Genomic impact of cigarette smoke, with application to three smoking-related diseases.

There is considerable evidence that inhaled toxicants such as cigarette smoke can cause both irreversible changes to the genetic material (DNA mutations) and putatively reversible changes to the epigenetic landscape (changes in the DNA methylation and chromatin modification state). The diseases that are believed to involve genetic and epigenetic perturbations include lung cancer, chronic obstructive pulmonary disease (COPD), and cardiovascular disease (CVD), all of which are strongly linked epidemiologically to cigarette smoking. In this review, we highlight the significance of genomics and epigenomics in these major smoking-related diseases. We also summarize the in vitro and in vivo findings on the specific perturbations that smoke and its constituent compounds can inflict upon the genome, particularly on the pulmonary system. Finally, we review state-of-the-art genomics and new techniques such as high-throughput sequencing and genome-wide chromatin assays, rapidly evolving techniques which have allowed epigenetic changes to be characterized at the genome level. These techniques have the potential to significantly improve our understanding of the specific mechanisms by which exposure to environmental chemicals causes disease. Such mechanistic knowledge provides a variety of opportunities for enhanced product safety assessment and the discovery of novel therapeutic interventions.

A further review of inhalation studies with cigarette smoke and lung cancer in experimental animals, including transgenic mice.

CONTEXT: The lack of an effective animal model for pulmonary carcinogenesis in smokers is a continuing problem for researchers trying to design Potentially Reduced Risk Products for those smokers who are either unwilling or unable to quit smoking. The major failing of inhalation assays with cigarette smoke in laboratory animals is that these assays produce only small percentages of animals with pulmonary tumors (e.g. adenomas, with the occasional adenocarcinoma), as opposed to the highly invasive carcinomas (e.g. small cell and squamous cell) seen in smokers. OBJECTIVE: To update previous reviews on animal models, and to add different types of transgenic (Tg) mice to the review. METHODS: Reviews were made of articles retrieved from PubMed and elsewhere. RESULTS: The addition of Tg mice to the arsenal of tests used for the evaluation of the carcinogenic potential of cigarettes did not result in any better understanding of the inability of such testing to reflect the epidemiological evidence for lung cancer in smokers. CONCLUSION: As in previous reviews on the subject, the best assay providing support for the epidemiology data is still the 5-month whole-body exposure of male A/J mice to a combination of mainstream/sidestream smoke, followed by a 4-month recovery.

A minireview of chronic animal inhalation studies with mainstream cigarette smoke.

This work was performed to verify whether or not the inhalation response to cigarette smoke in animal species for assessing carcinogenic potential in humans reflects the strong epidemiological evidence in human smokers. Significant increases in the numbers of malignant tumors of the respiratory tract were not seen in rats, mice, hamsters, dogs, or nonhuman primates exposed for long periods of time to very high concentrations of mainstream cigarette smoke. The results are clearly at variance with the epidemiological evidence in smokers, and it is difficult to reconcile this major difference between observational studies in humans and controlled laboratory studies.

A review of chronic inhalation studies with mainstream cigarette smoke, in hamsters, dogs, and nonhuman primates.

This paper is the continuation of previously published work, a review limited to studies on rats and mice. This paper makes an identical evaluation as before, but, restricting the species being evaluated to representative studies of smoke-exposed hamsters, dogs (both by tracheostomy and by direct inhalation), and nonhuman primates. As was seen previously, no statistically significant increase in the incidence of malignant tumors of the respiratory tract was found in any of the 3 species, even though very long exposures and high doses of smoke were used. All 5 of the species of laboratory animals commonly used to evaluate carcinogenic potential produce results with mainstream cigarette smoke that are at variance with the epidemiological evidence in smokers.

A review of chronic inhalation studies with mainstream cigarette smoke in rats and mice.

