Effects of fuel components and combustion particle physicochemical properties on toxicological responses of lung cells

<p>The physicochemical properties of combustion particles that promote lung toxicity are not fully understood, hindered by the fact that combustion particles vary based on the fuel and combustion conditions. Real-world combustion-particle properties also continually change as new fuels are implemented, engines age, and engine technologies evolve. This work used laboratory-generated particles produced under controlled combustion conditions in an effort to understand the relationship between different particle properties and the activation of established toxicological outcomes in human lung cells (H441 and THP-1). Particles were generated from controlled combustion of two simple biofuel/diesel surrogates (methyl decanoate and dodecane/biofuel-blended diesel (BD), and butanol and dodecane/alcohol-blended diesel (AD)) and compared to a widely studied reference diesel (RD) particle (NIST SRM2975/RD). BD, AD, and RD particles exhibited differences in size, surface area, extractable chemical mass, and the content of individual polycyclic aromatic hydrocarbons (PAHs). Some of these differences were directly associated with different effects on biological responses. BD particles had the greatest surface area, amount of extractable material, and oxidizing potential. These particles and extracts induced cytochrome P450 1A1 and 1B1 enzyme mRNA in lung cells. AD particles and extracts had the greatest total PAH content and also caused CYP1A1 and 1B1 mRNA induction. The RD extract contained the highest relative concentration of 2-ring PAHs and stimulated the greatest level of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNFα) cytokine secretion. Finally, AD and RD were more potent activators of TRPA1 than BD, and while neither the TRPA1 antagonist HC-030031 nor the antioxidant N-acetylcysteine (NAC) affected CYP1A1 or 1B1 mRNA induction, both inhibitors reduced IL-8 secretion and mRNA induction. These results highlight that differences in fuel and combustion conditions affect the physicochemical properties of particles, and these differences, in turn, affect commonly studied biological/toxicological responses.</p

Patient Comparisons with the 2006 CF Foundation Patient Registry^a.

a<p>Results are median (interquartile range) unless noted. CFFPR patients include all sputum-producing adult patients in 2006 but exclude those followed at the Intermountain Adult CF Center.</p>b<p>We used χ-square tests to determine statistical differences in Gender, Infections and Anti-inflammatory Therapy between the Intermountain CF Center and the CFFPR 2006. For all other variables shown, we used Kolmogorov-Smirnov tests because data were not normally distributed.</p

Study Design^a.

a<p>A total of 97 unique patients participated in the study including study and validation groups.</p>b<p>Validation Groups had no overlapping patients with Study Groups that underwent the same analysis with one exception (please see the next footnote). For Analysis 3, for example, no patients were found in both Study Group 2 and Validation Groups 3 and 4.</p>c<p>Eight of the 17 patients in Validation Group 2 were excluded from analysis 5 because they were already included with Study Group 3 leaving 9 patients for the validation and mutual consistency testing (see also text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042748#pone-0042748-t007" target="_blank">Table 7</a>).</p

Analysis Descriptions.

a<p>We examined between-biomarker correlations to help interpret results of multivariate models involving multiple potential biomarkers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042748#pone.0042748.s006" target="_blank">Table S4</a>).</p

Patient Characteristics^a.

a<p>Results are median (interquartile range) unless noted.</p>b<p>This group of patients provided two samples each, one from a stable state and one from an APE state at admission for a hospitalization. Data shown here are derived from the time point of the stable sample collection for each individual.</p>c<p>The 26 patients that gave paired samples necessarily suffered an APE during the study in order to give the necessary APE state sputums. This criterion selected patients with significantly lower lung function, <i>t</i>-test <i>p</i> = 0.005, increased incidence of CF-related diabetes, χ-square <i>p</i><0.001, decreased 5-year predicted survival, <i>t</i>-test <i>p</i> = 0.01 and more frequent APE (differences not tested due to confounding) than the other patients in the study.</p>d<p>Patients in Validation Group 1 had higher FEV₁% and 5-year predicted survival and remarkably no incidence of CF-related diabetes. Despite these differences, the coefficients for HMGB-1 reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042748#pone-0042748-t007" target="_blank">Table 7</a> for Validation Groups 1 and 2 are quite similar to those for Study Group 1 patients and pass testing for mutual consistency.</p>e<p>The 5-year predicted survival is a clinically useful composite estimate of overall disease state in CF but may be difficult to use in interpretation of inflammatory states. Similar to lung function and other clinical markers of disease, it may require years to see a change <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042748#pone.0042748-Liou1" target="_blank">[2]</a>.</p

Testing Validation Results for Mutual Consistency.

a<p>Weighted least squares analysis.</p>b<p>Study Group 2 patients, Analysis 3, n = 26.</p>c<p>Study Group 3 patients, Analyses 4 and 5, n = 76.</p>d<p>Validation Group 2 patients not included in Group 3, n = 9 with 1 death, 1 lung transplant.</p

Multivariate models for concurrent outcomes and APE-associated predictions.

