05. High smoking index otherwise COPD patients
may be a marker for an increased FEV1 decline and an
increased risk of deteriorating condition chronic

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Res.J.Recent.Sci
inflammatory lung diseases such as asthma and COPD,
indicating the importance of recognizing decreased FEV 1 in
targeting intervention efforts among smokers .

Another limitation of our study is the retrospective nature of
the FEV1 follow-up. The follow-up periods and numbers of
spirometry measurements varied among subjects. This
variation may have contributed to a biased estimation of the
annual decline in FEV1 among subjects; however, we used a
linear mixed-effects model to control for correlations among
repeated measures within each subject, and we limited the
analysis to include only subjects who had at least 4 separate
FEV1 measurements during 2 years follow-up. Nonetheless,
our results will need to be replicated in other, ideally
prospective, studies with large cohorts.

Our study has several limitations that require discussion.
Reversible obstructions, however, are mostly undiagnosed
asthma, and in this study, we carefully excluded asthma by a
detailed questionnaire on pulmonary symptoms (cough,
sputum, exertional dyspnea, wheeze), and physical
examinations including pulmonary auscultation8. In addition,
prebronchodilator lung function has been used in many
epidemiological studies and has been shown to have value in
predicting health outcomes on a population level.

Table – 2
Genotype profile of Asthmatics, COPD and control population in the studied cohort
Chromosomal
location

Gene

Polymorphisms

GSTP1

11q13

Ile105Val

GSTM1

1p13

null allele

GSTT1

22q11

null allele

Genotypes

Asthma

COPD

Control

Ile/Ile
Ile/Val
Val/Val
A(Ile)
G(Val)
null
WT
null
WT

9 (20.93)
5 (11.63)
6 (13.95)
23 (57.5)
17 (42.5)
6
14
8
12

4 (9.3)
11 (25.5)
8 (18.6)
19 (41.3)
27 (55.1)
2
21
4
19

22 (44)
15 (30)
13(26)
59
41
7
43
5
45

yGSTT1 = 0.142x + 49.33
R² = 0.001

% altered GST (s)

60
50

y GSTM1= 1.371x + 43.53
R² = 0.280

40

% GSTP1

30

% GSTM1
20

% GSTT1
yGSTP1 = -3.514x + 42.46
R² = 0.501

10

Linear (% GSTP1)
Linear (% GSTM1)

0
0

2

4

6

Linear (% GSTT1)
8

Smoking index/Radial distance of residence
from UCIL plant (kms)

Figure- 1
Linear Regression analysis of Percent GST (P1, M1 and T1) gene polymorphism versus smoking index and
Radial distance of residence from UCIL plant (giving an estimate of MIC exposure) among studied cohort in
Bhopal
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Vol. 1(ISC-2011), 219-223 (2012)
Res.J.Recent.Sci

Conclusion
Finally, although the GSTP1 genotype, smoking index and
FEV1/FVC ratio is a relatively sensitive index of mild
airflow obstruction, it seems that lower FEV1 in COPD and
asthmatic subjects may be of some help in identifying
subjects at risk for these chronic inflammatory pulmonary
diseases. Given the increasing societal burden of airflow
obstruction, early detection of an altered lung function in
asymptomatic adults is important 9. The demonstration of
increased decline of FEV1, even in healthy adults with lower
FEV1, should alert clinicians to the importance of case
detection in subjects at risk, irrespective of their smoking
status. Increased oxidative stress in COPD patients derive
from the increased burden of inhaled oxidants such as
cigarette smoke and other forms of particulate or gaseous air
pollution and from the increase in reactive oxygen species
(ROS) generated by several inflammatory, immune, and
structural airways cells10. There is increasing evidence that
genetic factors may also contribute to the pathogenesis if
COPD, particularly antioxidant genes, which may confer a
susceptibility to environmental insults such as cigarette
smoke and thereafter development of COPD. In addition, we
identified GSTP1 as associated with natural variation in lung
function, bringing new insight into our understanding of the
genetic basis of lung development and related airway
disorders, such as asthma and COPD.

Acknowledgement

3.

Hirvonen A., Saarikaski S.T., Linnainmaa K., Koskinen
K., Husgafvel-Pursiainen K., Mattson K., Vainio,H.
Glutathione 5-Transferase and /V-Acetyltransferase
Genotypes and Asbestos-Associated Pulmonary
Disorders, J.National Cancer Instt., (88), 24 (1996)

4.

Gilliland F.D., Li Y.F., Dubeau L., Berhane K. , Avol
E., McConnell R. , Gauderman W.J. and Peters J.M.
Effects of Glutathione S-Transferase M1, Maternal
Smoking during Pregnancy, and Environmental
Tobacco Smoke on Asthma and Wheezing in Children,
Am. J. Resp. Crit. Care Med, 166, 457-463 (2002)

5.

Zhao L. and Bracken M.B., Association of CD14 -260
(-159) C >T and asthma, a systematic review and metaanalysis. BMC Medical Genetics, 12,93 (2011)

6.

Hanene C., Jihene L., Jamel A., Kamel H., and Agn`es
H. Association of GST Genes Polymorphisms with
Asthma in Tunisian Children, Mediators of
Inflammation, 1-6 (2007)

7.

Nakamura Y., Miyata M. and Ohba T., et al. Cigarette
smoke extract induces thymic stromal lymphopoietin
expression, leading to T(H)2-type immune responses
and airway inflammation, J Allergy Clin Immunol,
122(6), 1208–1214 (2008)

8.

Mannino D.M., Buist A.S., Petty T.L., Enright P.L. and
Redd S.C. Lung function and mortality in the United
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Examination Survey follow up study, Thorax, 58(5),
388–393(2003)

9.

Svanes C., Sunyer J. and Plana E, et al, Early life
origins of chronic obstructive pulmonary disease,
Thorax, 65(1), 14–20 (2010)

The authors thank Bhopal Memorial Hospital and Research
Centre for financial support and providing necessary
laboratory facilities. We would also appreciate the
encouragement rendered by Dr(Brig) KK Maudar.

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