Association of Early-life Exposure to Household Gas Appliances and Indoor Nitrogen Dioxide With Cognition and Attention Behavior in Preschoolers

Abstract

The authors investigated the association of early-life exposure to indoor air pollution with neuropsychological development in preschoolers and assessed whether this association differs by glutathione-S-transferase gene (GSTP1) polymorphisms. A prospective, population-based birth cohort was set up in Menorca, Spain, in 1997–1999 (n = 482). Children were assessed for cognitive functioning (McCarthy Scales of Children's Abilities) and attention-hyperactivity behaviors (Diagnostic and Statistical Manual of Mental Disorders, 4th Edition) at age 4 years. During the first 3 months of life, information about gas appliances at home and indoor nitrogen dioxide concentration was collected at each participant's home (n = 398, 83%). Genotyping was conducted for the GSTP1 coding variant Ile105Val. Use of gas appliances was inversely associated with cognitive outcomes (β coefficient for general cognition = −5.10, 95% confidence interval (CI): −9.92, −0.28; odds ratio for inattention symptoms = 3.59, 95% CI: 1.14, 11.33), independent of social class and other confounders. Nitrogen dioxide concentrations were associated with cognitive function (a decrease of 0.27 point per 1 ppb, 95% CI: −0.48, −0.07) and inattention symptoms (odds ratio = 1.06, 95% CI: 1.01, 1.12). The deleterious effect of indoor pollution from gas appliances on neuropsychological outcomes was stronger in children with the GSTP1 Val-105 allele. Early-life exposure to air pollution from indoor gas appliances may be negatively associated with neuropsychological development through the first 4 years of life, particularly among genetically susceptible children.

Evidence of a link between air pollution and increased risk of cerebrovascular and neurodegenerative diseases is emerging (1, 2). Furthermore, indices of deleterious effects of air pollution on children's neurodevelopment have recently been reported (3, 4). Prenatal exposure to airborne polycyclic aromatic hydrocarbons has been associated with a lower mental development index at age 3 years (3). A long-term concentration of black carbon particles from mobile sources has been associated with decreases in cognitive test scores among children aged 8–11 years (4). In addition, some environmental factors have been related to development of attention deficit hyperactivity disorder (ADHD) symptoms (5), but no studies are known to have examined the influence of air pollution.

Although outdoor air pollution first brought the issue of the health effects of air pollution to the public's attention, indoor air pollution likely has the greatest impact on children's health and constitutes a public health problem in both developed and developing countries (6, 7). Among the major sources of indoor air pollution are combustion by-products from heating and cooking. Because the prevalence of gas stoves in developed countries is high (50%–70%), any evidence of a deleterious effect on health represents a major public health issue (8). Domestic use of gas-fueled cooking and heating appliances can produce high indoor levels of nitrogen dioxide, the most toxic of oxides of nitrogen and the most extensively studied indoor pollutant (9). Nitrogen dioxide cell damage is mediated by oxidant injury, inflammatory response, and lipid peroxidation (10). The glutathione S-transferase P1 gene, GSTP1, encodes major phase II xenobiotic metabolizing enzymes involved in antioxidant defenses to protect against oxidative stress (11). GSTP1 is the most strongly expressed glutathione S-transferase isoenzyme in the human brain during early life (12). Given the oxidative nature of nitrogen dioxide, we hypothesize that detoxification genes may modify the neurologic effect of nitrogen dioxide.

In this study, we aimed to investigate the effect of early-life exposure to household gas appliances and indoor nitrogen dioxide on cognitive functioning and inattention-hyperactivity symptoms in preschoolers. We also assessed whether these effects differ by polymorphisms of GSTP1.

MATERIALS AND METHODS

Study population

A population-based birth cohort was recruited on the island of Menorca (Spain) (13). Recruited were all women presenting for prenatal care in Menorca over 12 months starting in mid-1997. Subsequently, 482 children (94% of those eligible) were enrolled, and complete outcome data were provided for 422 (87%) up to 4 years of age. Of these 482 children, 411 (85%) were genotyped. The present paper is based on 398 children with complete information on neurodevelopmental assessment, gas appliances at home, and indoor nitrogen dioxide concentrations.

