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The heated cigarette (HC) generates mainstream smoke by vaporizing the components of the tobacco rod using a carbon heat source at the cigarette tip. Mainstream smoke of HC contains markedly less chemical constituents compared to combusted cigarettes. Mainstream smoke from HC was generated under Health Canada Intense regimen and its biological effects were compared to those of Reference (3R4F) cigarettes, using nose-only 5-week and 13-week inhalation studies. In the 13-week study, SD rats were necropsied following exposure to mainstream smoke from each cigarette at 200, 600 or 1000 µg wet total particulate matter/L for 1 h/day, 7 days/week or following a 13-week recovery period. Histopathological changes in the respiratory tract were significantly lesser in HC groups; e.g. respiratory epithelial hyperplasia in the nasal cavity and accumulation of pigmented macrophages in alveoli. After a 13-week recovery, the lesions were completely or partially regressed, except for accumulation of pigmented macrophages in alveoli, in both HC and 3R4F groups. In the 5-week study, SD rats were necropsied following exposure to mainstream smoke of either cigarette at 600 or 1000 µg/L for 1 h, two times/day (with 30 min interval), 7 days/week or following a 4-week recovery period. Bronchoalveolar lavage fluid (BALF) analysis of neutrophil percentages and enzyme levels like γ-GT, ALP and LDH indicated that pulmonary inflammation was significantly less in HC groups compared to 3R4F groups. In conclusion, HC demonstrated significantly lower biological effects compared to 3R4F, based on the BALF parameters and histopathology.

Cigarette smoke exposures in mice have been conducted under various exposure conditions using different strains as animal models of smoke-related diseases. We exposed cigarette smoke to two strains of mice [C57BL/6J (C57) and AKR/J (AKR)] under two different exposure regimens (1 h or 4 h/day) at equivalent daily exposure amount (concentration × time). After 2 weeks exposure, mice were evaluated using exposure markers and biological responses. Smoke exposure suppressed respiratory parameters dependent on exposure concentration. The 1-h regimen groups generally showed a greater degree of respiratory suppression and relatively lower exposure markers of urinary nicotine metabolites than the corresponding 4-h regimen groups. Tidal volume was more suppressed in AKR compared to C57, while respiratory rate was more suppressed in C57. Plasma exposure markers and respiratory parameters suggested that C57 inhaled more volume of smoke than AKR. Changes in bronchoalveolar lavage fluid (BALF) cytology and enzyme parameters were most noticeable in the 1 h AKR groups. In BALF cytokine concentration, TARC concentration in C57 was higher than AKR, while KC and MCP-1 in AKR were higher than C57. Relative lung/body weight ratio in smoke-exposed C57 was generally higher, as well as the incidence and severity of lesions in respiratory organs compared to AKR. In summary, C57 appeared to inhale relatively more smoke and displayed greater inflammatory changes in respiratory tract than AKR. Comparison of exposure regimens suggests that a longer exposure duration at lower WTPM concentration might deliver a larger dose of smoke than a shorter exposure duration at higher WTPM concentration.

A variety of dose x duration exposure regimens have been used in inhalation toxicity studies using rodents. We evaluated the effects of differences in smoke concentration and daily exposure duration under similar weekly cumulative exposures in rats to determine potential variation in type and severity of adverse effects in 13-week exposure studies. The weekly cumulative dosages were 2100 and 4200 μg wet total particle matter (WTPM)/L, and the daily exposure durations were 1 and 6 h. Weekly exposure duration was 5 or 7 days/week for groups exposed 1 h/day and 7 days/week for groups exposed 6 h/day. Recovery duration was 6 and 13 weeks. Mainstream smoke exposure suppressed body weight (BW) gain in both regimens. Lower dose groups exposed 1 h/day had a consistently greater of BW gain compared with corresponding 6 h/day groups. Respiratory rate, tidal volume, and minute volume (MV) were suppressed in a dose-dependent manner in both regimens. Higher MV in rats exposed for 6 h/day compared with rats exposed 1 h/day suggested that a lower concentration for longer duration resulted in a greater total inhaled mass (TIM) in rats exposed 6 h/day. Groups exposed for 6 h/day had lower blood carboxyhemoglobin and plasma nicotine levels than groups exposed 1 h/day, reflecting the lower carbon monoxide (CO) and WTPM concentrations in the 6 h/day groups. Data from examination of bronchoalveolar lavage fluids (BALF) and respiratory tract tissues indicated comparable effects between both regimens. Exposure-induced histopathological changes regressed similarly for both regimens after the recovery periods.

