Lung tumors in A/J mice exposed to environmental tobacco smoke: estimated potency and implied human risk
Kenneth T. Bogen1,3 and
Hanspeter Witschi2
1 Health and Ecology Assessment Division L-396, Lawrence Livermore National Laboratory, University of California, 7000 E. Ave., Livermore, CA, 94550 and
2 ITEH and Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA 95616, USA
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Abstract
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Directly inhaled tobacco smoke is a recognized human lung carcinogen, and epidemiological studies suggest relative risks of about 1.21.4 for nonsmoking spouses of smokers typically exposed to environmental tobacco smoke (ETS). While many individual ETS components have been shown experimentally to induce lung tumors, ETS itself was only recently shown to induce lung tumors in a series of studies in which strain A/J mice were exposed to well-defined ETS atmospheres. Data from these studies indicate that ETS exposure clearly can increase combined malignant and benign lung tumors in multiple experiments involving male and female A/J mice, and thus provide convincing evidence that ETS is a positive mouse carcinogen. Tumorigenic potencies estimated from these A/J mouse bioassay data predict a corresponding range of increased human risk (0.20.5%) that overlaps that implied by casecontrol studies showing increased lung cancer risks in lifelong nonsmokers married to smokers. In A/J mice exposed to a significantly tumorigenic ETS concentration, lung tumors were found to be significantly smaller than those in corresponding control mice, and mice so exposed for 9 months had significantly fewer tumors/animal than mice exposed for 5 months followed by 4 months in filtered ETS-free air. These findings support hypotheses that ETS does not promote growth of spontaneous neoplastic foci in A/J mice, and that ETS-induced lung-tumor risk in A/J mice occurs predominantly by genotoxic effects that can be suppressed by reduced cell proliferation associated with chronic, high-level ETS exposure. The results obtained add to evidence that A/J mouse lung tumors induced by ETS provide a relevant biological model of ETS-induced human lung tumors.
Abbreviations: ETS, environmental tobacco smoke; NHANES, National Health and Nutrition Examination Survey; RSP, respirable suspended; RR, relative risk; TSP, total suspended particulate matter; TWA, time-weighted average; UCL, upper confidence limit.
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Introduction
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Tobacco smoke has long been recognized as a human lung carcinogen based on strong epidemiological evidence (1) and supporting experimental cancer-bioassay and genotoxicity data (2,3). Recent reviews of the available chronic inhalation studies with mainstream cigarette smoke have concluded that the human disease had not been successfully duplicated in mice or rats, and that laryngeal tumors in hamsters were likely the best model system (2,3). However, a few studies did find a positive carcinogenic effect of mainstream cigarette smoke in lung tissue of albino mice (4,5) and BALB/c mice (6) exposed by inhalation. As discussed below, benign and malignant lung tumors are also produced in strain A mice exposed to a mixture of cigarette mainstream and sidestream smoke used to simulate environmental tobacco smoke (ETS) (7,8).
Casecontrol studies (including U.S. population-based casecontrol studies) have linked chronic workplace or residential ETS exposure with significantly increased risk of lung cancer incidence in never-smokers, with odds ratio estimates ranging from >1 to ~3 or even greater (~25) among women never-smokers deficient in glutathione-S-transferase-M1 activity and among never-smokers exposed to ETS during childhood (920). A US cohort study involving data on >106 men and women also reported a statistically significant increasing trend in estimated odds ratio (OR) for lung cancer in nonsmoking spouses versus spousal cigarettes smoked per day (21). Other casecontrol studies and a meta-analysis have shown no significant positive (or showed a significant negative) association between spousal, occupational, and/or childhood ETS exposure with increased lung cancer risk (18,2226). Both the statistical and biological significance of observed epidemiological associations involving ETS have been questioned (27). However, other meta-analyses and reviews of casecontrol data on lung cancer among ETS-exposed nonsmokers generally have concluded that ETS does cause lung cancer (2833). Best estimates of adjusted OR for ETS-induced lung cancer from all these studies range from about 1.2 to 1.4 for nonsmoking spouses (primarily women) married to smokers, similar or slightly lower ORs for occupational ETS exposure, and similar or somewhat higher ORs for childhood ETS exposure.
