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Guinea pigs were given single exposure (0.5\u20136 mg\/kg body weight) of 2\u2010chloroethyl ethyl sulfide (CEES) as a mustard analogue intratracheally and maintained for various lengths of time (1 h to 21 days). Within 1 h of CEES infusion at 4 mg\/kg, high levels of tumor necrosis factor \u03b1 (TNF\u2010\u03b1), ceramides, and nuclear factor \u03baB accumulated in lung and alveolar macrophages. Both acid and neutral sphingomyelinases were activated within 4 h. These signal transduction events were associated with alteration in the oxygen defense system. Within 1 h of exposure to CEES (6 mg\/kg body weight), there was 10\u2010fold increase in the <jats:sup>125<\/jats:sup>I\u2010BSA leakage into lung tissue, indicating severe lung injury. Although low level of CEES exposure (0.5 mg\/kg body weight) produced symptoms of chemical burn in lung as early as 1 h after exposure, the severity of edema, congestion, hemorrhage, and inflammation increased progressively with time (1 h to 21 days). Feeding of single dose of NAC (0.5 g) by gavage just before the CEES infusion was ineffective to counteract these effects. However, consumption of the antioxidant in drinking water for 3 or 30 days prior to CEES exposure significantly inhibited the induction of TNF\u2010\u03b1, activation of neutral and acid sphingomyelinases, production of ceramides, activation of caspases, leakage of <jats:sup>125<\/jats:sup>I\u2010bovine serum albumin (<jats:sup>125<\/jats:sup>I\u2010BSA) into lung tissue, and histological alterations in lung. Pretreatment with NAC for 3 and 30 days protected against 69\u201376% of the acute lung injury. Therefore, NAC may be an antidote for CEES\u2010induced lung injury. \u00a9 2003 Wiley Periodicals, Inc. J Biochem Mol Toxicol 17:177\u2013184, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). 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