The pharmacological activity of inhalation exposure to marijuana smoke in mice

Although the majority of cannabinoid users smoke marijuana, the preponderance of laboratory animal research is based on administration of Delta9-tetrahydrocannabinol (Delta9-THC) or other cannabinoid agents via injection. The aim of the present study was to evaluate the impact of inhaling marijuana, or ethanol-extracted placebo smoke in the mouse model of cannabinoid activity by assessing inhibition of spontaneous activity, antinociception, catalepsy, and body temperature. In order to determine dosimetry, blood levels of Delta9-THC were obtained following either marijuana exposure or intravenous injection of Delta(9)-THC. Inhalation exposure to marijuana produced dose-related increases in antinociception and catalepsy, with estimated ED50 doses of Delta9-THC of 2.4 and 3.8 mg/kg, respectively. However, hypothermia and locomotor depression occurred in both the placebo- and marijuana-exposed mice. The CB1 receptor antagonist, SR 141716A antagonized the antinociceptive effects of marijuana (AD50 = 0.6 mg/kg), but only slightly decreased marijuana-induced catalepsy, and failed to alter either the hypothermic or locomotor depressive effects. In contrast, SR 141716A antagonized the antinociceptive, cataleptic, and hypothermic effects of intravenously administered Delta9-THC in mice that were exposed to air alone, though all subjects exhibited locomotor depression, possibly related to the restraint. In accordance with reports of others, these data suggest that exposure to smoke alone has pharmacological consequences. Our findings also indicate that marijuana-induced antinociception is mediated through a CB1-receptor mechanism of action and are consistent with the notion that Delta9-THC is mainly responsible for this effect.     

No increase in carcinogen-DNA adducts in the lungs of monkeys exposed chronically to marijuana smoke.

Rhesus monkeys exposed to marijuana smoke either 7 or 2 days/weeks (HI and LO groups, respectively), or ethanol-extracted marijuana smoke for 7 days/week (EM) or sham treatment (SH) for 1 year were sacrificed 7 months following the last exposure. Pulmonary levels of carcinogen-DNA adducts were determined. Although mean or median adduct levels were not statistically different, 15 of 22 adduct measures were highest in the EM group and lowest 12 of 22 times in the SH group. The levels of aromatic carcinogen-DNA adducts seem no higher in the lungs of animals exposed to marijuana smoke than in untreated animals. Ethanol-extracted marijuana may have effects greater than marijuana itself.     

Spectrophotometric evaluation of carboxyhemoglobin in blood of mice after exposure to marijuana or tobacco smoke in a modified Walton horizontal smoke exposure machine

Carboxyhemoglobin (COHb) values were determined in mice exposed to varying amounts of marijuana and tobacco cigarette smoke utilizing a spectrophotometric technique. Mice were exposed to smoke inhalation in a modified Walton horizontal smoke exposure machine, whereby rodents can be exposed to multiples of 1-min smoke exposure cycles. Smoke exposure was intermittent; during the first 30 sec of each 1-min cycle, the subjects were exposed to smoke diluted either 1:10 or 1:5 with air. During the second half of the cycle the animals were given fresh air. There was a positive linear relationship between COHb values obtained and the number of puffs of marijuana smoke administered via either 2, 4, 6, or 8 "puffs" of marijuana smoke. COHb levels in plasma did not increase in animals given multiple 8-puff episodes of smoke daily as long as a 60-min period was interposed between smoking episodes. COHb values in mice exposed to tobacco smoke were significantly higher than those in mice receiving equal numbers of exposures to marijuana smoke. Mean COHb values of mice receiving 8 consecutive puffs of marijuana smoke were 18.6 and 22.0% saturation, but CO was rapidly cleared from the blood. This rapid clearance suggests that the binding affinity of CO for mouse hemoglobin may be be weaker than that of human hemoglobin. Mice similarly exposed to 6 or 8 puffs of tobacco smoke had mean COHb values of 24.6 and 28.5% saturation, respectively. No acute lethal effects were observed in mice receiving multiple daily episodes of 8 puffs per episode of marijuana smoke, whereas mice exposed to a single 8-puff episode of tobacco smoke suffered about 50% acute lethal effects.     

Induction of aryl hydrocarbon hydroxylase in rat lung by marijuana smoke.