In this paper, I review the results of a representative selection of chronic inhalation studies with rats and mice exposed to mainstream cigarette smoke and describe the inhalation exposures and the histopathological changes reported by various authors. Many of the studies used nose-only exposure systems, whereas others simply used large whole-body chambers. Smoke-induced epithelial hypertrophy, hyperplasia, and squamous metaplasia were reported in the conducting airways in most of the studies, along with increased numbers of intra-alveolar macrophages that were occasionally associated with alveolar metaplasia. Lung adenomas and adenocarcinomas were reported in only a few of the studies. No statistically significant increase in the incidence of malignant lung tumors was seen in either species as a result of smoke exposure, a finding that does not agree with the results of epidemiological studies in humans. Possible reasons for this lack of correlation are given.

Relevant exposure to environmental tobacco smoke surrogate does not produce or modify secretory otitis media in the rat.

Parental smoking is a possible risk factor in the development of secretory otitis media (SOM) in children. This experiment was designed to determine, using rats as an experimental model, whether exposures to environmental tobacco smoke (ETS) produce SOM and whether ETS exposure affects the rate of clearance of an experimentally induced effusion. Male Sprague-Dawley rats were exposed to 3 different concentrations of aged and diluted sidestream smoke, a surrogate for ETS, from IR4F research cigarettes for 6 hr per day for 5 days. Experimental SOM was induced bilaterally in subgroups of animals from each group, by cold air exposure to the external auditory canals. Ears of rats were examined during the in-life portion of the study. Histopathologic examination of the middle ear was conducted at the termination of the 5-day period. The production of SOM was not induced by ETS exposure, nor were there differences noted between the groups in the rates of clearance of the experimentally induced SOM. Short-term exposure to ETS did not affect the acquisition or clearance of SOM in the rat.

Environmental tobacco smoke: experimental facts and societal issues.

Involuntary exposure to environmental tobacco smoke (ETS) in public or in working places is considered to be a serious risk to human health. This symposium addressed several issues of toxicological interest that are associated with exposure to ETS. Epidemiologic evidence obtained in human studies suggests that "passive smoking" increases the risk of developing lung cancer in nonsmokers and favors the development of respiratory tract infections in children. Comparatively few data are available from animal studies that provide experimental support of the observations. Exposure of pregnant or neonate rats to cigarette sidestream smoke (SS) affects developmental patterns of drug metabolizing enzymes that may persist up to 90 days. In young roosters, SS accelerates the development of arteriosclerotic plaques. On the other hand, exposure of adult rats for up to 90 days induces only transient signs of damage in the nasal passages, but not in the deep lung, and this only at extremely high concentrations of ETS. So far, experimental toxicology has provided comparatively few data on the correlation between exposure to ETS and adverse health effects. yet, such data are needed, particularly since many conclusions drawn from the epidemiological studies remain open to criticism and questions.

Effects of low humidity on the rat middle ear.

Secretory otitis media is common in the winter, and the possible risk factors are numerous. This study examines the effect of low humidity on the middle ear using a Sprague-Dawley rat model: 23 test rats housed for 5 days in a low-humidity environment (10% to 12% relative humidity) and 23 control rats housed at 50% to 55% relative humidity. Microscopic ear examinations were graded for otitis media with effusion (OME) before testing and on test days 3 and 5. The mucosa of the middle ears and eustachian tubes was examined histopathologically. Significantly more effusions were observed in the low-humidity group on test days 3 (P = .003) and 5 (P = .01), but no intergroup histopathologic differences were noted. We conclude that a low-humidity environment contributed to the development of OME in the test animals, and that low-humidity warrants further investigation as a contributing factor in childhood middle ear disease.

Design, construction, and evaluation of an inhalation system for exposing experimental animals to environmental tobacco smoke.

An inhalation system was designed to expose experimental animals to aged and diluted sidestream smoke (ADSS), used as a surrogate for environmental tobacco smoke (ETS). The construction of the smoke generator and of the smoke dilution systems is described. Target ADSS concentrations in a 90-day inhalation study were 0.1, 1, and 10 mg/m3 of respirable suspended particulates (RSP). Data is presented on the physical and chemical composition of the smoke presented to animals at or near these target RSP concentrations. The design of the inhalation laboratory was shown to result in highly reproducible respirable aerosols that were effective surrogates of ETS.

