Short-term biomarkers of cigarette smoke condensate tumor promoting potential in mouse skin.

Previous studies demonstrated that cigarette smoke condensates (CSCs) possessing significantly different tumorigenic potentials according to a standardized 30-week mouse skin tumor-promotion protocol could likewise be discriminated utilizing short-term indices of sustained hyperplasia and/or inflammation (G. M. Curtin et al., 2004, Toxicol. Sci. 81, 14-25). The current study employed a truncated initiation-promotion protocol to further evaluate CSC-induced hyperplasia, examining issues related to time course of induction, existence of a threshold and suitable dynamic range for detectable responses, and reversibility. Condensate application (9-36 mg "tar"/200-microl application, thrice-weekly for 3-15 weeks) induced treatment-related increases for epidermal thickness, proliferative index as assessed by 5-bromo-2'-deoxyuridine (BrdU) labeling, and ornithine decarboxylase (ODC) expression. Interestingly, observed increases for interfollicular BrdU labeling and ODC expression were partially reversed but still elevated upon cessation of promotion, while increases within the perifollicular epidermis remained elevated at a level similar to that observed during CSC application. In particular, assessments based on perifollicular ODC expression would appear to provide a greater opportunity for test article discrimination based on a rapid time to induction, a low threshold and expanded dynamic range of responses, and the potential to account for irreversible changes. These findings are particularly intriguing based on reports suggesting that ODC expression may be necessary for tumor promotion and that mouse skin tumors originate primarily within the perifollicular epidermis.

Short-term in vitro and in vivo analyses for assessing the tumor-promoting potentials of cigarette smoke condensates.

Previous studies found that repeated application of smoke condensate from tobacco-burning reference cigarettes to chemically initiated SENCAR mouse skin promoted the development of tumors in a statistically significant and dose-dependent manner, while condensate from prototype cigarettes that primarily heat tobacco promoted statistically fewer tumors. Based on the recognized correlation between sustained, potentiated epidermal hyperplasia and tumor promotion, we conducted tests to examine the utility of selected short-term analyses for discriminating between condensates exhibiting significantly different promotion activities. In vitro analyses assessing the potential for inducing cytotoxicity (ATP bioluminescence) or free radical production (cytochrome c reduction, salicylate trapping) demonstrated significant reductions when comparing condensate collected from prototype cigarettes to reference condensate. Short-term in vivo analyses conducted within the context of a mouse skin, tumor-promotion protocol (i.e., comparative measures of epidermal thickness, proliferative index, myeloperoxidase activity, leukocyte invasion, mutation of Ha-ras, and formation of modified DNA bases) provided similar results. Reference condensate induced statistically significant and dose-dependent increases (relative to vehicle control) for nearly all indices examined, while prototype condensate possessed a significantly reduced potential for inducing changes that we regarded as consistent with sustained epidermal hyperplasia and/or inflammation. Collectively, these data support the contention that selected short-term analyses associated with sustained hyperplasia and/or inflammation are capable of discriminating between smoke condensates with dissimilar tumor-promotion potentials. Moreover, our results suggest that comparative measures of proliferative index and myeloperoxidase activity, both possessing favorable correlation coefficients relative to tumor formation (i.e., > or = 0.95 after 8 or 12 weeks of promotion), may constitute reasonable end points for further investigation.

Lung tumorigenicity in A/J and rasH2 transgenic mice following mainstream tobacco smoke inhalation.

Hypothesizing that their respective genetic backgrounds would confer an increased sensitivity to lung tumorigenesis, the plausibility of selected rodent models for the inhalation testing of mainstream tobacco smoke (MTS) was evaluated. Strain A/J and rasH2 transgenic (Tg) mice were exposed to MTS from Kentucky 1R4F research cigarettes using either a whole-body or nose-only exposure regimen. The whole-body regimen consisted of a 20-week exposure period [0.200 mg wet total particulate matter/liter (WTPM/l), 6 h/day, 5 days/week]; nose-only dosing proceeded for 28 weeks [0.040, 0.125, or 0.400 mg WTPM/l, 3 h/day, 5 days/week]. Both regimens included a 16-week recovery period. Gross and microscopic examinations of the lungs were used to evaluate tumor formation, with experimental results supporting the following conclusions: 1. Evaluation of MTS-induced tumorigenicity based on gross evaluation versus microscopic confirmation provides strikingly disparate results, indicating that serial sectioning is necessary for a definitive assessment of lung tumors. 2. While the dosing regimens employed do not allow for a definitive comparison, whole-body exposure appeared to be more effective for inducing statistical changes in tumor multiplicity and incidence compared to nose-only exposure. 3. Exposure-related stress, evidenced as reductions in both body weight gain and background tumor formation, represents a potential confounder during inhalation testing of MTS tumorigenicity, with additional investigation warranted to validate the specificity of exposure-related responses. 4. Comparative findings between A/J and rasH2 Tg mice suggest that the former may be overly sensitive to exposure-related stress, potentially influencing tumorigenic responses.