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally-accepted nomenclature for proliferative and non-proliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in the respiratory tract of laboratory rats and mice, with color photomicrographs illustrating examples of some lesions. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous developmental and aging lesions as well as lesions induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for respiratory tract lesions in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

AKR/J mice were exposed to cigarette smoke (CS) and/or lipopolysaccharide (LPS) via inhalation for 3 wk and pulmonary responses were evaluated. The objective was to explore the feasibility of coexposing LPS with cigarette smoke under a subacute exposure, as a surrogate for viral or bacterial insults, that would mimic the pathogenesis of infection-related chronic obstructive pulmonary disease (COPD) exacerbations. The study was the first step in an effort to develop a rodent COPD model in which morphologic lesions of COPD develop in a shorter period of exposure and more closely simulate human COPD. Mice were exposed 6 h/day, 5 days/wk for 3 wk to one of the following: (1) sham control: filtered air; (2) CS: 250 microg/L wet total particulate matter (WTPM) for 5 h/day followed by 1 h/day air; (3) LPS: 0.5 microg/L LPS (055:B5 Escherichia coli; 3,000,000 EU/mg) for the last 1 h/day 2 day/wk (following 5 h/day of filtered air); and (4) CS/LPS: CS 5 h/day followed by air or LPS (2 days/wk) for 1 h/day. After the last exposure, animals were necropsied and subjected to bronchoalveolar lavage (BAL) or histopathology. The BAL neutrophil counts were highest in the LPS group, while macrophage counts were higher in the CS/LPS group than other exposed groups. The LPS group displayed the greatest increases in BAL cytokines, while KC (keratinocyte-derived chemokine) and TARC (thymus and activation-regulated chemokine) were highest in the CS group. The CS/LPS group had generally lower cytokine levels relative to the LPS or CS groups, except for the levels of RANTES and G-CSF (granulocyte-colony stimulating factor) comparable to the LPS group. At microscopic examination of lung sections, cellular inflammatory infiltrates were most notable in the CS/LPS group, which had a diffuse, predominantly macrophage infiltrate with fewer neutrophils. The LPS group had predominantly neutrophils in the pulmonary infiltrate and the CS group had a predominantly macrophage infiltrate in alveolar ducts and adjacent alveoli. Apoptotic labeling of lung cells was highest with the CS/LPS group. In summary, the CS/LPS group displayed greater cellular infiltration and apoptotic responses in the lung with an indication of immunosuppressive effects (lower BAL cytokines) than the CS or LPS group, suggesting that the CS/LPS model shows promise to be further explored as an animal model for studying pathogenesis of COPD exacerbations. A longer term study with interim assessments is needed to confirm that the subacute responses observed in the CS/LPS group will result in greater severity of COPD-related pulmonary lesions following prolonged exposures.

A series of in vitro and in vivo studies evaluated the potential effects of tobacco flavoring and casing ingredients. Study 1 utilized as a reference control cigarette a typical commercial tobacco blend without flavoring ingredients, and a test cigarette containing a mixture of 165 low-use flavoring ingredients. Study 2 utilized the same reference control cigarette as used in study 1 and a test cigarette containing eight high-use ingredients. The in vitro Ames Salmonella typhimurium assay did not show any increase in mutagenicity of smoke condensate from test cigarettes designed for studies 1 and 2 as compared to the reference. Sprague-Dawley rats were exposed by nose-only inhalation for 1 h/day, 5 days/wk for 13 wk to smoke from the test or reference cigarettes already described, or to air only, and necropsied after 13 wk of exposure or following 13 wk of recovery from smoke exposure. Exposure to smoke from reference or test cigarettes in both studies induced increases in blood carboxyhemoglobin ((COHb)) and plasma nicotine, decreases in minute volume, differences in body or organ weights compared to air controls, and a concentration-related hyperplasia, squamous metaplasia, and inflammation in the respiratory tract. All these effects were greatly decreased or absent following the recovery period. Comparison of rats exposed to similar concentrations of test and reference cigarette smoke indicated no difference at any concentration. In summary, the results did not indicate any consistent differences in toxicologic effects between smoke from cigarettes containing the flavoring or casing ingredients and reference cigarettes.