Ninety-day inhalation study in rats, using aged and diluted sidestream smoke from a reference cigarette: DNA adducts and alveolar macrophage cytogenetics.

To study the genotoxic effects of subchronic exposure to environmental tobacco smoke, Sprague-Dawley rats were exposed to 0, 0.1, 1.0, and 10 mg total particulate matter (TPM)/m3 of aged and diluted sidestream smoke (ADSS) from 1R4F reference cigarettes 6 hr per day, 5 days a week for 13 weeks. DNA from lung, heart, larynx, bladder, and liver was tested for adduct formation by the 32P-postlabeling assay after 28 (except bladder) and 90 days of exposure and 90 days after cessation of exposure. In addition, alveolar macrophages from animals exposed for 28 or 90 days were examined for chromosomal aberrations. Exposure-related DNA adducts were not observed in any tissue in any of the animals exposed to 0.1 or 1.0 mg TPM/m3. However, increased levels of DNA adducts with diagonal radioactive zones were observed in lung, heart, and larynx DNA of animals exposed to the highest concentration of ADSS (10 mg TPM/m3). Adduct analyses with varying amounts of DNA from lungs of mid- and high-exposure animals clearly indicate that the dose-response for DNA adduct formation is nonlinear. The adduct levels were highest after 90 days of exposure and were significantly reduced in all target tissues 90 days after cessation of exposure. Chromosomal aberrations in alveolar macrophages were not elevated in any group after 28 or 90 days of exposure. These results indicate a no-observed-effect-level (NOEL) of at least 1.0 mg/m3 for DNA adduct formation in lung, heart, and larynx, and a NOEL of at least 10 mg/m3 for the induction of chromosome aberrations in alveolar macrophages, under the conditions of this study.

Fourteen-day inhalation study in rats, using aged and diluted sidestream smoke from a reference cigarette. I. Inhalation toxicology and histopathology.

Sprague-Dawley rats were exposed 6 hr per day for 14 consecutive days to aged and diluted sidestream smoke (ADSS), used as a surrogate for Environmental Tobacco Smoke (ETS), at concentrations of 0.1 (typical), 1 (extreme), or 10 (exaggerated) mg of particulates per cubic meter. Animals were exposed nose-only, inside whole-body chambers, to ADSS from the 1R4F reference cigarette. End-points included histopathology, CO-oximetry, plasma nicotine and cotinine, clinical pathology, and organ and body weights. The only pathological response observed was slight to mild epithelial hyperplasia and inflammation in the most rostral part of the nasal cavity, in the high-exposure group only. No effects were noted at medium or low exposures. The minimal changes noted were reversible, using a subgroup of animals kept without further treatment for an additional 14 days. Overall, the end-points used in the study demonstrated that there was no detectable biological activity of ADSS at typical or even 10-fold ETS concentrations and that the activity was only minimal at very exaggerated concentrations (particle concentrations 100 times higher than typical real-world concentrations).

Fourteen-day inhalation study in rats, using aged and diluted sidestream smoke from a reference cigarette. II. DNA adducts and alveolar macrophage cytogenetics.