Rats were exposed to the smoke produced by burning four cigarettes of marijuana or of marijuana placebo material. Six hours later, maximally stimulated aryl hydrocarbon hydroxylase (AHH) activity was observed in the lung. Marijuana placebo material also induced pulmonary AHH activity. Actinomycin D (1 mg/kg) and cycloheximide (2 mg/kg), given ip 1 hr before exposure to marijuana smoke, partially blocked AHH induction. If rats were exposed repeatedly to marijuana smoke, they showed higher activities of pulmonary AHH within 6 hr of the last exposure than did animals exposed for the first time. Inhalation of marijuana smoke increased the number and size of debris-filled vacuoles in alveolar macrophages and in the ciliated cells of the bronchiolar epithelium. Smoke condensate from the marijuana material used in these studies contained 0.45 ng/mg of benzo(a)pyrene.     

Effects of single and repeated marijuana smoke exposure on fetal EEG.

The effect of single and repeated marijuana smoke exposure on fetal EEG was investigated in the chronic fetal lamb model using power spectral analysis. Maternal inhalation of marijuana smoke (n = 9) resulted in a significant reduction in total power and power distribution in the delta (1-4 Hz) band, and an increase in power distribution in the faster frequencies in the first h after smoke inhalation. These EEG changes were not observed following maternal inhalation of placebo smoke (n = 5), nor in animals with 3-5 prior exposures to marijuana smoke (n = 5). These results suggest that the effects of marijuana smoke exposure on fetal EEG is short-lived and tolerance develops rapidly with repeated exposure.     

The experimental investigations of the toxic influence of tobacco smoke affecting progeny during pregnancy.

1. Tobacco smoke contains around 4000 substances, most of which are described as toxic, and they may have an influence on the development of progeny. 2. The present studies concentrate on the measurement and calculation of indices describing the new-born's survival, rearing of pups, weight of foetuses, young animals, placenta and females in relation to different doses of tobacco smoke (carbon monoxide levels). The morphological studies of placenta, foetal and newborn lungs were done as a supplement. Biochemical placenta study was also done. 3. The results of the experiment proved that some indices for animals in groups which were passively exposed to the highest concentrations of tobacco smoke were lower, others fluctuated (4 day, 12 day and total survival) and some did not reveal any changes (rearing). Direct correlation between maternal passive exposure to tobacco smoke and the presence of acute respiratory distress syndrome symptoms in new-borns was observed. A decrease of body weight of pregnant females passively exposed to tobacco smoke was also observed. An increase of placenta-foetal factor was found. A decrease of rat weight was observed after passive exposure to tobacco smoke. 4. We concluded that there is correlation between passive exposition to tobacco smoke during pregnancy and delayed lung maturation in the offspring. Exposure of the pregnant rats to cigarette smoke increases the activity of isocitric and glucose-6-phosphate dehydrogenases in placenta.     

Inhalation experiments with 14C-labelled cigarette smoke III. Body size dependent distribution of particulate matter in small rodents during cigarette smoke inhalation

Syrian golden and European hamsters were exposed to ¹⁴C-labelled cigarette smoke. The deposition of the ¹⁴C activity in the respiratory and digestive tracts was determined immediately after the exposure. From the results obtained it can be seen that the distribution of particulate matter between the upper and lower respiratory tract, as well as, between the respiratory and digestive tracts is related to the body weight of the experimental animals.     

Inhalation bioassay of cigarette smoke in rats

Groups of 80 female rats were exposed to cigarette smoke from three types (code 13 = high tar, low nicotine; code 27 = low tar, medium nicotine; code 32 = high tar, high nicotine) of cigarettes in Maddox-ORNL smoking machines, eight cigarettes per day, 7 days per week, for up to 24 months. An additional group received sham exposures and a fifth group served as untreated controls. The sham-exposed animals had significantly lower body weights than the untreated controls. The smoke-exposed animals had significantly lower weights than the sham-exposed controls; the weights were lower for the code 27 and code 32 animals than for the code 13 animals during the second year of exposure. The survival of the code 13 animals was similar to that for the sham-exposed and untreated control group; survival times of the code 27 and code 32 animals were shorter. Body weight and survival reflected the high- and low-nicotine dose groups indicated by in vivo dosimetry measurements. Smoke-induced histopathologic lesions consisted primarily of pulmonary smoke granulomas; the smoke granulomas were less severe in the code 27 exposure group than in the groups exposed to smoke from code 13 or code 32 cigarettes. Additional changes included pulmonary alveolar epithelial hyperplasia, and squamous metaplasia and basal cell hyperplasia of laryngeal and tracheal epithelium. One primary epidermoid carcinoma was found in the lung of a code 27 rat. The rats tolerated the chronic exposures relatively well and certain of the smoke-induced lesions allowed differentiation between the different types of cigarettes.     