Comparative study of smoke condensates from 1R4F cigarettes that burn tobacco versus ECLIPSE cigarettes that primarily heat tobacco in the SENCAR mouse dermal tumor promotion assay.

Numerous chemical and toxicological studies indicate that smoke from ECLIPSE, a cigarette that primarily heats rather than burns tobacco, is simplified and reduced in specific chemicals believed to be associated with smoking-related diseases, and demonstrates reduced smoke toxicity and biological activity in vitro when compared to conventional tobacco burning cigarettes. These data led to the hypothesis that cigarette smoke condensate (CSC) from ECLIPSE should have lower tumorigenicity than 1R4F condensate in the SENCAR mouse dermal tumor promotion assay. Female SENCAR mice were initiated with a single topical application of 7,12-dimethylbenz[a]anthracene (DMBA) followed by promotion with ECLIPSE or 1R4F CSC. Dermal application of 10, 20, or 40 mg ECLIPSE or 1R4F CSC three times/week for 29 weeks did not alter body weights, survival or other indicators of subchronic toxicity. In DMBA-initiated mice, there were significant increases in both the number of microscopically confirmed tumor-bearing animals and total number of microscopically confirmed dermal tumors at all 1R4F CSC doses and the high-dose ECLIPSE CSC. However, the number of ECLIPSE tumor-bearing animals were reduced 83%, 93% and 67% at the low-, mid- and high-doses, respectively, compared to the 1R4F. Similarly, the total number of dermal tumors was reduced 91%, 94% and 87% at the low-, mid- and high-dose, respectively, compared to the 1R4F CSC. ECLIPSE CSC demonstrated dramatic reductions in dermal tumor promotion potential compared to 1R4F CSC.

A responsive, sensitive, and reproducible dermal tumor promotion assay for the comparative evaluation of cigarette smoke condensates.

The mouse dermal initiation/promotion bioassay has been used for several decades to study cigarette smoke condensates (CSCs). However, these studies have used highly variable methodologies that differ in the manner of CSC collection, duration of treatment, mouse strain, number of mice and endpoints measured. In this report, a protocol that uses female SENCAR mice and standardizes many of the procedures is presented. A reference cigarette (University of Kentucky 1R4F), readily available to researchers, was used. This report presents the combined data from four independent studies. Female, SENCAR mice (40/group) were treated with a single dose (75microg) of dimethylbenz[a]anthracene (DMBA) as an initiator, followed 1 week later by treatment (three times/week) with 10, 20 or 40mg "tar"/application of 1R4F CSC for 29 weeks. There were no treatment-related effects on body weights. Histological diagnosis of all masses at study termination indicated a dose-dependent increase in the number of tumor-bearing mice and total tumor number. These studies support the conclusion that the 1R4F cigarette is suitable for use as a reference standard and the protocol presented is an appropriate and standardized model suitable for the comparative evaluation of CSC.

Toxicological evaluation of propane expanded tobacco.

A tiered testing strategy has been developed to evaluate the potential for tobacco processes, ingredients, and other technological developments to increase or decrease the biological activity resulting from burning tobacco. The strategy is based on comparative chemical and biological testing. Propane expanded tobacco is an example of a processed tobacco used in the modern manufacture of cigarettes. Test cigarettes containing propane expanded tobacco were compared to control cigarettes containing tobacco expanded with a traditional expansion agent (Freon-11). The toxicological evaluation included chemistry studies using mainstream cigarette smoke (determination of selected constituent yields), in vitro studies using cigarette smoke condensate (Ames study in Salmonella typhimurium and sister chromatid exchange study in Chinese hamster ovary cells) and in vivo studies (13-week inhalation study of mainstream cigarette smoke in Sprague-Dawley rats and 30-week dermal tumor promotion study of cigarette smoke condensate in SENCAR mice). Although statistically significant differences in several smoke constituents were observed, most constituents from cigarettes containing 100% propane expanded tobacco were within market survey ranges. Furthermore, biological tests indicated that the cigarettes containing propane or Freon-11 expanded tobacco were not significantly different.

Toxicological evaluation of dry ice expanded tobacco.

A tiered testing strategy has been developed to evaluate the potential of tobacco processes, ingredients, or technological developments to change the biological activity resulting from burning tobacco. The strategy is based on comparative chemical and biological testing. Dry ice expanded tobacco (DIET) is an example of a common tobacco expansion process currently used in the manufacture of cigarettes to increase tobacco filling capacity. As part of the toxicological evaluation of DIET, test cigarettes containing DIET were compared with control cigarettes containing tobacco expanded with a traditional expansion agent (Freon-11, also known as trichlorofluoromethane). Testing included mainstream cigarette smoke chemistry studies, genotoxicity studies (Ames and sister chromatid exchange, SCE), a 13-week inhalation study in Sprague-Dawley rats, and a 30-week dermal tumor promotion study in SENCAR mice. Cigarettes containing DIET or Freon-11 expanded tobacco were similar in biological activity.