After the study was explained to the parents, their written informed consent was obtained. The ethics committee of the Institut Municipal d'Investigació Mèdica approved this study.

Data collection procedures

In the longitudinal study, detailed data on maternal and paternal years of education, maternal and paternal social class (using the United Kingdom Registrar General's 1990 classification according to parental occupation by ISCO-88 (International Classification of Occupations) code), maternal marital status, maternal health and obstetric history, parity, dietary intake during pregnancy, child's sex, maternal alcohol consumption during pregnancy, parental tobacco smoking habits, and type and duration of breastfeeding were obtained through questionnaires administered in person. Gestational age and anthropometric measures at birth were collected from clinical records.

Outcomes of interest

Two certified psychologists performed the neuropsychological testing of the children at age 4 years, including assessment of cognitive functioning, motor abilities, and ADHD symptoms, as described in detail previously (14–16). This process was supervised by the project's consulting psychologist (including intra- and interpsychologist validity at the beginning, midpoint, and end of the study), who rescored the tests. Staff members involved in the neuropsychological testing were blinded to a child's exposure.

Neurocognitive functions and motor skills were measured with the Spanish version of the McCarthy Scales of Children's Abilities test, consisting of 6 different scales (general cognitive, verbal, perceptual-performance, quantitative, memory, and motor) (17). To further improve our understanding of the neurocognitive functions, we reorganized the items from the McCarthy test into a new outcome, the executive function subscale, according to present neuropsychological assessment standards. The executive function involves coordinating complex behaviors such as attention and working memory. The new subscale showed good psychometric characteristics (14). Extensive information about the outcome assessments, including scale and subscale descriptions, evaluation procedures, and validity of the instruments, has been published elsewhere (14).

ADHD was evaluated by using Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, criteria (18). This internationally recognized questionnaire comprises 18 items designed to evaluate attention-deficit (items 1–9), hyperactivity (items 10–15), and impulsivity (items 16–18) symptoms in children. The original Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, list was used, which rates items as yes or no (15, 16). The ADHD Diagnostic and Statistical Manual of Mental Disorders, 4th Edition questionnaire was administered to children's teachers and mothers at the same time to assess interobserver reliability. We found a moderate, but significant association (Spearman r = 0.27; P < 0.001) between mothers and teachers, similar to that reported in other studies (19).

Exposure assessment

During a subject's first 3 months of life, a trained field technician visited the home to complete a questionnaire on household characteristics (cooking appliances, heating and cooling systems, number of hours of ventilation, and size of the house), to collect dust samples, and to measure nitrogen dioxide, as previously described (20). Briefly, average 2-week nitrogen dioxide concentrations were measured by passive diffusion tubes installed in the living room wall at a height of 2 m and away from any window or air conditioner (21). Nitrogen dioxide concentrations were measured in a single laboratory by colorimetric reaction, as previously described (20).

Genotyping methods

DNA was extracted from blood (87%) and saliva (13%) samples. Two semiautomated assays were implemented to facilitate detection of the coding variant Ile105Val in GSTP1; that variant was analyzed by using pyrosequencing technology (Biotage, Uppsala, Sweden) in a single assay. All assays were performed by technicians blinded to neurodevelopmental outcomes and exposure information. GSTP1 genotypes were in Hardy-Weinberg equilibrium in the total analyzed cohort (P > 0.05).

Statistical analysis

Continuous cognitive outcomes were standardized to a mean of 100 with a standard deviation of 16 to homogenize all the scales. The criterion for ADHD was the presence of either 6 or more symptoms of inattention or 6 or more symptoms of hyperactivity-impulsivity. Both components (inattention and hyperactivity) were also studied separately. To limit exposure misclassification, we decided to use all available exposure predictors and estimate indoor nitrogen dioxide concentration by using a regression model, regressing the 2-week average values of nitrogen dioxide against type of stove (electric, gas), gas fire (no, yes), use of extractor fan when cooking (always, sometimes, never), number of gas appliances at home, and season (winter, spring, summer, autumn). Predicted nitrogen dioxide derived from the regression model was used as the exposure variable. To evaluate the linearity of the relation between predicted nitrogen dioxide as continuous and McCarthy Scales of Children's Abilities's cognitive outcomes, we used adjusted general additive models (through nonparametric depiction of the predictor when the effects of the other variables had been taken into account) (22).

