Page 1 of 44 AJRCCM Articles in Press. Published on October 22, 2009 as doi:10.1164/rccm.200812-1887OC
In utero smoke exposure and maternal and infant GST genes on airway responsiveness and lung function in infancy.
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Jasminka Murdzoska, 1Sunalene G Devadason, 1Siew-Kim Khoo, 1Louis I Landau, 1Sally
Young, 1Jack Goldblatt, 1Guicheng Zhang, 1Peter N Le Souëf, 1Catherine M Hayden 1
School of Paediatrics and Child Health, University of Western Australia, Australia
Descriptor Number: 58
Running Title: Maternal and infant GST genes
Word Count: 4,413
At A Glance Commentary Scientific Knowledge on the subject The effects of in utero smoke exposure on infant lung health may be modulated by maternal and infant GST detoxifying genes. What This Study Adds to the Field Associations between maternal and infant GSTT1 and GSTP1 in combination with in utero smoke exposure on infant airway responsiveness and lung function suggests an important role for GST genes in detoxifying tobacco smoke constituents in utero, thereby influencing infant respiratory health.
Copyright (C) 2009 by the American Thoracic Society.
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Funding Supported by the National Health & Medical Research Council of Australia
Corresponding author: Ms. Jasminka Murdzoska Doctoral Candidate School of Paediatrics and Child Health, University of Western Australia Princess Margaret Hospital Roberts Road, Subiaco WA 6008, AUSTRALIA
This article has an online data supplement, which is accessible in this issue’s table of content online at www.atsjournals.org
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ABSTRACT
Rationale: Xenobiotics in the maternal circulation are capable of crossing the placental barrier so a reduction in the mother and fetus’s detoxification ability due to genetic variation in the Glutathione S-transferases (GSTs) could expose the fetus to higher levels of toxins.
Objective: To investigate the interactive effects of maternal smoking during pregnancy with maternal and infant GST genotypes on airway responsiveness (AR) and lung function in infancy.
Methods: GSTT1, GSTP1 and GSTM1 were genotyped in infants and mothers, in utero smoke exposure was evaluated by questionnaire, AR was assessed by histamine challenge and V’maxFRC was measured using the rapid thoracoabdominal compression technique.
The
interactive effects of maternal smoking during pregnancy with maternal and infant GST genes on AR and lung function at 1, 6 and 12 months and longitudinally throughout the first year were investigated.
Main Results: Infant and/or maternal GSTT1 non-null was associated with reduced AR at 12 months and throughout the first year and increased V’maxFRC at 6 months. Maternal GSTP1 Val/Val or Ile/Val was associated with increased V’maxFRC at 6 months. In infants exposed to in utero smoke, infant and/or maternal GSTT1 non-null was associated with reduced AR at 1 month and throughout the first year and increased V’maxFRC throughout the first year. Maternal GSTP1 Val/Val or Ile/Val was associated with increased V’maxFRC at 6 months.
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Conclusions: GST genes may be especially important during fetal development as they may modify, through proficient detoxification, the effects of in utero maternal smoke exposure on AR and lung function in infants.
Word Count: 251
Key Words: Infant lung function, airway responsiveness, GST genes, maternal smoking during pregnancy
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INTRODUCTION
In utero exposure to tobacco constituents has been shown to have profound deleterious effects on lung maturation and the development of the respiratory system in the fetus (1). Normal lung development in utero is essential in order to attain maximum lung health in adulthood (2, 3) and involuntary exposure to tobacco smoke during this period may contribute to serious respiratory health problems later in childhood (3-5). A single inhaled breath of cigarette smoke contains over one trillion free radicals which directly contribute to the formation of reactive oxygen species and subsequent oxidative stress (6). If a mother smokes from the time of conception, the level of fetal exposure to these reactive oxygen species is comparable to that of an active smoker (7). Tobacco smoke constituents need to be metabolised and detoxified to minimise oxidative stress and toxic effects to the mother and fetus. A combination of genetic and environmental factors is likely to determine the variability in individual susceptibility to exogenous toxins in utero.
The glutathione S-transferases (GSTs) are an important family of enzymes involved in the detoxification of xenobiotics, including tobacco by-products. Eight cytosolic GSTs have been identified in humans: alpha (A), mu (M), pi (P), theta (T), kappa (K), omega (O), Sigma (S) and Zeta (Z); each class consists of several distinct subclasses with some overlap in tissue expression (8, 9) which suggests some redundancy in the GST system. Human GSTT1 has been shown to be predominately expressed in erythrocytes (8) with some expression in the placenta (10). GSTP1 has also been shown to be expressed in the placenta (10) as well as in the lung epithelium (11), and GSTM1 is predominately expressed in the liver (8). Deletions of the GSTT1 and
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GSTM1 genes result in the complete loss of enzyme function.
Approximately 50% of
Caucasians are homozygous for a deletion in GSTM1 and 20-25% for a deletion for GSTT1 (12). GSTP1 is a polymorphic gene that contains a substitution at codon 105 (Ile105Val), which has been shown to result in altered enzyme activity (13).
Thus, inherited variations in these
detoxification genes may modify the metabolism and excretion of xenobiotics from the body, which may explain the varying susceptibility to the adverse health effects of cigarette smoke.
Since almost all xenobiotics present in the maternal circulation are capable of crossing the placental barrier and accumulating in fetal organs (14), a reduction in the mother’s natural ability to detoxify xenobiotics will expose the fetus to higher levels of these toxins. GST enzymes are present in large amounts in embryonic and fetal tissues throughout gestation, suggesting that both have the ability to detoxify exogenous pollutants which may cross the trophoblastic tissue (15-17). As a result, in addition to the mother’s detoxification capabilities, the fetus’s own xenobiotic defence system may play a role in protection against maternally inhaled toxic compounds. To our knowledge, there are no reports in the literature examining the effects of in utero exposure to tobacco smoke in combination with maternal and infant GST genotypes on lung function and airway responsiveness in early infancy.