a<p>Data from study group 1, n = 56. We found no evidence of two-way interactions or non-linear effects using squared terms for these models. Age, gender, CF-related diabetes, airway infection with either <i>Pseudomonas aeruginosa</i> or <i>Staphylococcus aureus</i> and chronic azithromycin, oral or inhaled steroid use had no significant interactions with any inflammatory marker terms in any multivariate model. Log transformed values of biomarkers were used for modeling outcomes. Concurrent FEV₁% and Weight-for-age <i>z</i>-score models used linear regression. The model for the number of APE occurring in the year prior to initial sputum collection used quasi-Poisson regression.</p>b<p>Data from study group 2, n = 26. Additional adjustment for the stable FEV₁% measurement, sequence of stable and APE time point collections, airway infection with either <i>Pseudomonas aeruginosa</i> or <i>Staphylococcus aureus</i>, use of azithromycin or steroids had no significant effect in these models.</p>c<p>Estimates of the mean change in FEV₁% per unit change in log scale biomarkers. Results from a linear regression model for the associations between difference in FEV₁% between stable and APE time points and GM-CSF (log scale) measured at the APE onset time point. Each univariate representing measurements obtained during clinically stable and APE time points were added in turn to a model containing GM-CSF measured at the APE time point, the only statistically significant univariate. IL-5 (<i>p</i> = 0.006) and IL-10 (<i>p</i> = 0.015) measured at the APE time point and TCC (<i>p = </i>0.012) measured at the stable time point were found to be positively associated with FEV₁% decline independently of GM-CSF. Backward selection of a multivariate model containing GM-CSF (APE), IL-5 (APE), IL-10 (APE), and TCC (Stable) produced the final model presented here.</p>d<p>Estimates of the predicted total number of APE during 5 years of follow up per unit change in log scale biomarkers measured during clinical stability. Results show a quasi-Poisson regression model for the association with number of APE during 5 years of follow-up. HMGB-1 (log scale) was the only significant univariate (<i>p</i><0.05), but CRP, IFN-α and IL-8 (all log scale) had trends toward significance (<i>p</i><0.2). Backwards multivariate model selection retaining adjustment variables for follow-up time and low or high number of APE in the year prior to stable sputum collection (low  =  0 or 1 (reference group), high >1) as an indicator of baseline inflammation, retained only HMBG-1. A 1 unit change in log scale HMGB-1 is associated with a mean change in number of APE of 0.34.</p

Proportional Hazards Models of Time-to-Event^a.

a<p>The table shows results from proportional hazards models for the association between time-to-first APE following sputum collection and HMBG-1 (log scale) measurement from clinically-stable time points, Study Group 2, n = 26, and the association between time-to-lung transplant or death following initial sputum collection and HMGB-1 (log scale) measurements for all patients in the study with sufficient sample to measure HMGB-1, Study Group 3, n = 76. Both analyses shown met the assumption of proportionality for proportional hazards modeling <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042748#pone.0042748-Grambsch1" target="_blank">[31]</a>. Among the 76 patients, there were 15 events: 9 deaths and 6 listings for lung transplantation. All listed patients were subsequently transplanted. Adjustments for number of APE in the year prior to stable sputum collection were non-significant, and inclusion of variables for use of azithromycin or steroids had no effect on these models. Concurrent FEV₁% and airway infection with either <i>Pseudomonas aeruginosa</i> or <i>Staphylococcus aureus</i> had non-significant associations with time-to-first APE. FEV₁% is confounded as a predictor of time-to-transplant or death (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042748#s4" target="_blank">Discussion</a>). <i>P aeruginosa</i> and <i>S aureus</i> infection are not primarily considered in selection of candidates for transplant and are not potential confounders; they had no effect on time-to-transplant or death. Approximately a 10% increase in HMGB-1 is associated with a 4% increase in the hazard rate for time-to-first APE and a 5% increase in hazard rate for time-to-lung transplant or death.</p

Kaplan-Meier Curves for the Time from Stable Sputum Collection to First Event.

<p>The curves illustrate the difference in time to A) first APE and B) death or censoring by listing for lung transplantation for patients with HMGB-1 measurements higher and lower than the value of 6.0 (log ng/ml). The value is the rounded median of the actual HMGB-1 data for both the 26 patients in A and the 76 patients in B. P-values shown are the results of log rank testing <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042748#pone.0042748-Peto1" target="_blank">[52]</a>. These graphs show the results of evaluation of HMGB-1 simplified to high or low values, which are consistent with the proportional hazards modeling <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042748#pone.0042748-Cox1" target="_blank">[29]</a> of the effects of HMGB-1 as a continuous variable. Models were tested for consistency with proportionality <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042748#pone.0042748-Grambsch1" target="_blank">[31]</a> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042748#pone-0042748-t006" target="_blank">Table 6</a>).</p

Key Univariate Relationships.

<p>HMGB-1 had strong statistically significant associations with A) concurrent FEV1% and B) number of APE suffered in the year prior sputum sample collection. These results illustrate the immediate clinical relevance of HMGB-1. C) GM-CSF measured at APE time-points had an extremely strong univariate association with the size of the APE-associated decline in FEV₁% for each of 26 patients in Group 2. D) Although the univariate relationship with APE-associated FEV₁% decline was weak (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042748#pone.0042748.s005" target="_blank">Table S3</a>), the addition of IL-5 as a covariate to GM-CSF significantly strengthened the multivariate linear regression model of APE-associated decline in FEV₁% (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042748#pone-0042748-t005" target="_blank">Table 5</a>).</p