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.

Chronic inhalation study in rats, using cigarettes containing different amounts of Cytrel tobacco supplement.

The paper describes a chronic (18-month) inhalation study in rats, comparing respiratory tract lesions produced by exposures to cigarettes with different inclusions of Cytrel, a tobacco supplement. Histopathological examinations indicated lower responses in animals exposed to Cytrel-containing cigarettes than in those exposed to all-tobacco cigarettes, despite similar total deliveries of smoke. The decrease in response was approximately proportional to the level of Cytrel inclusion.

Cigarette smoke induced pathology of the rat respiratory tract: a comparison of the effects of the particulate and vapour phases.

Changes in the rat respiratory tract produced by a 12-week exposure to the particulate or vapour phases of cigarette smoke are described and compared with the changes produced by combined exposure to both phases. The most extensive changge in the upper respiratory tract was a pronounced squamous metaplasia of the laryngeal epithelium, along with extensive deciliation and squame cell production. In the lung, smoke exposure produced marked increase in the numbers of bronchial goblet cells and intra-alveolar brown-gold macrophages. None of the above changes could be directly attributed to exposure to vapour phase alone, and in many cases the lesions produced by whole smoke and by carbon filtered smoke were directly comparable. For lesions to occur in some ciliated areas of the tract both phases of the smoke were required.

The tumourigenicity of smoke condensates from cigarettes containing different amounts of cytrel, as assessed by mouse skin painting.

Smoke condensates derived from cigarettes containing different amounts of the tobacco supplement Cytrel were painted onto the banks of mice 3 times a week for 104 weeks. Cigarette samples contained 0, 25, 50 or 100% Cytrel, blended with tobacco, and 3 dose levels were used for each sample. Statistical analyses were made of tumour incidence using a mathematical model based on the Weibull distribution. Good dose relationships were observed for each product; and for samples containing Cytrel there was a reduction in smoke condensate production and in tumourigenicity (per unit weight of smoke condensate) approximately proportional to the level of inclusion. Histopathological examinations of tumours indicated a reduction in malignancy of approximately 50% for all-Cytrel samples when compared with all-tobacco samples.

Changes in the minute ventilation of rats exposed to different concentrations of cigarette smoke.

Measurements of tidal volume and breathing frequency were made in rats during a 30-min period of exposure to cigarette smoke, and were compared with those obtained during a 10-min pre-exposure period. All measurements were made in animals previously habituated to smoke exposure. Both tidal volume and breathing frequency were decreased by low-dose exposure to smoke, resulting in a 34% decrease in minute volume. At high-dose exposures (double the low-dose smoke concentration) tidal volumes showed a 23% increase over pre-exposure values with little change in breathing frequency, giving a net 20% increase in minute ventilation. This apparent inverse dose-relationship is discussed.

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.

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.

Subchronic nose-only inhalation study of propylene glycol in Sprague-Dawley rats.

Groups of nineteen Sprague-Dawley rats of each sex were exposed by a nose-only inhalation to 0.0, 0.16, 1.0 or 2.2 mg propylene glycol/litre air, for 6 hr/day, 5 days/wk for 90 days. There were no significant differences in respiratory rates, minute volumes or tidal volumes between any of the groups during aerosol exposure. The uniformity of respiratory parameters between dose groups implied that the delivered doses were proportional to the exposure concentrations. The mean terminal body weights were not significantly different from controls for any group of male animals. The mean body weights of the females exposed to 2.2 mg/litre were significantly less than those of female controls from day 50 onwards. This effect, in female rats, was consistent with a decrease in feed consumption for the high-exposure female rats beginning on study day 43. Statistically significant differences between the treated and control groups in certain haematological parameters, serum enzyme activities, other serum chemistry parameters and organ weights did not show clear dose relationships. There was a significant increase in the number of goblet cells or an increase in the mucin content of the existing goblet cells in the nasal passages of the medium- and high-exposure animals. Exposure to the above concentrations of propylene glycol caused nasal haemorrhage and ocular discharge in a high proportion of animals, possibly as a result of dehydration of the nares and eyes.

A method for producing forced expirograms in laboratory animals.

A method is described for the production of forced expirograms in anaesthetized laboratory animals, using a whole-body plethysmograph and a Hewlett-Packard (HP) 1000-F computer.

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.

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 program for handling data obtained by 'blind' histopathological evaluations.

An interactive data input, storage, retrieval and treatment system (LAMES) is described for use in histopathological studies where pathologists evaluate tissues without any identification (termed evaluations which are 'blind'). The system produces the mean values of individual groups within an experiment, and results of statistical comparisons between the groups. The system was implemented on a Hewlett-Packard (HP) 1000-F minicomputer operating under RTE-6/VM, and was written using FORTRAN IV.

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.

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.

On-line data acquisition and control in a physiology laboratory.

A series of programs are described for use in estimating the pulmonary function of laboratory animals. Data acquisition and control are through a Hewlett-Packard (HP) 1000-F minicomputer. The system allows the estimation of respiratory parameters similar to those obtained in laboratories estimating pulmonary function in humans.

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.