Multivariate linear regression models were used to examine the association between gas appliances or indoor nitrogen dioxide concentration (both continuous and in quartiles) and cognitive outcomes at age 4 years. We used logistic regression to measure the associations of indoor air pollution with ADHD. All the variables significantly related to the outcomes of interest (P < 0.20) were included in the multivariate model, and they were retained only if they modified the coefficient of predictor variables during pregnancy by more than 10%. Final multivariable models were adjusted for maternal social class, maternal education, school trimester at testing, evaluator (neuropsychologist), maternal smoking during pregnancy, number of smokers at home, maternal alcohol consumption during pregnancy, and home location (urban vs. nonurban). Allowance for other possible confounding variables such as methylmercury, prematurity, and low birth weight did not materially alter the estimates. To evaluate whether the effect of the evaluated exposures differed by polymorphisms of GSTP1, we stratified the analysis according to GSTP1 genotypes. The presence of gene-environment interactions between indoor nitrogen dioxide and GSTP1 polymorphisms was assessed by including interaction terms in the regression model. All statistical analyses were conducted with Stata 8.0 statistical software (Stata Corporation, College Station, Texas).

RESULTS

Compared with nonparticipants, participants had higher general cognitive scores and were of a higher social class (Table 1). There were nonstatistically significant differences between participants and nonparticipants regarding ADHD symptoms prevalence, gas appliances at home, and indoor nitrogen dioxide concentrations. The prevalence of cooking with gas was 71.1% and of heating with gas fires was 23.6%. A total of 213 (53.5%) homes had 1 gas appliance, and 82 (20.6%) had 2 gas appliances. GSTP1 allelic frequencies did not differ between participant and nonparticipant preschoolers. The GSTP1 genotype prevalences of the polymorphic Ile105Val were 43.6% for Ile/Ile, 47.5% for Ile/Val, and 8.9% for Val/Val.

Concentrations of indoor nitrogen dioxide were, as expected, higher in homes in which gas was used for cooking, when an extractor fan was not used, and in which individual gas fires were used (Table 2). Indoor nitrogen dioxide concentrations significantly increased with increasing number of gas appliances. Although indoor nitrogen dioxide concentration increased with number of parental smokers at home, no statistically significant association was found. Seasonal variations were observed, with winter measurement levels being higher; however, no significant differences were found by home location.

Predicted indoor nitrogen dioxide concentration increased significantly with low social class and low level of education. Sex, gestational age, birth weight and height, and breastfeeding of the child as well as maternal smoking and maternal alcohol consumption during pregnancy were not associated with indoor nitrogen dioxide concentration (Table 3).

Exposure to gas appliances at home was inversely associated with general cognitive, memory, verbal, and executive function scores, with an increasing relation between number of gas appliances and cognitive scores (Table 4). Children exposed to gas appliances were at higher risk of ADHD symptoms. The 2 components of ADHD were analyzed separately, and results showed that exposure to gas appliances was associated with inattention but not with hyperactivity. Adjustment by parental social class, parental educational level, breastfeeding, maternal alcohol consumption and tobacco smoking during pregnancy, and number of smokers at home did not confound these associations.

We observed a linear relation between predicted nitrogen dioxide concentration and the different cognitive outcomes of interest (P gain > 0.30), indicating a linear relation with neurodevelopment outcomes at age 4 years and indoor nitrogen dioxide concentration (Figure 1). A statistically significant negative dose-response effect was found between indoor nitrogen dioxide levels and cognitive outcomes at age 4 years, including general cognitive function, verbal function, and executive function (Table 4). Moreover, children exposed to higher levels of indoor nitrogen dioxide were at higher risk of developing ADHD symptoms (odds ratio = 1.04, 95% confidence interval: 1.00, 1.09; P = 0.04). The negative effect was restricted to the inattention component (odds ratio = 1.06, 95% confidence interval: 1.01, 1.12; P = 0.024). Similar results were obtained for children in the highest quartile of indoor nitrogen dioxide: ADHD symptoms (odds ratio = 2.52, 95% confidence interval: 0.96, 6.64; P = 0.06) and inattention (odds ratio = 2.88, 95% confidence interval: 0.91, 9.11; P = 0.07).