Strain A/J mice have a relatively high spontaneous adenoma/adenocarcinoma incidence and, following exposure to a carcinogen, readily develop additional lung tumors that show multiple similarities in genetic alterations and signal transduction pathways to human lung adenocarcinoma (34). The strain A/J mouse lung-tumor assay is currently the most widely used method to assess agents for chemopreventive effects on lung carcinogenesis (35). Recent studies by Witschi et al. (7,3539) involving this assay indicate that ETS is also a lung carcinogen in A/J mice. These experiments all dealt with various aspects of ETS toxicity, such as whether ETS would be tumorigenic or at least `promote' lung tumor development (36), whether the gas phase of ETS alone was as toxic as unfiltered tobacco smoke (37), or whether ETS-induced lung tumors could be inhibited by chemopreventive agents (35,38). Most of these experiments (3539) used nearly identical 5-month-ETS-exposure/4-month-recovery protocols involving an ETS-exposure system previously described (40,41) in which ~9 parts sidestream to 1 part mainstream smoke was drawn from reference cigarettes, aged, and then diluted to maintain a target ETS-like concentration of total suspended particulate matter (TSP) for exposed animals, while filtered clean air was supplied simultaneously to identically housed control animals. The most recent study was similar, except that ETS exposure continued for nine months without a four-month recovery period (7).
Uncertainty has persisted concerning the key mechanism(s) underlying ETS-induced lung tumorigenesis in strain A/J mice, as well as how the observed potency of this response compares with ETS potency estimates consistent with human epidemiological data. The present analysis was undertaken to assess the tumorigenic `potency' (i.e. increased lung-tumor risk per unit intake) of ETS, based on combined bioassay data from previous studies by Witschi et al. (7,3539). Lung tumor sizes observed in ETS-exposed and corresponding control male A/J mice were also compared using a recent subset of these bioassay data (7). Resulting estimates of ETS potency in A/J mice were compared to that of tobacco smoke in humans based on a previous analysis of epidemiological data. Corresponding estimates were made of the increased risk of lung tumors due to human exposure to ETS, based on published ETS-exposure estimates combined with a new analysis of California and U.S. nationwide (NHANES III) survey data on residential cigarette use. The plausibility of these risk estimates is discussed in view of mechanistic implications of the data obtained on lung tumor sizes in ETS-exposed versus unexposed A/J mice, as well as related casecontrol data discussed above.
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Materials and methods
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Animal experiments
The conduct of the animal studies and generation of an ETS atmosphere has been described in detail in previous publications (36,37,40). Briefly, male or female strain A/J mice (Jackson Laboratory, Bar Harbor, ME) were exposed for 6 h/day, 5 days/week, to an atmosphere of ETS consisting of 89% sidestream smoke and 11% mainstream smoke produced by burning Kentucky 1R4F reference cigarettes. After a gradual increase of ETS concentrations during the first 5 weeks, final chamber TSP concentrations (measured daily) ranged from 80 to 90 mg/m3 in three studies and from 130 to 140 mg/m3 in two studies (Table I
). Control animals were kept in similar chambers ventilated with HEPA-filtered ETS-free air. After 5 months of exposure, mice were transferred to conventional animal holding facilities, and thus allowed to recover from ETS exposure, for 4 months. Nine months after the beginning of each experiment, the animals were killed (at 1112 months of age). After a suitable fixation time, the number of tumor nodules visible on the lung surface was counted under a dissecting microscope. Selected tumors were cut and examined under the light microscope. About 80% of these tumors were classified as alveolor-bronchiolar adenomas, and 20% as alveolarbronchiolar adenocarcinomas. Several of the carcinomas arose within the adenomas, indicating the potential of adenomas to progress to malignancy (37).
