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Short-Term Exposure to Aged and Diluted Sidestream Cigarette Smoke Enhances Ozone-Induced Lung Injury in B6C3F1 Mice.

Reference: Yu, M., Pinkerton, K.E., and Witschi, H. Toxicological Sciences 65, 99-106 (2002)

The purpose of this study was to evaluate the effects of exposure of mice to ADSS (aged and diluted cigarette smoke) followed by exposure to ozone. ADSS with a concentration of 30 mg/m³ of TSP (total suspended particulates) represents ETS (environmental tobacco smoke) in this study. The mice inhaled 0.5 ppm ozone for 24 h following three days of exposure to ADSS for 6 hr a day. Both these exposure levels are higher than levels to which humans are normally exposed. However 0.5 ppm ozone is the same as the level in the air during a stage-III smog alert in CA.

The endpoints studied included cellular and protein composition of BAL (bronchoalveolar lavage fluid) as well as histopathological and cytokinetic changes in the centriacinar region of the lung. Alveolar macrophages from the BAL were assessed for their ability to release the cytokines, interleukin (IL)-6 and TNFµ under LPS (lipopolysaccharide) stimulation. The centriacinar region defined as the bronchiolar alveolar duct junction including the terminal bronchiole and adjacent alveolar region was chosen specifically for histological study because this is the area in which ozone has been shown to cause changes.

Methods:

Exposure protocol: There were 4 groups of experimental B6C3F1 mice:

	Exposed to filtered air
	Exposed to ADSS alone
	Exposed to ozone alone
	Exposed to ADSS (6 hr/d for 3 d) followed immediately by 24 hr of 0.5 ppm ozone

Sacrifice

Single sacrifice on day 5 with BAL immediately after Ozone while BrdU animals were necropsied one day following the end of exposure to ozone.

Exposure system:

A cigarette smoking machine designed by Steve Teague at UC Davis generated cigarette smoke. Mainstream and sidestream smoke were mixed, aged and diluted before mice were exposed.

Cigarettes:

These were 1R4F Kentucky reference cigarettes

Animals:

Male B6C3F1 mice (10 wks) were used and housed under standard conditions.

Endpoints studied:

	BALF: The lungs of anesthetized mice were lavaged with HBSS. Total protein was measured in the supernatant. Centrifuged cells were used to determine the cell differential in the BALF.
	Immunohistochemistry: Before exposure, osmotic pumps containing BrdU were implanted in each mouse. Mice were sacrificed one day after completion of the ozone exposure. Lung tissue from the centriacinar region was embedded in paraffin and standard 5 m sections prepared. Cell proliferation was determined by positive staining with BrdU and labeling indices calculated.
	Cytokine release: ELISA kits were used to determine LPS-stimulated cytokine release.

Results:

	BALF: Only mice exposed to smoke followed by ozone had significant numbers of cells in the BALF as well as total protein levels compared to control animals. The differential count of cells in BALF showed increased neutrophils and lymphocytes.
	Cell kinetics: Both animals exposed to ozone alone and smoke followed by ozone had elevated labeling indices in the centriacinar region of the lung.
	Cytokine release: Cell cultures of alveolar macrophage supernatant contained a decrease in spontaneous release of both IL-6 and TNF-µ in animals exposed to both ozone alone and ozone followed by smoke and ozone. IL-6 release from macrophages stimulated by LPS was also decreased in both these groups of animals although levels of TNF-µ were increased in the same animal groups.

Conclusions:

The authors conclude that this study shows that lungs of mice exposed to ADSS followed by ozone have more pronounced signs of injury than either mice exposed to ADSS or ozone alone. Thus ADSS exposure enhances the sensitivity to ozone-induced lung injury in this mouse model. The pertinent evidence for the above discussion is summarized above. For an excellent discussion of the ramifications of the findings and more detailed related findings, the manuscript itself should be consulted. It is available in PDF format from the authors: mailto:kepinkerton@ucdavis.edu.

Editorial note: Two common air pollutants are ozone and cigarette smoke. Many studies concerning the effects of ozone on a variety of animal species are found in the scientific literature. These studies were initiated as early as the 1960’s. A large variety of endpoints were studied during and after ozone exposure. Studies of the effects of cigarette smoke on the lungs on animals are relatively recent by comparison. Finally investigators have only recently published studies of the effects of multiple air pollutants. These studies are significant because they more closely reflect exposures humans are likely to receive. The report by Yu, Pinkerton, and Witschi is an example of such a multiple exposure study. In this case, mice exposed to cigarette smoke followed by ozone were more sensitive to the oxidant effects than either alone. Such a protocol has similarities to initiation-promotion assays for carcinogenesis.

By: Susan G. Shami, ScD

Science Editor