Stress and hypothermia in mice in a nose-only cigarette smoke exposure system

In nose-only exposure systems, animals need to be restrained inside a tube, which leads to stress. Stress is known to cause hyperthermia in rodents. Chronically repeated episodes of hyperthermia could be detrimental to animal health and influence results of nose-only exposure studies. Therefore we investigated whether hyperthermia occurred in male C57BL/6J mice that were restrained for increasing lengths of time, using nosepieces held at room temperature, preheated at 37 degrees C, or thermostat controlled at different temperatures, with and without exposure to different concentrations of cigarette smoke. Body temperature, body weight, plasma corticosterone levels, and adrenal weights were recorded. Restraint using nosepieces at room temperature caused a time-dependent decrease in body temperature, which could be reversed by preheating the nosepieces to 37 degrees C. Cigarette smoke dose-dependently caused an additional decrease, which was counteracted by controlling nosepiece temperature at 38 degrees C. During 3 mo of exposure using heated nosepieces, Delta body temperature remained constant. Body weight gain did not differ between smoke-exposed and room air-breathing animals exposed using either heated or room-temperature nosepieces, but both groups gained significantly less weight, while adrenal weights were significantly and similarly increased, when compared to unrestrained littermates. Plasma corticosterone levels did not differ between the three groups. In conclusion, during restraint in nose-only exposure tubes with room temperature metal nosepieces, mice suffer a pronounced hypothermia. Preventing this by heating the nosepieces does not reduce the stress experienced by the animals.     

Weight and activity in male mice after daily inhalation of cannabis smoke in an automated smoke exposure chamber

An automatic smoking machine capable of delivering marihuana smoke to groups of 10 mice at a time at a constant concentration of Δ⁹-THC is described. In all cases the flow rate (50 ml s^?1 or 100 ml ^s-1) of a smoke: air mixture, and the dilution factor (1:20 or 1:10 smoke:air) was adjusted to give the same equilibrium concentration of Δ⁹-THC in the chamber atmosphere. At a concentration of 0–123 mg Δ⁹-THC litre^?1 of exposure chamber atmosphere (1:20 smoke: air; 100 ml s^?1 flow rate), a seven day schedule of 20 min daily treatments significantly decreased activity of the marihuana-treated animals by the sixth day over both air and placebo smoke controls. This concentration and exposure schedule failed to affect weight gain during this same period. A significant decrease in activity, although not as great as in the marihuana-treated group, was also seen in the placebo control group compared with the air control animals, indicating the need for a smoke control group in all experiments involving the administration of marihuana via inhalation. An exposure period of 102 min, with marihuana smoke diluted with air 1:10 (50 ml ^s-1 flow rate), at the same equilibrium concentration of 0·123 mg Δ⁹-THC litre^?1, resulted in a mortality of 90% following administration or within 48 h. 102 min of exposure to an atmosphere of placebo smoke at a smoke:air dilution of 1:10 (50 ml ^s-1 flow rate), resulted in no mortality.     

Inhibition of immunological function mediated DNA damage of alveolar macrophages caused by cigarette smoke in mice

Exposure to cigarette smoke impairs the pulmonary immune system, including alveolar macrophage function, although the mechanisms by which this occurs are not fully elucidated. This study investigates the effect of cigarette smoke exposure on the antigen-presenting activity of alveolar macrophages, which is required for antigen-specific response to T cells. C57BL/6 mice were exposed to cigarette smoke for 10 days using a Hamburg II smoking machine, and alveolar macrophages were obtained by bronchoalveolar lavage. The antigen-presenting activity of alveolar macrophages was significantly inhibited in mice exposed to cigarette smoke compared with mice not exposed to cigarette smoke. Major histocompatibility complex class II cell surface molecule–positive cells, B7-1 molecule–positive cells, and interleukin-1β messenger RNA gene expression in alveolar macrophages were significantly decreased in mice exposed to cigarette smoke compared with mice not exposed to cigarette smoke. In contrast, DNA damage and generation of superoxide and hydrogen peroxide in alveolar macrophages were significantly increased by cigarette smoke exposure. These results suggest that inhibition of the antigen-presenting activity of alveolar macrophages may result from decreased expression of major histocompatibility complex class II and B7-1 molecules and interleukin-1β messenger RNA gene expression following cigarette smoke exposure. Furthermore, inhibition of antigen presentation in alveolar macrophage may result from DNA damage induced by excessive amounts of reactive oxygen species being generated by alveolar macrophages following cigarette smoke exposure. These findings suggest that cigarette smoke impairs the immunological function of alveolar macrophages and, as a result, increases the risk for pulmonary infection.     