Bioassay data selection
Bioassay data used to assess the tumorigenic potency of ETS in strain A/J mice are summarized in Table I
. Studies 15 listed in Table I
involved a comparison of lung-tumors in A/J mice exposed to ETS or filtered air for
10 weeks, followed by exposure to filtered air until death or sacrifice at an approximate age of about 50 weeks. Data involving exposures to test compounds other than ETS (and associated control experiments), as well as studies involving a more brief experimental duration, were excluded from the present assessment. Thus, all six control data sets (five with male and one with female mice) involved an identical 50-weeks filtered-air exposure protocol; five of the six ETS-exposure data sets (four with male and one with female mice) involved an identical 22-weeks ETS-exposure protocol; and the remaining ETS-exposure data set involved a 10-weeks ETS-exposure protocol (applied to male mice only) otherwise identical to that of the other five involving ETS exposure (Table I
). Additional data (corresponding to Study 6 in Table I
) involved similarly exposed male A/J mice, except that exposures continued for 9 months and did not include the 4 month recovery period (7).
Tumor response measures
Based primarily on human epidemiological evidence, chemicals in primary and environmental tobacco smoke are known to cause lung cancer in humans (28,42). Data from studies summarized in Table I
involve benign or malignant alveolar or bronchiolar tumors (i.e. adenomas or carcinomas). In these studies, ~1025% of all tumors counted were malignant, and progression appeared to occur along a continuum from hyperplasia to adenoma to carcinoma, with the latter occasionally observed to arise within adenomas in support of the hypothesis that these adenomas can undergo malignant progression (36,37). Data summarized in Table I
therefore reflect measures of tumor response in which malignant and benign tumors were combined.
Tumor data were tabulated both in terms of multiplicity (average tumors/lung including animals without any tumor) and incidence (fraction of exposed animals with any tumor). The second measure is one traditionally used for chemical carcinogen identification and for cancer potency assessment (4345). However, it may be appropriate (and, in the context of linear risk-extrapolation models, is preferable) to base potency analysis on the first measure (tumor multiplicity) rather than the second (fraction of tumor-bearing animals) if tumors counted each represent a statistically independent occurrence (46). The reason for this is that, if the tumors are independent, then rare additional tumors would be expected to exhibit Poisson occurrence statistics, and so would almost certainly occur in different individuals. Consequently, potency assessment based on tumor-bearing animals tends to underestimate low-level risk, whereas that based on tumors/animal more accurately estimates low-level risk if increased risk is linearly proportional to dose. In the case of lung tumors in studies listed in Table I
, such independence is reasonable insofar as each observed tumor was a distinctly separated nodular/neoplastic mass within the surface area of excised lung tissue examined, with no evidence of metastatic origin.
Model of tumorigenic potency and risk
The `linearized' multistage model was used to estimate potency and risk from tumor incidence data (43,44). This model defines cancer risk R(d) as the following function
 | (1) |
of equivalent lifetime, time-weighted average (TWA) dose d (in mg/kg per day), where qi denote coefficients in dose to be estimated, and g + 1 is the number of bioassay-animal groups. The linear term q1 in dose denotes tumorigenic `potency,' defined here as the limiting value of dose-induced increase A(d) in tumor risk R(d) above the (presumed independent) background risk R(0) per unit d as d
0, where
 | (2) |
Tumor multiplicity R(d), and its corresponding potency q1, were estimated from corresponding tumor multiplicity data using only the (bracketed) polynomial term in Eq. (1). A linear-no-threshold dose-response allowed by Eq. (1) is plausible for any carcinogenic agent known to induce misrepaired or unrepaired DNA damage yielding mutations in somatic stem cells (4346). A linear-no-threshold dose-response is thus plausible for ETS because its constituents include many known or suspected human carcinogens (e.g. benzo[a]pyrene, other polycyclic aromatic hydrocarbons, and tobacco-specific nitrosamines) that induce such genotoxic damage, including mutation of lung-tumor-specific oncogenes (3,36,37,41,47,48).