To evaluate residual confounding by socioeconomic factors and parental characteristics, a sensitivity analysis was carried out. Exposure to gas appliances at home was also associated with a decrease (in points) in children's global cognitive score at age 4 years for children of a high social class (β = −8.73, 95% confidence interval: −14.04, −3.41) and from families with a high educational level (β = −10.92, 95% confidence interval: −25.11, 3.26) as well as for children not exposed to active maternal smoking during pregnancy (β = −5.21, 95% confidence interval: −10.03, −0.38). The results were essentially the same for indoor nitrogen dioxide exposure.

Adverse effects of exposure to gas appliances and indoor nitrogen dioxide on cognitive functioning and ADHD symptoms were found in children with any GSTP1 Val-105 allele but not in children with the Ile/Ile genotype (Table 5). Exposure to gas appliances was associated with a decrease of 9.72 (95% confidence interval: −17.13, −2.30) points in general cognitive score and with an increased risk of inattention for children with any GSTP1 Val-105 allele. In addition, indoor nitrogen dioxide exposure was negatively associated with general cognitive (P for interaction = 0.04), verbal (P for interaction = 0.30), and executive (P for interaction = 0.13) function scores. An increased risk of developing inattention symptoms was found for children with any GSTP1 Val-105 allele (P for interaction = 0.26).

DISCUSSION

We found that early-life exposure to household gas appliances is associated negatively with general cognitive functioning and with a higher risk of developing ADHD symptoms at age 4 years. A negative dose-response effect was found even after adjustment for social class, educational level, and tobacco smoke and alcohol exposures. A higher effect was found for some cognitive subareas, specifically verbal and executive function, and for the inattention component of ADHD. Moreover, we found a decrease in cognitive functioning and an increased risk of developing inattention symptoms in children exposed to higher levels of indoor nitrogen dioxide. Children with the GSTP1 Val-105 allele were at higher risk of the adverse effects of gas appliances and indoor nitrogen dioxide exposure on cognitive function and ADHD symptoms. A statistically significant interaction between indoor nitrogen dioxide concentration and GSTP1 polymorphisms was found for general cognitive functioning.

Several biologic mechanisms could explain the negative association between exposure to gas appliances at home and neuropsychological outcomes in preschoolers. Gas appliances produce complex mixtures of volatile organic compounds, sulfur dioxide, water vapor, particulates, carbon dioxide, and oxides of nitrogen (8). It has been shown that urban particulate materials induce proinflammatory cytokines in human bronchial epithelial cells (23), lung epithelial cells (24), and macrophages (25). The interaction of macrophages with epithelial cells amplifies cytokine production in those cells, and these cytokines are also present in the blood of subjects during an episode of acute atmospheric air pollution. Furthermore, there is evidence that oxidative stress and induced inflammation translate systemically (26, 27). We postulate that an inflammatory systemic response associated with indoor pollution derived from use of gas appliances might adversely affect child neurodevelopment.

Inhalation of nitrogen dioxide can induce structural and functional changes in lungs; alteration of the body's defense system; and abnormal biochemical, physiologic, and enzymatic reactions (28). Epidemiologic studies have shown a relation between indoor nitrogen dioxide exposure and respiratory and allergic diseases (29, 30). The inflammatory airway response to nitrogen dioxide was not assessed in our study. However, it is well established that nitrogen dioxide–mediated lung injury may be causally related to generation of higher levels of proinflammatory mediators including nitric oxide and other cytokines, such as interleukin-8, interleukin-1β, and tumor necrosis factor-α. In addition, early changes in bronchial cellular events may lead to a cascade of more widespread systemic inflammatory responses of the lung, which may cause further damage to the surrounding cells and tissue and even may have systemic effects in other organs including the heart and brain.

Information about neurologic effects of nitrogen dioxide exposure is limited. In guinea pigs, nitrogen dioxide intoxication (exposure to 5 ppm and 10 ppm for 2 hours daily for 5 weeks) has been reported to induce significant depletion of the total lipids and phospholipids content as well as the cholesterol diminution content of the cerebral hemisphere, cerebellum, and midbrain (31). Interestingly, the rate of lipid peroxidation was found to be enhanced in all brain areas investigated, and, with increasing dosage, the effect was correspondingly pronounced (31). It is noteworthy that lipids are essential components of all cellular elements of the central nervous system. Biomembranes and subcellular organelles are the major sites of the lipid peroxidation damage. The peroxidation changes triggered by free radicals in the brain lipids may be of importance in the development of brain cell damage.

