Even though the need to identify asthma endotypes has grown within the last decade, specific environmental toxicants, except for secondhand cigarette smoke, have not been directly examined as their causal contributors to date. Here, we characterize children with either non-atopic or atopic asthma, in terms of main exposure of interest, B[a]P, and multiple indicators of pathophysiology, including clinical history, lung function deficit diagnosis, and two biomarkers, 15-Ft2-isoP and 8-oxodG. To the best of our knowledge, our analysis demonstrates for the first time that not only the childhood exposure level to B[a]P but also the roles of two systemic oxidant markers, 15-Ft2-isoP and 8-oxodG, are markedly divergent between the non-atopic asthmatic versus atopic asthmatic children. Our postulate of endotypes is further supported by overall dissimilar pattern of co-morbid events during the children’s first three years of life, preceding the present diagnosis. Namely, the so-called atopic march (e.g. allergic rhinitis, upper respiratory infection) is absent among the non-atopic asthmatic children. On the other hand, the atopic control children are associated with highest prevalence of the atopic march diagnoses. Furthermore, contrary to current body of evidence supporting adulthood onset of the non-atopic asthma , our data suggest for the first time that the lung function deficit during early childhood as critical sentinel event preceding non-atopic asthma. Collectively, following lines of evidence suggest that childhood exposures to B[a]P contributes toward non-atopic asthma, while the atopic one arises through B[a]P-independent mechanisms among the lean children.
First, among the lean children, B[a]P is not associated with elevated odds of atopic asthma, while it is associated with robust increase in the odds of non-atopic asthma. For example, a unit B[a]P exposure is not associated with asthma among the lean atopic boys, while the same exposure predicts 10-times greater odds of asthma in the lean non-atopic boys. The non-atopic asthmatic boys with highest exposure to B[a]P (median, 20 ng/m3) were also positively diagnosis with lung function deficit, compared to the non-atopic controls (median, 4.3 ng/m3). In contrast, in the lean atopic boys, median B[a]P was uniformly low in those with or without the deficit and with or without asthma. Such trends suggest that childhood exposure to elevated B[a]P level contributes toward a development of non-atopic asthma, while the atopic asthma occurs through B[a]P-independent mechanisms in lean children.
Second, B[a]P is associated with plasma 15-F2t-IsoP and urinary 8-oxodG, respectively, in an opposite fashion between the non-atopic and the atopic children. While B[a]P and F2t-isoP are inversely associated among the non-atopic OV/OB girls, the same association is null among the atopic OV/OB girls (Fig. 4). Among the non-atopic lean girls, the same association is also weakly positive. Such non-linear dose-response suggests the ambient B[a]P concentration might pose pro-inflammatory risk at low concentration, which subsequently switches to anti-inflammatory mechanism activation beyond a certain threshold B[a]P level . Earlier investigations support hierarchical oxidative stress phases, posed by multiple oxidants within air pollution exposures . At low exposure concentration, the ambient B[a]P pose pro-inflammatory risk, which subsequently switches to anti-inflammatory mechanism activation beyond a certain threshold . Moreover, the adjustment for F2t-isoP and 8-oxodG, respectively, in the regression models are associated with large decrements in B[a]P-asthma effect sizes for the non-atopic girls (Table 3). Conversely, the atopic girls are associated with an increased B[a]P-asthma associations, following the same adjustments. Such divergent trends suggest that B[a]P might initiate and/or exacerbate distinct mechanisms for the non-atopic versus atopic asthma. Our earlier analyses have shown robust activation of anti-inflammatory mechanisms in children with high B[a]P exposure as well as a severe outcome . Thus, while F2t-isoP and 8-oxodG concentrations seem suppressed among those who develop non-atopic asthma, the same oxidants appears to pose mildly pro-inflammatory role among the atopic girls and corresponding increased odds of atopic OV/OB asthma (Table 3, Figs. 3 and 4).
