Chronic and Binge Alcohol Ingestion Increases Truncated Oxidized Phosphatidylcholines in Mice Lungs Due to Increased Oxidative Stress

Abstract

Heavy alcohol drinking has negative health effects in multiple organs. It predisposes lungs to inflammatory conditions associated with acute lung injury and increased incidence of pneumonia and sepsis, which may lead to death due to acute respiratory distress syndrome in some individuals with alcohol use disorder (AUD). In general, rodent models of alcohol exposure either do not recapitulate multiple organ injuries as seen in humans or require longer duration to establish tissue injury and inflammation. The recently introduced NIAAA model of alcohol-induced liver injury, characterized by a marked increase in steatosis and liver damage with 10 days of a liquid diet containing 5% ethanol followed by a single ethanol binge (5 g/kg). Therefore, we employed this model to explore the status of surfactant phospholipids, oxidative stress, tissue injury markers and inflammatory cytokines in lungs. In lungs of C57BL/6J mice, the alcohol feeding significantly increased levels of the surfactant phospholipid dipalmitoyl phosphatidylcholine (DPPC) as well as the truncated oxidized phosphatidylcholines palmitoyl oxovaleryl phosphatidyl-choline (POVPC), palmitoyl glutaryl phosphatidyl-choline (PGPC), palmitoyl oxo-nonanoyl phosphatidyl-choline (ALDO-PC), and palmitoyl azelaoyl phosphatidyl-choline (PAzePC) at 9 h post-binge. Additionally, gene expression of the enzymes catalyzing lipid oxidation, such as arachidonate 15-lipoxygenase (Alox15), prostaglandin synthase 2 (Ptgs2), Cytochrome P450 2E1 (Cyp2E1) and NADPH oxidase 1 (Nox1) were significantly increased. Furthermore, ethanol increased levels of the inflammatory cytokine Interleukin-17 in bronchoalveolar lavage fluid. In conclusion, the NIAAA alcohol feeding model might be suitable to study alcohol-induced lung injury and inflammation.

Keywords: IL-17; LPS; PGPC; POVPC; alcohol; lung injury; oxidized phospholipids.

FIGURE 1

Experimental paradigm of the NIAAA alcohol feeding model in mice. (A) Schematic presentation of the NIAAA alcohol feeding model. (B) Levels of alanine transaminase (ALT) and aspartate transaminase (AST) in serum. Data represent mean +S.E.M from 4 mice per group. Data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test. * (p < 0.05), ** (p < 0.01) indicates significant difference from the pair-fed control group.

FIGURE 2

Chronic and single binge ethanol feeding increased expression of oxidative enzymes in mouse lungs. Gene expression of oxidative enzymes arachidonate 15-lipoxygenase (Alox15) (A), Prostaglandin synthase 1 and 2 (Ptgs1 and Ptgs2) (B), NADPH oxidase 1, 2 and 4 (Nox1, Nox2 and Nox4) (C), Alcohol dehydrogenase 1 (Adh1) and Alcohol dehydrogenase 2 (Aldh2) (D), Cytochrome P450 2E1 (Cyp2E1) (E) in lungs either pair-fed or ethanol fed mice. Data represent mean +S.E.M from 4-5 mice per group. Data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test. * (p < 0.05), **** (p < 0.0001) indicates significant difference from the pair-fed control group.

FIGURE 3

Chronic and single binge ethanol feeding induced elevation of truncated oxidized phospholipids in muse lungs. (A) Levels of surfactant phospholipid dipalmitoyl phosphatidyl choline (DPPC), and cellular phospholipids palmitoyl-arachidonoyl phosphatidyl choline (PAPC), palmitoyl-oleoyl phosphatidyl choline (POPC) in lungs. (B) Levels of truncated oxidized phosphatidyl cholines, Palmitoyl Glutaryl Phosphatidyl Choline (PGPC), Palmitoyl Oxovaleryl Phosphatidyl Choline (POVPC), Palmitoyl Oxo-nonanoyl Phosphatidyl Choline (ALDO-PC), and Palmitoyl Azelaoyl Phosphatidyl Choline (PAzePC). Platelet activating factor C16:0 (PAF C16:0) (C). Lyso-PAF C16:0 (D). Data represent mean +S.E.M from 4-5 mice per group. Data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test. * (p < 0.05), ** (p < 0.01) indicates significant difference from the pair-fed control group.

FIGURE 4

The NIAAA ethanol feeding model did not alter vascular permeability, and epithelial cell membrane integrity but did increase inflammatory cytokines in BALF (A) Schematic presentation of ethanol feeding and oropharyngeal LPS (50 ug/mice) induced lung injury model. Levels of (B) Albumin, (C) Total protein, (D) Lactate dehydrogenase (LDH) activity (E) MCP-1, (F) TNFα, (G) IL-1β and (H) IL-17 in bronchoalveolar lavage fluid (BALF) from mice 9 h after either pair-fed or ethanol binge following 10 days chronic feeding or 24 h after oropharyngeal LPS instillation. Data represent mean +S.E.M from 4-5 mice per group. Data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test. * (p < 0.05), ** (p < 0.01), *** (p < 0.001), **** (p < 0.0001) indicates significant difference from the pair-fed control group.