Toxicological evaluation of honey as an ingredient added to cigarette tobacco.

A tiered testing strategy has been developed to evaluate the potential for new ingredients, tobacco processes, and technological developments to increase or reduce the biological activity that results from burning tobacco. In the manufacture of cigarettes, honey is used as a casing ingredient to impart both aroma and taste. The primary objective of this document is to summarize and interpret chemical and toxicological studies that have been conducted to evaluate the potential impact of honey on the biological activity of either mainstream cigarette smoke or cigarette smoke condensate. As part of ongoing stewardship efforts, cigarettes produced with honey (5% wet weight) as an alternative to invert sugar in tobacco casing material were subjected to extensive evaluation. Principal components of this evaluation were a determination of selected mainstream smoke constituent yields, Ames assay, sister chromatid exchange assay in Chinese hamster ovary cells, a 30-wk dermal tumor promotion evaluation of cigarette smoke condensate in SENCAR mice, and a 13-wk inhalation study of cigarette smoke in Sprague-Dawley rats. Comparative analytical evaluations demonstrated that the substitution of honey for invert sugar as a casing material in cigarettes had no significant impact on mainstream smoke chemistry. In addition, in vitro and in vivo studies demonstrated that cigarettes containing tobacco cased with honey had comparable biological activity to cigarettes containing invert sugar. Collectively, these data demonstrate that the use of honey as an alternative casing material in the manufacture of cigarettes does not alter the potential toxicity of cigarette smoke condensate (CSC) or cigarette smoke; therefore the use of honey as an ingredient added to cigarette tobacco is acceptable from a toxicological perspective.

Rat subchronic inhalation study of smoke from cigarettes containing flue-cured tobacco cured either by direct-fired or heat-exchanger curing processes.

A subchronic, nose-only inhalation study compared the effects of mainstream smoke from a cigarette containing 100% flue-cured tobacco cured by a direct-fired process to that of a cigarette containing 100% flue-cured tobacco cured by a heat exchanger process. The tobaccos and mainstream smoke from tobaccos cured by the heat exchanger process have been shown to have significantly lower levels of tobacco-specific nitrosamines than tobaccos cured by a direct-fired process. Male and female rats were exposed for 1 h/day, 5 days/wk, for 13 wk to mainstream smoke at 0, 0.06, 0.20, or 0.80 mg wet total particulate matter per liter of air. Clinical signs, body and organ weights, clinical chemistry, hematology, carboxyhemoglobin, serum nicotine, plethysmography, gross pathology, and histopathology were determined. When histologic changes resulting from exposure to smoke from the two types of cigarettes were compared, the only significant difference was increased epithelial hyperplasia of the anterior nasal cavity in males in the high-exposure group for the heat-exchanger cigarette. At the end of the exposure period, subsets of rats from each group were maintained without smoke exposures for an additional 13 wk (recovery period). At the end of the recovery period, there were no statistically significant differences in histopathological findings observed between the heat-exchanger-cured tobacco cigarette when compared to the direct-fired cured tobacco cigarette. The complete toxicological assessment in this study of heat exchanger and direct-fired tobaccos suggests no overall biologically significant differences between the two cigarettes.

Quantification of continuous glass filaments on eclipse cigarettes retrieved from the test market.

ECLIPSE cigarettes utilize a special form of continuous glass filament (CGF) as an insulator around the carbon heat source. The average numbers of CGFs on the external barrel and cigarette filter end were determined subsequent to manufacture, subsequent to real-world consumer handling and subsequent to simulated consumer handling. The following were not statistically significantly different: the average number of CGFs on the external barrel of cigarettes retrieved from the test market compared to the average external barrel counts from cigarettes subsequent to manufacture or when subjected to simulated consumer handling, and the average number of CGFs on the external barrel of cigarettes subsequent to manufacture compared to the average external barrel counts from cigarettes subjected to simulated consumer handling. The average number of CGFs on the filter end of cigarettes retrieved from the test market was statistically significantly higher than average cigarette filter end counts from cigarettes subsequent to manufacture. The average number of CGFs on the cigarette filter end of cigarettes retrieved from the test market was statistically significantly lower than average cigarette filter end counts from cigarettes subjected to simulated consumer handling. Overall, results from this study suggest that consumer handling does increase the average numbers of CGFs on the external surfaces of the cigarette. Further, the results of this study demonstrate that for the purpose of CGF quantification, the simulated consumer handling protocol used in this study (i.e., based on laboratory measurements of forces) is a reasonably good model for actual consumer handling of cigarettes. Based on the minimal number of CGFs that could be transferred to the smoker and the deposition pattern governed by their physical characteristics, the potential to deposit CGFs from these cigarettes to the lungs of smokers is extremely remote. Therefore, no convincing information exists to suggest that smokers would be exposed to CGFs from any ECLIPSE-related source at a biologically significant level.