Equivalent lifetime TWA TSP concentration and dose
For interspecies scaling of equitoxic dose, dose d traditionally has been measured as mass per unit of either body weight, body surface area, or more recently body weight to the 0.75 power (43,44,49). A comparison made of estimated potencies for chemical carcinogens in humans versus rodents based on epidemiological versus bioassay data, respectively, indicates that all these animal-to-human scaling methods have roughly comparable predictivity (49,50). In the present study, potency assessment was based on equivalent lifetime TWA dose d in units of mg/kg per day, the average equivalent daily dose that would have been incurred had animals in a lifetime bioassay been exposed continuously over the duration (Le) of experimental exposure, provided that: (a) Le = Lb, where Lb is the total bioassay duration (including the period prior to exposure and observation period post-exposure); and (b) Le = La, where La is the typical lifespan of the experimental animals used. It is evident from Table I
that Le = 10 weeks for the dose group in Study 1 for which Ce = 52.6 mg, and Le = 22 weeks for all other (non-control) exposure levels, where Ce denotes experimental TSP concentration (in mg/m3). In Studies 16, Lb = 50 weeks (Table I
), and La is ~102 weeks. Using standard methods (43), Ce thus corresponds to the following equivalent lifetime TWA concentration C (mg/m3):
 | (3) |
and conversion of estimated tumorigenic potency Q1 in units of m3/mg to equivalent potency q1 in units of kg d mg1 was done by assuming
 | (4) |
Wa is mean sex-specific mouse weight in kg, and Va is mouse inhalation rate under the simplifying assumption that respired TSPor any other tumorigenically effective volatile ETS component with a concentration proportional to that of TSPis fractionally retained by equal amounts in the mouse lung and in the human lung.
Tumor size analysis
Sustained induction of cell proliferation may indicate a basis for expecting low-dose nonlinearity in cancer dose-response, whereas a purely or predominantly genotoxic mechanism of action would imply a linear-no-threshold low-dose dose-response relation (5156). Chronic exposure to a carcinogenic agent that acts solely or predominantly by a promotional mechanism, involving accelerated growth of spontaneously induced neoplastic foci, is expected to increase the size (e.g. diameter) of such foci after any specified duration of exposure to that agent. Carcinogenic agents that act by purely genotoxic mechanisms without affecting cell proliferation rates are expected to increase the number, but not the size, of observed neoplasia. (Of course, a carcinogen mightand perhaps most doact via one or more pathways that are jointly genotoxic as well as promotional.) ETS does not induce sustained cell proliferation within target alveolar/bronchial stem cells in exposed A/J mice, but rather (presumably via cytotoxicity) induces substantial proliferation only during the first 23 weeks of a 10-week ETS-exposure (41,57). To further assess whether proliferative promotion involved in ETS-induced lung tumors in A/J mice, tumor diameters in exposed versus control animals from two recent bioassays (7,39) were compared. Tumor diameters were measured under a magnifying glass using a metric ruler with a minimum detection size of 0.2 mm.
Calculation of implied risk due to lifetime residential ETS exposure
Increased cancer risk associated with lifetime residential exposure to ETS was estimated by applying the risk-extrapolation model given by Eq. (2). This model was applied to tumorigenic potency estimates derived from A/J mouse bioassay data, using an estimated lifetime TWA dose rate d corresponding to typical ETS exposure of never-smokers in U.S. households. The ETS exposure estimate used was based on the mean excess concentration of 0.0349 mg/m3 measured by Heavner et al. (58) for respirable suspended particulate matter in 29 homes containing a smoking spouse compared to 58 nonsmoking homes monitored for an average of 14.1 h/day, excluding one measure five standard deviations above the smoker-home mean (58). Assuming a metabolically realistic mean breathing rate of 14 m3/day per 70 kg for a reference person (59), and an estimate of 50% for the fraction of residential time typically spent either outside the home (60) or at home under conditions of negligible ETS exposure, the corresponding lifetime ETS exposure corresponds to a TWA dose d = 0.00349 N mg TSP/kg per day, where N is the number of smokers per household (which was 1 in the study by Heavner et al. (58)).