SR 141716 (Rimonabant) precipitates withdrawal in marijuana-dependent mice

Repeated marijuana use is known to lead to physical dependence in humans; however, its dependence liability has yet to be adequately assessed in laboratory animals. The goals of the present study were to: assess whether the CB(1) antagonist SR 141716 (rimonabant) precipitates withdrawal in mice that had been repeatedly exposed to marijuana smoke, and to compare these precipitated withdrawal effects to those elicited following intravenous administration of its chief psychoactive component Delta(9)-tetrahydrocannabinol (Delta(9)-THC). SR 141716 elicited a significant increase in paw tremors in mice that were repeatedly dosed with either marijuana or Delta(9)-THC. Unexpectedly, the blood and brain concentrations of Delta(9)-THC following marijuana exposure were considerably lower than those found following Delta(9)-THC injection when comparing an equivalent magnitude of paw tremors in both conditions. Finally, Delta(9)-THC dose-dependently alleviated SR 141716-induced paw tremors in marijuana-dependent mice, but marijuana itself failed to reverse the precipitated withdrawal effect. It is likely that marijuana exposure generated insufficient Delta(9)-THC brain levels (i.e., 203+/-19 ng/g) to reverse the withdrawal signs compared with the brain levels following intravenous injection (i.e., 1862+/-82 ng/g). These findings taken together indicate that mice exposed repeatedly to marijuana smoke exhibit similar precipitated withdrawal effects as Delta(9)-THC-injected mice.     

Chronic marijuana smoke alters alveolar macrophage morphology and protein expression.

Male rhesus monkeys were subjected to chronic exposure to marijuana smoke. High dose animals (HI) were exposed 7 days/week to 1 MJ cigarette/day; low dose animals (LO) were exposed on 2 consecutive weekend days to 1 MJ cigarette/day; placebo animals (EM) were exposed to 1 ethanol-extracted MJ cigarette/day for 7 days/week; sham animals (SH) were exposed to sham smoking conditions 7 days/week. This regimen was maintained for 1 year and was followed by a 7 month rest period. Alveolar macrophages of animals exposed to the LO and HI dose smoking regimens exhibited irregular cell surface morphology, increased vacuolization, and a spherical conformation upon adherence to plastic. Gel protein profiles of purified macrophages from HI and LO animals showed marked differences in both constitutive and bacterial lipopolysaccharide-elicited protein expression when compared with those of macrophages from the EM or SH animals. These results indicate that chronic THC exposure alters macrophage morphology and protein expression to external stimuli even after a 7 month rest period.     

Passive inhalation of marijuana smoke: urinalysis and room air levels of delta-9-tetrahydrocannabinol

In two separate studies, 5 drug-free male volunteers with a history of marijuana use were passively exposed to the sidestream smoke of 4 and 16 marijuana cigarettes (2.8% delta-9-tetrahydrocannabinol [THC]) for 1 h each day for 6 consecutive days. A third study was similarly performed with 2 marijuana-naive subjects passively exposed to the smoke of 16 marijuana cigarettes. Passive smoke exposure was conducted in a small, unventilated room. Room air levels of THC and CO were monitored frequently. All urine specimens were collected and analyzed by EMIT d.a.u. assay, Abuscreen radioimmunoassay and GC/MS. The studies show that significant amounts of THC were absorbed by all subjects at the higher level of passive smoke exposure (eg., smoke from 16 marijuana cigarettes), resulting in urinary excretion of significant amounts of cannabinoid metabolites. However, it seems improbable that subjects would unknowingly tolerate the noxious smoke conditions produced by this exposure. At the lower level of passive marijuana-smoke exposure, specimens tested positive only infrequently or were negative. Room air levels of THC during passive smoke exposure appeared to be the most critical factor in determining whether a subject produced cannabinoid-positive urine specimens.     

