Acute inhalation exposure to vaporized methamphetamine causes lung injury in mice

Abstract

Methamphetamine (MA) is currently the most widespread illegally used stimulant in the United States. Use of MA by smoking is the fastest growing mode of administration, which increases concerns about potential pulmonary and other medical complications. A murine exposure system was developed to study the pulmonary affects of inhaled MA. Mice were exposed to 25-100 mg vaporized MA and assessments were made 3 h following initiation of exposure to model acute lung injury. Inhalation of MA vapor resulted in dose-dependent increases in MA plasma levels that were in the range of those experienced by MA users. At the highest MA dose, histological changes were observed in the lung and small but significant increases in lung wet weight to body weight ratios (5.656 +/- 0.176 mg/g for the controls vs. 6.706+/- 0.135 mg/g for the 100 mg MA-exposed mice) were found. In addition, there was 53% increase in total protein in bronchoalveolar lavage (BAL) fluid, greater than 20% increase in albumin levels in the BAL fluid, greater than 2.5-fold increase in lactate dehydrogenase levels in the BAL fluid, and reduced total BAL cell numbers (approximately 77% of controls). Levels of the early response cytokines tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 were dose-dependently increased in BAL fluid of MA-exposed mice. Exposure to 100 mg MA significantly increased free radical generation in the BAL cells to 107-146% of controls and to approximately 135% of the controls in lung tissue in situ. Together, these data show that acute inhalation exposure to relevant doses of volatilized MA is associated with elevated free radical formation and significant lung injury.

FIG. 1

Heated methamphetamine exposure system. MA was heated over a propane heat source in the heating vessel. The resultant MA vapor was moved through the animal chamber by constant air flow of 1.5 L/min generated by an air pump and a vacuum source.

FIG. 2

MA plasma levels with increasing doses of MA. Plasma MA levels were determined by LC/MS. Following exposure to the vapors from increasing amounts of heated MA, MA plasma levels were increased in the exposed mice. Values are means ± SEM from four mice in each group. Correlation between MA dose and plasma levels was calculated using a Pearson correlation test (r = .7450,p <.001).

FIG. 3

Assessment of lung injury following acute exposure to vaporized MA. Mice were exposed to vapors from 25 to 100 mg of heated MA. Tissues were collected 3 h following exposure to assess parameters of lung injury. (A) Total protein in the BAL fluid was assessed (controls, n = 15; 25–100 mg MA, n = 5 each). (B) Albumin in the BAL fluid was assessed (n = 4 for all groups). (C) Cell numbers from whole lung lavage was determined (controls, n = 15; 25 mg MA, n = 8; 50 mg MA, n = 11; 100 mg MA, n = 9). (D) LDH in BAL fluid was measured (n = 8 for all groups). Asterisk indicates significant at p < .05 versus controls. Data are shown as means ± SEM.

FIG. 4

Assessment of lung histopathology following acute exposure to vaporized MA. Lung morphology was evaluated by H&E staining of paraffin sections. Representative sections from control mice (n = 4; panels A—B) and mice 3 h after MA exposure (n = 4; panels C—D). Panels A and B show representative images of lung tissue (original magnification, 20×). In panel A, the capillaries (black arrows) are apparent throughout the tissue. In panel C, no clear capillaries could be identified, but what appears to be the remains of a small arteriole is shown (black arrow). Higher magnification (original magnification 100×) of the airway epithelial cells are shown in panels B and D. The black arrows in panel D point to areas of disruption of normal cellular architecture.

FIG. 5

Levels of early response cytokines in BAL fluid following acute exposure to vaporized MA. Three hours following 25–100 mg MA exposure, TNF-α production (A) and IL-6 production (B) in BAL fluid was determined (n = 8 for all groups). Asterisk indicates significant at p < .05 versus controls. Data are shown as means ± SEM.

FIG. 6

Determination of oxidative stress in lung cells and tissue following exposure to vaporized MA. Frozen sections were stained with DHE ± PEG-SOD (see Materials and Methods), imaged, and analyzed by laser scanning cytometry (LSC). (A) DHE staining in lung sections. Representative images from control and MA-exposed mice are shown. (B) The maximum pixel values and percentages of DHE-positive cells are graphed (n = 4). Asterisk indicates significant at p < .05 versus controls. Data are shown as means ± SEM.