To estimate human risk posed by residential ETS exposure based on estimated ETS potency in A/J mice, a typical value for N was estimated using 6-year survey data from the Third National Health and Nutrition Examination Survey (NHANES III), which were collected during 19881994 (61). The range of risks estimated from A/J mouse data was compared with that implied by the estimated OR range (~1.21.4) from casecontrol data on lung cancer in nonsmoking spouses referred to above (Introduction), combined with additional estimates of lung cancer risk posed by active tobacco smoking based on 198084 US lifetime lung cancer mortality rates. Lifetime background risk (R0) of lung cancer mortality was about 0.85% for all never-smokers during this period, compared to a relative risk (RR) of about 11 incurred by smokers of ~20 cigarettes per day for ~two-thirds of their lives (62,63). The range of increased (i.e. excess) lifetime lung cancer risk to nonsmoking spouses was thus estimated to be R0(RR 1) = 0.180.36%, where (by definition) RR = OR(R0(OR 1) + 1)1. Because this risk range is based on studies involving ETS exposures each due to a single smoking spouse, it was multiplied by N to characterize increased ETS-related risk typical of U.S. homes, in which multiple smokers may reside (e.g. together with nonsmokers).
Data analysis
Positive carcinogenicity was evaluated by criteria established for the strain A mouse lung tumor assay (64,65), as well as by traditional criteria used in the context of risk assessment for chemical carcinogens (44,45). According to the first set of criteria, an A/J mouse lung tumor assay is positive if tumor multiplicity in treated animals is significantly increased above that in controls of similar age, preferably to
1 tumor/mouse. Additionally, for marginally positive compounds, positive results in an initial test must be repeatable in a second test. According to the second set of criteria, positivity for a compound is indicated if multiple data sets each reflect a statistically significant increased tumor incidence in treated compared to control animals.
An estimate (
1) of tumorigenic potency q1 was obtained corresponding to each bioassay data set considered using iteratively reweighted
2 minimization, and Monte-Carlo simulation to estimate each corresponding one-tail 95% upper confidence limit (q*1), as explained in the Appendix. Potency assessment for female mice was based on data listed in the last two rows of Table I
. Only one measure of response was used in potency assessment for female mice, namely, total tumors per animal, because only this measure was significantly elevated in female ETS-exposed mice compared with controls (Table I
and Results). Separate potency analyses were not conducted for study-specific positive sets of male data obtained from Studies 1, 3, 4, and 5. Rather, in view of nearly identical experimental conditions used in all these studies, all ETS-exposure and corresponding control data pertaining to male mice were combined to allow a single potency analysis for each of the two measures of tumor response considered. A separate analysis was conducted for each measure because each measure indicated a positive tumorigenic response (Table I
and Results). Analyses done for male mice were repeated after including data derived from Study 6 involving 9-month-exposed male mice. Each potency estimate was obtained together with corresponding
2 goodness-of-fit test, using a sequential fitting procedure in which degrees of freedom (d.f.) = (no. of data points) (no. of nonzero estimated parameters) (43).
Characterization of US and California residential cigarette use was based on NHANES III `ADULT.DAT' data, which include a total of 20 051 records each with a corresponding 6-year sampling weight recommended for use in data analyses intended to reflect US population or population subsets (61). All analyses were conducted using these sampling weights applied to those US or California adult-survey records containing non-blank and non-Unknown entries for questions concerning: (1) whether or not cigarettes are smoked in the surveyed household, (2) the number of smokers residing in that residence, (3) the number of cigarettes/day smoked by the surveyed adult, and (4) the total number of cigarettes smoked in that residence by any/all smokers.
Additional calculations and statistical tests were performed as indicated in Results, including Fisher exact tests of difference in tumor incidence, standard (or, as appropriate, Welch's) 2-tailed T-tests of difference in means, and Hommel's Bonferroni-type adjusted P-value (Padj) that accounts for multiple tests (6668). All potency, NHANES III, and additional tests and calculations were performed using the computer programs Mathematica 4.0® and RiskQ 4.0 (69,70).
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Results
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Only data from Studies 2 and 3 listed failed to indicate a statistically significant (P < 0.05) increase in tumor incidence among ETS-exposed versus either study-specific-control or pooled-control mice (Table I
). Data from all studies indicate statistically significant (P < 0.02) ETS-related increases in tumor multiplicity when compared with pooled controls (Table I
). Tumor multiplicity in male mice exposed in Study 6 to ETS for 9 months was significantly less (P = 0.00043) than that in male mice exposed in Study 5 to a nearly identical ETS concentration for only 5 months (Table I
).