A strong, adverse effect of indoor nitrogen dioxide exposure on cognitive function was observed regarding executive function (i.e., cognitive tasks performed by a predominant activation of the prefrontal cortex). This neurologic area, including its neural circuitry, is innervated by the monoamines dopamine, noradrenaline, and serotonin. Alterations in brain concentrations of catecholamines have been reported in relation to other pollutant gases. Experimental studies have found that prenatal ozone exposure disrupts the cerebellar catecholamine system, showing a decrease in dopamine and noradrenaline mainly in the early postnatal period (32). When ozone is inhaled, it produces reactive oxygen species that can reach the central nervous system through the bloodstream, producing oxidative stress. Oxidative stress caused by ozone exposure increases lipid peroxidation in different brain structures. Dopamine has an oxidative metabolism that makes dopaminergic neurons and their fibers especially sensitive to oxidative stress. Tyrosine hydroxylase is the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine. Tyrosine hydroxylase is inhibited and nitrated at tyrosine residues in vitro by nitrogen dioxide and in vivo by drugs that damage dopamine neurons (33). In addition, the dopaminergic system plays a role in the pathogenesis of ADHD (34). Nitrogen dioxide could probably impair dopaminergic neuron functioning by inducing oxidative stress and by inhibiting dopamine biosynthesis, thus resulting in a poor executive function score and development of ADHD symptoms.

We found a stronger adverse effect of indoor nitrogen dioxide concentration in children with any GSTP1 Val-105 allele. GSTP1 is involved in antioxidant defenses to protect against oxidative stress (11), detoxifying lipid peroxidation products and DNA oxidation products, and it represents the most strongly expressed glutathione S-transferase isoenzymes in the human brain during early life (12). The GSTP1 Ile105Val substitution is located near the substrate binding site of the enzyme, resulting in a less active enzyme (35). Our results suggest that, for those children with the less active GSTP1 Val-105 variant, brain cells are more susceptible to biochemical changes induced by early-life exposure to nitrogen dioxide.

Our study has several strengths, including a population-based birth cohort with scales individually administered to parents and teachers. Additionally, we validated the McCarthy Scales of Children's Abilities new scale (executive function) and applied a strict protocol to reduce interobserver variability during testing, increasing robustness of the psychometric measures. Thus, we found similar effects in related areas measured with 2 different methods (the executive function from the McCarthy test performed by a neuropsychologist and the inattention symptoms from the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, test reported by teachers). Moreover, measurement of exposure was obtained from the model of a long-term mean based on variables regressed against 2-week nitrogen dioxide measurements collected at each participant's home, and the neurodevelopmental areas affected were the same as those found to be related to gas appliances when measured through questionnaire. Finally, the gene-environment interaction we found reinforces the plausibility of neurotoxic effects of nitrogen dioxide.

Children included in the study did not differ from nonparticipants regarding gas appliances and indoor nitrogen dioxide exposure. Higher cognitive scores and social class of participants in comparison with nonparticipants, if anything, would have diminished the strength of our findings. Moreover, data on individual exposure to household gas appliances, nitrogen dioxide measures, and the presence of GSTP1 polymorphisms were obtained without knowledge of neuropsychological outcomes. Thus, differential information bias is unlikely to have affected these associations.