Third, question whether OV/OB asthma represents a unique endotype or phenotypic consequence remains unanswered [4, 30]. While multiple endotype might exist within so-called OV/OB asthma, a unit B[a]P exposure is associated with vastly different estimated between non-atopic and the atopic girls within our case-control children. Among the non-atopic girls, a unit B[a]P exposure is associated with a modest increase in the odds of lean asthma (aOR, 27.4; 95% CI: 3.2 to 237.1) and OV/OB asthma (aOR, 46.1; 95% CI: 1.7 to 1271.4), respectively. In contrast, the same unit exposure is associated with markedly lower odds of OV/OB asthma (aOR, 17.1; 95% CI: 1.8 to 165.6) among the atopic girls. Overall, the B[a]P effect sizes differ more dramatically between the non-atopic and atopic children, than between the lean and OV/OB children within either the non-atopic or atopic group. Our data suggest OV/OB asthma as a severe outcome, nested within the non-atopic asthma endotype, rather than constituting a unique endotype.
Fourth, the lung function deficit, which only appear among those with highest median value of B[a]P, appears to be a particularly important predictor of non-atopic asthma only. Overall, non-atopic boys with a highest median B[a]P exposure are associated with lung function deficit, as well as elevated odds of lean asthma. In contrast, the lean atopic boys and girls with low median B[a]P exposure are neither associated with the lung function deficit diagnosis, nor asthma. Such trend suggests that processes underlying non-atopic asthma might be distinct from those for the atopic asthma among the boys. That is, lung function impairment appears to be a ‘meet-in-the-middle’ biomarker of B[a]P and asthma association. Above a certain threshold for exposure, B[a]P are associated with asthma, regardless of the atopy status.
In our investigation, we could not directly examine whether non-atopic asthma represents T-helper 2 or type 2 low asthma, while atopic asthma captures T-helper 2 or type 2 high asthma, two known endotypes [3, 30]. This is because we did not measure typical cells and cytokines associated with T-helper 2 or type 2 low vs. T-helper 2 or type 2 high endotypes. Thus, more comprehensive characterization of the two asthmas, in terms of the repertoire of cytokines, cells, and clinicals traits of the children are warranted.
Strengths and limitations of the present study has been discussed [10–13]. Briefly, B[a]P is used as a representative PAH compound here, while a more realistic exposure scenario involves exposure to complex mixture of air pollutants. At the same time, robust body of evidence, including our own, have demonstrated B[a]P as a etiologically pertinent and representative PAH compound [9, 10, 13, 19, 31–36]. Both the laboratory and epidemiologic evidence have shown that PAHs could induce or enhance allergic sensitization, exacerbate pre-existing asthma, and enhance the risk of de novo asthma development [33, 37, 38]. In particular, B[a]P has been shown to directly target hematopoietic stem cells by binding to aryl hydrocarbon receptors, and subsequently impart a wide array of adverse effects, including mitochondrial functional deficit . Furthermore, B[a]P represents an efficient indicator of child’s exposure to ambient pollutant mixture, due to its extremely high correlation with other traffic-related air pollutants [40–43]. At the same time, other constituents of complex mixtures could increase multiple types of oxidative injury, respiratory system inflammation, and alteration in lung structure and function . Thus, other unmeasured, yet correlated air pollutants (e.g. metals), may pose a threat of residual confounding.
Our earlier investigation has shown the ambient monitored PAH concentrations as apt marker of chronic exposure. For example, the interquartile range of ambient B[a]P levels during our exposure window of interest (i.e. November, 2008) is representative of the ambient concentrations from the earlier years . At the same time, using ambient monitoring as primary marker of chronic exposure might underestimate exposure from other routes (e.g. oral and dermal). Our earlier sensitivity analysis has shown that while both the dietary intake and the inhalation exposure to PAHs contribute to human body burden, inhalation represents predominant route of exposure during the heating season .
As our goal was to estimate a proof-of-principle dose-response function of asthma associations under extreme variations in ambient B[a]P concentrations, our case-control children are not representative of general Czech population.