Heterozygosity in the glutathione synthesis gene Gclm increases sensitivity to diesel exhaust particulate induced lung inflammation in mice

Abstract

Context: Inhalation of ambient fine particulate matter (PM₂.₅) is associated with adverse respiratory and cardiovascular effects. A major fraction of PM₂.₅ in urban settings is diesel exhaust particulate (DEP), and DEP-induced lung inflammation is likely a critical event mediating many of its adverse health effects. Oxidative stress has been proposed to be an important factor in PM₂.₅-induced lung inflammation, and the balance between pro- and antioxidants is an important regulator of this inflammation. An important intracellular antioxidant is the tripeptide thiol glutathione (GSH). Glutamate cysteine ligase (GCL) carries out the first step in GSH synthesis. In humans, relatively common genetic polymorphisms in both the catalytic (Gclc) and modifier (Gclm) subunits of GCL have been associated with increased risk for lung and cardiovascular diseases.

Objective: This study was aimed to determine the effects of Gclm expression on lung inflammation following DEP exposure in mice.

Materials and methods: We exposed Gclm wild type, heterozygous, and null mice to DEP via intranasal instillation and assessed lung inflammation as determined by neutrophils and inflammatory cytokines in lung lavage, inflammatory cytokine mRNA levels in lung tissue, as well as total lung GSH, Gclc, and Gclm protein levels.

Results: The Gclm heterozygosity was associated with a significant increase in DEP-induced lung inflammation when compared to that of wild type mice.

Discussion and conclusion: This finding indicates that GSH synthesis can mediate DEP-induced lung inflammation and suggests that polymorphisms in Gclm may be an important factor in determining adverse health outcomes in humans following inhalation of PM₂.₅.

Figure 1

Images of lung cross sections 1 h after intranasal instillation of DEP (10μL of 10mg/mL DEP per nostril) in WT C57bl6 mice. Panels A and B are a 4x image and a 40x image of a single cross section from the upper region of the right lung.

Figure 2

The FACS analysis of percentage neutrophils (F4/80^lo/Gr1^hi) from 5000 cells collected in BAL in Gclm WT, Gclm^−/+, and Gclm^−/− mice 6 h after either PBS or DEP intranasal instillation. The n values for treatments are: Gclm WT, N = 13-PBS: 10-DEP, Gclm^−/+, n = 12-PBS: 10-DEP and Gclm^−/−, n = 8-PBS: 9-DEP. Error bars represent SEM; * = Significant difference at p-values of <0.05.

Figure 3

Concentration of inflammatory cytokines TNFα (3a) and IL6 (3b) in BAL fluid in Gclm WT, Gclm^−/+, and Gclm^−/− mice 6 h after either PBS or DEP intranasal instillation. The n = 6 for all genotypes and treatments. Error bars represent SEM; * = Significant difference at p-values of <0.05.

Figure 4

Linear regression analysis between percentage neutrophils and BAL fluid concentration of inflammatory cytokines TNFα (4a) and IL6 (4b) within all genotypes 6 h after either PBS or DEP intranasal instillation. The R² values are given for regression performed within treatment condition.

Figure 5

Real-time PCR assessment of mRNA levels for TNFα, IL6, (5a) and GMCSF, IL1β, and MCP1 normalized to β-actin in the lung of Gclm WT, Gclm^−/+, and Gclm^−/− mice 6 h after either PBS or DEP intranasal instillation. The n = 6 for all genotypes and treatments. Error bars represent SEM; * = Significant difference at p-values of <0.05.

Figure 6

Linear regression analysis between real-time PCR assessment of mRNA levels for TNFα (6A) and IL6 (6B) normalized to β-actin and BAL fluid concentration of inflammatory cytokines TNFα and IL6 in Gclm WT, Gclm^−/+, and Gclm^−/− mice 6 h after DEP intranasal instillation.

Figure 7

The MPO protein level (A) and MMP activity (B) in Gclm WT, Gclm^−/+, and Gclm^−/− mice 6 h after either PBS or DEP intranasal instillation. The n = 6 for all genotypes and treatments. Error bars represent SEM.

Figure 8

Total GSH level in whole lung homogenate in Gclm WT, Gclm^−/+, and Gclm^−/− mice 6 h after either PBS or DEP intranasal instillation. The n values for treatments are: Gclm WT, n = 9-PBS: 8-DEP, Gclm^−/+, n = 13-PBS: 15-DEP, and Gclm^−/−, n = 6-PBS: 7-DEP. Error bars represent SEM.

Figure 9

Total GCLC and GCLM protein levels normalized to β-actin measured by western blot in Gclm WT, Gclm^−/+, and Gclm^−/− mice 6 h after either PBS or DEP intranasal instillation; n = 6 for all genotypes and treatments. Error bars represent SEM; * = Significant difference from the PBS-treated WT control at p-value of <0.05; *** = Significant difference between Gclm^−/− PBS and DEP treated mice at p-value of < 0.001.