Pharmacodynamic effects of chronic cigarette smoke exposure in spontaneously hypertensive rats

In investigating the influence of chronic cigarette smoke exposure on hypertension, we compared the pharmacodynamic effects of enforced exposure to smoke on spontaneously hypertensive rats (SHR) with those on Wistar-Kyoto (WKY) rats. Chronic cigarette smoke exposure for 8 weeks decreased the elevated heart rate of mature male SHR to approximately the rate in WKY rats 24 h after smoke exposure. Both systolic and diastolic blood pressures also decreased slightly. However, WKY rats showed a marked rise in heart rate soon after exposure to cigarette smoke began, with no change in blood pressure, while the heart rate of SHR in the early stage remained similar to that of animals without exposure, although their blood pressure was clearly reduced. The body weight of both strains tended to decrease during smoke exposure, but the effect was more severe in SHR. Moreover, the effects of chronic smoke exposure were observed using retired, aged female SHR breeders. A decrease in body weight and heart rate, but not in blood pressure, was also recognized even in these mature animals. These effects gradually recovered after withdrawal from exposure. On the basis of these results, a profile of chronic cigarette smoke exposure under hypertension is discussed in this study.     

Cigarette smoke enhances abdominal aortic aneurysm formation in angiotensin II-treated apolipoprotein E-deficient mice

Cigarette smoke, hyperlipidemia, and hypertension with the risk of development and progression of atherosclerosis and associated pathologies such as abdominal aortic aneurysm (AAA) are correlated. We examined the interaction of cigarette mainstream smoke (MS) and angiotensin-II (Ang II)-induced hypertension in the atherosclerotic process using hyperlipidemic apolipoprotein E-knockout (ApoE(-/-)) mice. ApoE(-/-) mice were treated with Ang II for 4 weeks and then further exposed to MS or to fresh air for 4 weeks. AAA formation was observed in all mice treated with Ang II, regardless of smoke exposure; however, smoke exposure increased the incidence of AAA in these mice. Ang II treatment resulted in higher gene expression of matrix metalloproteinases (MMP)-2, -3, -8, -9, and -12 in the abdominal aortas, which was further increased by MS exposure. The proteolytic activity of MMP-2 and MMP-9 was also enhanced in Ang II-treated mice exposed to MS, but only minor changes were seen with either smoke exposure or Ang II treatment alone. This study shows for the first time that both formation and severity of AAA in hypertensive ApoE(-/-) mice are accelerated by exposure to MS and that the proteolytic activity of MMPs is enhanced by the combination of Ang II and MS.     

Cerebral and cerebellar neurochemical changes and behavioral manifestations in rats chronically exposed to marijuana smoke.

Groups of male and female Fischer rats were exposed to marijuana cigarette smoke via an automatic smoking machine. Inhaled Δ⁹-tetrahydrocannabinol doses of 0.7, 2, and 4 mg/kg were relevant to man and were given for 12, 18, 27, 57, and 87 days. Another group of rats treated for 87 days was studied after a recovery of 20 days. Control animals inhaled smoke produced by placebo cigarettes. In the first week of exposure, 20% of lower-dosed rats were hyperactive and 50% at the high dose were prostrate or ataxic upon removal from the inhalator. Behavioral aberrations ameliorated within a few hours except for the depression exhibited by males at the high dose. Tolerance to CNS inhibition developed in 1–2 weeks. CNS stimulation, as manifested by hypersensitivity and hyperactivity, progressively involved more animals, primarily females, in all groups during Days 27–57. Tolerance to CNS stimulation developed thereafter. Fighting was displayed by 90% of females and 50% of males at 4 mg/kg by weeks 6–7. Neurotoxicity was expressed by involuntary vertical jumping, predominantly among high-dosed males in weeks 3 and 8. Normal behavior was displayed after cessation of treatment. At necropsy, homogenates of cerebrum and cerebellum were prepared and were analyzed for protein, RNA and acetylcholinesterase (AChE) activity. Cerebral AChE activity in females increased 33–71% after 12 exposures, decreased 10–23% after 57 exposures and rose 12% after 87 exposures. Cerebellar enzyme activity initially increased 15–35% in animals of both sexes during the subchronic phase but declined in females after 27 exposures. The extent of change in enzyme activity was generally reduced with continued treatment. Cerebellar RNA increased approximately 20% in rats of both sexes, but at different time intervals during the subchronic phase, and remained elevated in females at 87 days. Neurochemical changes were sex related and coincided with behavioral manifestations, and some changes extended into the recovery period. Inhalation findings were similar to those obtained earlier by the oral route; however, females demonstrated a greater facility to adapt to the cumulative toxic effects of marijuana smoke.     