The chemical constituents of cigarette smoke are greatly diluted in environmental tobacco smoke (ETS). In the typical indoor environment where cigarettes are smoked, the mean value of respirable suspended particles is approximately 0.1 mg/m3. In this study, we used aged and diluted sidestream smoke (ADSS) of 1R4F University of Kentucky research cigarettes as a surrogate for ETS and exposed Sprague-Dawley rats nose-only to 0, 0.1, 1.0, and 10 mg wet total particulate matter (WTPM)/m3 for 6 hr per day for 14 consecutive days. DNA from lung, heart, larynx, and liver was tested for adduct formation after 7 and 14 days of exposure and after 14 days of recovery. In addition, alveolar macrophages from animals exposed for 7 days were examined for chromosomal aberrations. Exposure-related DNA adducts were not observed in any of the animals at 0.1 or 1.0 mg WTPM/m3, which represent ambient and 10-fold exaggerated ETS concentrations, respectively. Slight diagonal radioactive zones, characteristic of adducts observed in human smokers and in animals exposed to mainstream smoke, were observed, but only in lung and heart DNA of animals exposed to the highest concentration of ADSS (10 mg WTPM/m3), a 100-fold exaggeration of typical field measurements of ETS. The mean relative adduct labeling values (+/- SE) were 8.7 (+/- 0.2) adducts per 10(9) nucleotides for lung DNA and 5.7 (+/- 0.7) adducts per 10(9) nucleotides for heart DNA after 14 days of exposure. No elevation in chromosomal aberrations was observed in alveolar macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

Histological sectioning of the rodent larynx for inhalation toxicity testing.

In rodents, the larynx is a major site of histopathologic alteration following inhalation exposure to particulates, vapors, and aerosols. Specifically, the epithelial lining of a narrowly delineated region on the ventral floor of the larynges of rats and mice appears to be especially vulnerable to inhaled materials, and is recognized as a preferred site for histopathological evaluation in inhalation studies. This site is located at the base of the epiglottis, cranial to the ventral laryngeal diverticulum (ventral pouch). The presence of underlying seromucinous glands is critical for histologic identification of this site. We report a histologic sectioning technique, using the ventral laryngeal diverticulum as the anatomical landmark, to obtain tissue sections from this area of predilection in rats and in mice.

Analysis of cytogenetic effects in bone-marrow cells of rats subchronically exposed to smoke from cigarettes which burn or only heat tobacco.

The genotoxic effects of 90-day nose-only exposures to smoke from new cigarettes, which heat but do not burn tobacco (New), or from reference cigarettes, which burn tobacco, were evaluated in Sprague-Dawley rats by examining the cytogenetic endpoints of sister-chromatid exchanges (SCE), chromosome aberrations, and micronuclei in bone-marrow cells. The concentrations of wet total particulate matter (WTPM) and carbon monoxide in the smoke from both cigarette types were similar. The mainstream smoke from both New and reference cigarettes was adjusted to WTPM concentrations of approx. 200 and 400 micrograms/l for low and high smoke exposure. Rats were exposed to smoke 1 h per day, 5 days per week for 13 consecutive weeks. Inhalation of smoke by the exposed animals was confirmed by analysis of blood carboxyhemoglobin and plasma nicotine. Examination of bone-marrow cells following the final day of exposure showed that smoke from neither the New nor reference cigarette induced a positive response in the SCE, chromosome aberration, or micronucleus assays in rats.

Histologic changes in the respiratory tract induced by inhalation of xenobiotics: physiologic adaptation or toxicity?

Toxicologists and pathologists are often faced with the dilemma of categorizing changes observed in the respiratory tract of laboratory animals as either "adaptive" or "toxic." However, it is often difficult to interpret the nature of a given change as either "adaptive" or "toxic." Certain lesions or changes in the respiratory tract are to be expected from the concentration of materials given or the experimental design of a study. Careful analysis suggests that some of these changes may be more properly described as adaptive rather than toxic within the context of a given study or situation. Tissue changes discussed in this paper include squamous metaplasia of laryngeal epithelium, goblet cell change in respiratory epithelium, macrophage accumulation within alveoli, and bronchiolization of alveolar epithelium. Examples provided show that some of these changes observed in inhalation studies are similar in severity but slightly increased in frequency over sham control animals. The introduction of exogenous material into the respiratory tract of laboratory animals in an experimental setting should be expected to result in certain changes. The challenge scientists must accept is to interpret these changes so that toxic events may be separated from adaptive changes. In order to meet this challenge, studies incorporating several species and novel technologies may have to be utilized.