However, our study has some limitations. The small numbers of subjects in each subgroup limit the conclusions about interactions between indoor nitrogen dioxide concentrations and GSTP1 genotypes in a single study, although control of missing observations by using the method of multiple imputations did not change the results. We included a large number of covariates, but we were unable to control for parental IQ, mental health, or problematic family functioning, which might be linked to assessed exposures. Moreover, it is still possible that the associations we found in this study with nitrogen dioxide could be attributable to unmeasured indoor air contaminants, such as particulates derived from gas appliances. This is supported by the fact that, in Menorca, the principal source of gas appliances is butane bottles, a fuel more toxic than natural gas (36). In addition, information about the contribution of outdoor nitrogen dioxide to indoor nitrogen dioxide levels was lacking. However, Menorca is an island with little industry and traffic, and the association with nitrogen dioxide was invariable after we adjusted for urban home location. In fact, an increase in hours of ventilation in our study population was associated with a statistically significant decrease in levels of measured indoor nitrogen dioxide (20), indicating that nitrogen dioxide in our population mostly originated from indoor sources. Furthermore, adjustment for organochlorinated compounds and methylmercury, as well as maternal smoking or alcohol consumption during pregnancy, did not confound the observed associations (data not shown). Finally, exposure assessment was limited to early life only, and conclusions could not be extended to further time periods of exposure, although we expected the larger effect during early life, when children spend most of their time at home and critical brain growth occurs. To our knowledge, this study is the first to analyze the effect of indoor air pollution on cognitive functioning and ADHD symptoms in preschoolers and the influence of glutathione S-transferase polymorphisms. We cannot exclude the possibility that findings may have been confounded by other genetic variants in linkage disequilibrium with the variant under study or by population stratification. However, GSTP1 is the most strongly expressed of the glutathione S-transferase isoenzymes in the human brain, its expression is observed as early as 12 weeks of gestation, and it is highly expressed in the blood-brain barrier, which supports the hypothesis that GSTP1 may be the susceptibility gene rather than a linkage disequilibrium marker. Moreover, the role of maternal genotypes in modifying the adverse effects of early-life exposure to household gas appliances and indoor nitrogen dioxide, as well as maternal-fetal gene interaction, should be assessed in future studies.

In conclusion, current data provide preliminary evidence that early-life exposure to indoor air pollution from gas appliances may be related to impaired cognitive functioning among preschoolers and may increase their risk of developing ADHD symptoms. Children with the GSTP1 Val-105 allele appear more susceptible to adverse neuropsychological effects of indoor air pollution from gas appliances, suggesting oxidative stress as a potential mechanism. In view of widespread use of gas appliances, confirmation of the present results in coming studies could have important public health implications.

Abbreviations

Abbreviations
 
• ADHD

  attention deficit hyperactivity disorder

 
• GSTP1

  glutathione S-transferase P1

Author affiliations: Center for Research in Environmental Epidemiology (CREAL), Barcelona, Catalonia, Spain (Eva Morales, Jordi Julvez, Mònica Guxens, Nino Künzli, Jordi Sunyer); Municipal Institute of Medical Research (IMIM-Hospital del Mar), Barcelona, Catalonia, Spain (Eva Morales, Jordi Julvez, Mònica Guxens, Nino Künzli, Jordi Sunyer); Preventive Medicine and Public Health Educational Unit IMAS-UPF-ASPB, Barcelona, Catalonia, Spain (Eva Morales); Area de Salud de Menorca, IB-SALUT, Menorca, Spain (Maties Torrent); Genes and Disease Program, Centre for Genomic Regulation (CRG), Barcelona, Catalonia, Spain (Rafael de Cid, Mariona Bustamante); Centro Nacional de Genotipado (CeGen), Barcelona, Catalonia, Spain (Rafael de Cid, Mariona Bustamante); CIBER Epidemiología y Salud Pública (CIBERESP), Barcelona, Catalonia, Spain (Eva Morales, Mònica Guxens, Mariona Bustamante, Nino Künzli, Jordi Sunyer); and Department of Experimental Sciences, Universitat Pompeu Fabra (UPF), Barcelona, Catalonia, Spain (Jordi Sunyer).

This work was supported by grants from the Spanish Ministry of Health ((FIS-PI041436, FIS-PI041705, and FIS-PI051187), Instituto de Salud Carlos III (Red INMA G03/176 and CB06/02/0041), and CIBER en Epidemiología y Salud Pública (CIBERESP)); the Generalitat de Catalunya-CIRIT (1999SGR 00241); and Genome Spain.

The authors acknowledge all teachers and parents of the children from the island of Menorca for patiently responding to the questionnaires; the psychologists who coordinated the fieldwork; and the nurses and administrative personnel from the Primary Health Care Center of Maó, who provided administrative, technical, and material support.

Conflict of interest: none declared.

References