Exposure to waterpipe smoke induces renal functional and oxidative biomarkers variations in mice

Context: Waterpipe smoking (WPS) has been known for over 400 years. It has been spread widely especially between youth because of the addition of pleasant flavor and because it was misconsidered to be less harmful than cigarette.

Objective: In this study, we investigated the effect of waterpipe smoking on renal oxidative and functional parameters and compared that at acute and chronic exposure time in mice.

Materials and methods: Mice were divided into three groups, namely acute, chronic and fresh air control. Acute group was exposed to waterpipe smoke for one hour daily for six days using whole-body exposure system, while chronic group was exposed to waterpipe smoke for one hour daily for 30 days using whole-body exposure system.

Result: Exposure to waterpipe smoke has shown significant changes on the mice kidney functional parameters such as creatinine and blood urea nitrogen. Both exposures (acute and chronic) has shown a significant reduction in superoxide dismutase (SOD) activity (p?<?0.05), whereas the activity of other antioxidant enzymes (catalase and GPx) reduced only with chronic exposure to waterpipe smoke (p?<?0.05). Additionally, the level of thiobarbituric acid reactive substances (TBARS) in mice kidney homogenates has shown a significant elevation following chronic exposure to waterpipe smoke (p?<?0.05).

Discussion and conclusion: In conclusion, chronic waterpipe smoke affects the kidney parameter and antioxidant markers, therefore affecting its functionality of detoxifying and removal of poisonous material from the body.     

Functional and biochemical effects on the lung following inhalation of cigarette smoke and constituents. I. High- and low-nicotine cigarettes in mice

Techniques for measuring pulmonary function in mice have been developed. Daily inhalation of cigarette smoke for 5 or 10 wk elicited the following effects: (1) increase in pulmonary compliance; (2) decrease in functional residual capacity; (3) decrease in pulmonary compliance; (4) decrease in tidal volume; (5) no change in phospholipid content of the lung; and (6) increase in wet weight of the lung relative to body weight, which was reduced. The increase in pulmonary resistance and the decrease in functional residual capacity were elicited by nonfiltered smoke as well as by the vapor phase, and their appearance was related to the nicotine content of the cigarettes and the duration of exposure. These results indicate that these two effects are elicited by a combination of the nicotine contained in particulate material and constituents of the vapor phase. The decrease in pulmonary compliance was elicited by inhalation of nonfiltered smoke but not by the vapor phase, indicating that the causative factor is in the particulate matter, probably nicotine, because the appearance of decreased compliance depended on the nicotine content. The decrease in tidal volume as well as the increase in pulmonary resistance, or bronchospasm, occurred more readily in ICR strain mice than in the Swiss strain. Both strains developed tolerance to bronchospasm after 10 wk of exposure. There was no increase in functional residual capacity and, hence, no functional sign of pulmonary emphysema in mice that had been exposed to cigarette smoke for 5 or 10 wk.     

Lung cell reactions in guinea pigs exposed to tobacco smoke and silica dust or bacterial lipopolysaccharides

In order to investigate the possibility of the synergistic effects of tobacco smoke and/or silica dust (SiO2) or bacterial endotoxins (LPS), guinea pigs were exposed to combinations of these agents. A 15-day exposure to SiO2 alone caused a decrease in intracellular lysosomal enzymes of alveolar macrophages (AM) and an increase of lysosomal enzymes detected in lung lavage fluid which was present 16 weeks after exposure. The effect was the same in animals which received SiO2 in combination with tobacco smoke. Exposure to LPS caused an increase in the number of neutrophils recovered in lavage fluid. The increase in neutrophils was less in animals previously exposed to tobacco smoke alone or in combination with LPS. Acute exposure to LPS also caused an increase in lactate dehydrogenase, N-acetyl-beta-D-glucosaminidase and acid phosphatase activity detectable in lung lavage fluid. The increase was less pronounced in animals previously exposed to smoke. Cathepsin D was increased in AM after tobacco smoke exposure alone and was decreased to below control values of the animals which received an acute LPS exposure.