Glutathione (GSH) and the GSH synthesis gene Gclm modulate plasma redox and vascular responses to acute diesel exhaust inhalation in mice

Abstract

Context: Inhalation of fine particulate matter (PM₂.₅) is associated with acute pulmonary inflammation and impairments in cardiovascular function. In many regions, PM₂.₅ is largely derived from diesel exhaust (DE), and these pathophysiological effects may be due in part to oxidative stress resulting from DE inhalation. The antioxidant glutathione (GSH) is important in limiting oxidative stress-induced vascular dysfunction. The rate-limiting enzyme in GSH synthesis is glutamate cysteine ligase and polymorphisms in its catalytic and modifier subunits (GCLC and GCLM) have been shown to influence vascular function and risk of myocardial infarction in humans.

Objective: We hypothesized that compromised de novo synthesis of GSH in Gclm⁻/⁺ mice would result in increased sensitivity to DE-induced lung inflammation and vascular effects.

Materials and methods: WT and Gclm⁻/⁺ mice were exposed to DE via inhalation (300 μg/m³) for 6 h. Neutrophil influx into the lungs, plasma GSH redox potential, vascular reactivity of aortic rings and aortic nitric oxide (NO•) were measured.

Results: DE inhalation resulted in mild bronchoalveolar neutrophil influx in both genotypes. DE-induced effects on plasma GSH oxidation and acetylcholine (ACh)-relaxation of aortic rings were only observed in Gclm⁻/⁺ mice. Contrary to our hypothesis, DE exposure enhanced ACh-induced relaxation of aortic rings in Gclm⁻/⁺ mice.

Discussion and conclusion: THESE data support the hypothesis that genetic determinants of antioxidant capacity influence the biological effects of acute inhalation of DE. However, the acute effects of DE on the vasculature may be dependent on the location and types of vessels involved. Polymorphisms in GSH synthesis genes are common in humans and further investigations into these potential gene-environment interactions are warranted.

Figure 1

Alveolar macrophages collected by BAL of mice treated with either filtered air (FA) or diesel exhaust (DE) for 6 h. Images reveal diesel exhaust particulate taken up into the cell in DE-exposed mice.

Figure 2

Percent neutrophils (Gr1^hi/F4/80^lo/CD11b^vhi) as measured by the FACS analysis of BAL cells collected from FA and DE-exposed male and female mice. N = 12 for each genotype and treatment with equal number of each sex in each group. *Significantly different (p < 0.05) compared to genotype specific FA control by ANOVA and Dunnett’s t-test.

Figure 3

Stratification of percent neutrophils in BAL by sex. The data reveal nearly identical trends for each sex.

Figure 4

Plasma [GSH] (A), plasma [GSSG] (B), %GSSG of total glutathione (C), and GSH reduction potential (D) in male FA or DE exposed mice. N = 6 for each genotype and treatment. *Significantly different (p < 0.05) compared to genotype specific FA control by ANOVA and Dunnett’s t-test.

Figure 5

Measurement of phenylephrine (PE)-stimulated contraction of aortic rings from WT and Gclm^−/+ mice following 6 h FA or DE inhalation. WT FA and DE: N = 8, Gclm^−/+ FA and DE: N = 9.

Figure 6

Measurement of acetylcholine (ACh)-stimulated relaxation of aortic rings from WT and Gclm^−/+ mice following 6 h FA or DE inhalation. (A) WT FA versus WT DE, (B) Gclm^−/+ FA versus Gclm^−/+ DE, (C) WT FA versus Gclm^−/+ FA, (D) WT DE versus Gclm^−/+ DE. Curves represent nonlinear regression model used for EC50 and significance testing. Significance (*p < 0.05, **p < 0.001) determined by nonlinear regression fit and EC50 determination, as well as by two-way ANOVA. WT FA and DE: N = 8, Gclm^−/+ FA and DE: N = 17.

Figure 7

Measurement of aortic NO^• production by Fe(DETC)₂ spin trap and ESR. NO-Fe(DETC)₂ signal relative to dry weight of aortic tissue (A) within genotypes, (B) with both genotypes combined, (C) delta of percentage increase in NO signal of DE versus FA in WT and Gclm^−/+ mice. *Significantly different (p < 0.05) compared to genotype specific FA control by ANOVA and Dunnett’s t-test.