In the present study, we aimed to investigate the possible interactive effects of maternal smoking during pregnancy with maternal and infant GSTT1, GSTM1 and GSTP1 genotypes on lung function and airway responsiveness throughout the first year of life. We hypothesized that if infants and their mothers both have deletions for the GSTT1 and GSTM1 genes, and the GSTP1 105 substitution those infants would be more vulnerable to in utero smoke exposure, resulting in
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decreased lung function and increased airway responsiveness in infancy. Some of these data have been previously reported in the form of an abstract (Am. J. Respir. Crit. Care Med., Apr 2009; 179: A5408.)
METHODS
THE PERTH INFANT ASTHMA FOLLOW-UP (PIAF) STUDY POPULATION
The PIAF cohort consisted of 253 unselected Caucasian children recruited before birth from expectant mothers attending a local metropolitan antenatal clinic (Osborne Park Hospital, Perth, Australia) between June 1987 and November 1990. At the time of enrolment, information on parental history of asthma and smoking during pregnancy were collected. The exclusion criteria included premature birth, congenital abnormalities and respiratory symptoms in the first month after birth (18). The study was approved by the Ethics Committee of Princess Margaret Hospital for Children.
ANTENATAL AND POSTNATAL SMOKING ASSESSMENT
Information on the mother’s smoking status before and during each trimester of pregnancy was assessed via parental questionnaire at each study visit. Fetal exposure to maternal tobacco smoke was classified as exposed if the mother reported ever smoking during the pregnancy and non-exposed if the mother had never smoked while pregnant. No objective measurements of maternal smoking during pregnancy (eg. cotinine levels), were available for the cohort.
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LUNG FUNCTION AND AR ASSESSMENTS
At 1 month, 6 months and 12 months, maximal flow at functional residual capacity (V’maxFRC) was measured using the rapid thoraco-abdominal compression technique during tidal breathing (18). To determine AR, histamine was administered via a nebuliser until there was either a 40% reduction in V’maxFRC or the final dose of 16 mg/ml had been administered.
AR was
expressed as a dose-response slope (DRS) and calculated by the percent reduction in V’maxFRC per administered dose of histamine (18).
GENOTYPING
DNA was extracted from blood samples that were obtained from the participants at the 6 and 12 year follow-up. The presence of the deletion in GSTT1 and GSTM1, in the mothers and their children, was determined using a polymerase chain reaction (PCR) procedure (19).
For
confirmation of genotype, a second PCR was performed on a subset of samples using different sets of primers (20, 21). In addition, primers for an internal positive control (CYP1A1) were included with each PCR. Individuals with at least one GSTT1 or GSTM1 allele were categorized as gene non-null and individuals with a homozygous deletion were categorized as gene null. The substitution polymorphism in GSTP1 was also determined using a PCR based procedure (22).
STATISTICAL ANALYSIS
The V’maxFRC data was approximately normally distributed and hence parametric tests were utilised.
However, the distribution of the DRS values was skewed so the data were loge
transformed prior to analysis. A constant of 1 was added to each measurement to allow for the loge transformation when DRS was 0. Analysis-of-variance and independent sample T tests
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were used to assess for statistical significance between V’maxFRC and DRS at 1, 6 and 12 months of age and the maternal and infant’s GSTT1, GSTM1 and GSTP1 genotypes. The analysis was repeated using linear regression models, adjusting for potential confounders such as gender, weight and length at 1, 6 and 12 months of age (expressed as padj). Genotypes from the 3 genes were stratified by maternal smoking during pregnancy and the effect on V’maxFRC and DRS was investigated. To further investigate the interactions between in utero exposure to maternal smoke, GST genotypes and V’maxFRC and DRS throughout the first year of life, V’maxFRC and DRS measurements from 1, 6 and 12 months were analysed longitudinally and a Generalized Estimating Equations (GEE) technique was utilized, accounting for within individual covariance and adjusting for random effects (details in on-line supplement). We used a general linear model for the longitudinal data analyses with a categorical variable representing the different time points in the model. Likelihood-ratio tests were employed to examine the interaction terms by comparing an unrestricted (with interaction) and a restricted model (without interaction) (23).
Analyses were performed using the statistical software package SPSS
(Version 15) except for GEE regression analyses and interaction tests using the STATA software package (Intercooled Stata 9).
RESULTS POPULATION CHARACTERISTICS
Of the 253 infants originally recruited, DNA was available for 180 of whom 179 (58% male) were genotyped for GSTT1, GSTM1, and 180 for GSTP1, and 163 mothers of whom 162 were genotyped for the 3 genes. Infant and maternal GST genotype frequencies are reported in Table 1. Data on genotyped infants, V’maxFRC and AR at 1, 6 and 12 months are shown in Figure 1.
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There were no significant differences in maternal smoking, gender, V’maxFRC or AR between the genotyped and non-genotyped infants (data in online supplement).
With regards to smoking during pregnancy, 32% of infants were classified as being exposed to maternal smoke in utero, with 5% of mothers smoking in the first trimester only, 2% in the second trimester only, 1% in the third trimester only, 1% at some stage during the pregnancy then quitting and 23% throughout the entire pregnancy.
AR, LUNG FUNCTION AND IN UTERO SMOKE EXPOSURE
As reported previously by Young et al (2000) in this same cohort, our respective analyses confirmed that in utero exposure to maternal smoke had a significant effect on lung function throughout the first year of life (p