Table II
lists the estimates of tumorigenic potency (
1 and q*1) obtained based on the bioassay data summarized in Table I
. Best fits obtained for the tumor-incidence and tumor-multiplicity functions used to model corresponding combined male A/J mouse bioassay data are shown in Figures 1 and 2
, respectively, together with goodness-of-fit statistics. Data on tumor incidence including that among exposed males from Study 6 (involving a 9-month exposure to ETS without a recovery period) yielded a multistage fit somewhat (but not significantly) less consistent than that obtained to corresponding data excluding Study 6 (Figure 1
). However, the corresponding fits obtained to data on tumor multiplicity differed markedly: the fit to data including Study 6 was inconsistent (P = 2.4 x 105) with the combined data, whereas an excellent fit (P = 0.89) was obtained to data on all other exposed groups excluding Study 6 (Figures 1 and 2
). Therefore, tumor multiplicity data on exposed mice only from Study 6 were combined with pooled male control data to obtain potency estimates involving Study 6 listed in Table II
. Best estimates of ETS potency (
1) listed in Table II
range from 0.310.98 kg day per mg; the largest value corresponds to Study 2 involving a single ETS-exposure group of female mice. Upper-bound (q*1) estimates of potency listed in Table II
are from 1.3 to 2.1-fold greater than the corresponding best (
) estimates.
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Table II. Tumorigenic ETS potency, and corresponding increased lifetime risk due to typical US residential ETS exposure
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Results of the analysis of NHANES III household smoking information are summarized in Table III
. The NHANES III data reveal a marked (~40%) difference between the number (2.6) of nonsmokers per smoking household in California versus that number (1.9) throughout the US, which may be important to consider when extrapolating national smoking statistics to a California context. This difference reflects the substantially larger mean size (4.1) of smoking households in California compared to the mean size (3.4) of smoking households in the US. The mean number (N) of smokers per smoking household is ~1.5 both in California and throughout the US, which implies that typical ETS levels experienced by nonsmokers in smoking households were underestimated by ~50% in the study by Heavner et al. (58), which measured household exposures of adult nonsmoking women to ETS from a single smoking spouse.
The estimated ETS potencies for A/J mice were combined with a lifetime TWA residential ETS concentration of 34.9 µg/m3 based on data reported by Heavner et al. (58) (Materials and methods) and with the NHANES III-based estimate (N = 1.5) of the mean number of smokers per typical smoking household, to obtain corresponding estimates of increased lifetime risk of lung cancer due to typical US residential ETS exposure, which are listed in Table II
. These estimated values of increased risk range from 0.160.51%, in proportion to corresponding potency (
1) estimates listed. This range overlaps that of 0.270.53% implied by the range of odds ratios now considered most consistent with data from many casecontrol studies indicating significantly more lung cancer in nonsmoking women living with smoking versus nonsmoking spouses (see Introduction, Materials and methods).
Neither tumor incidence (P = 0.84) nor tumor multiplicity (P = 0.47) differed significantly between the two Study-5 control groups, and their mean tumor diameters were only marginally significantly different after adjusting for making three comparisons (Padj = 0.048), so these control groups were pooled. Likewise, none of these measures differed significantly between the two Study-5 exposed groups (P > 0.05), so they were pooled. Distributions characterizing diameters of lung tumors in mice from Studies 5 and 6 are shown in Figure 3
. The mean (±1 standard error) tumor diameter of 0.95 (±0.051) mm observed in Study-5 mice (exposed to ~130 mg/m3 ETS for 5 months and then to clean air for 4 months) did not differ significantly (P = 0.75) from that of 1.0 (±0.051) mm observed in Study-6 mice (exposed to the same ETS concentration for 9 months). The mean tumor diameters of ETS-exposed mice in both Studies 5 and 6 were significantly smaller (P = 0.00051 and P = 0.031, respectively) than that (1.0 ± 0.12 mm) of pooled Study-5 control mice unexposed to ETS.

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Fig. 3. Cumulative relative frequencies of mean diameters of lung tumors in three groups of male A/J mice: pooled ETS-exposed mice from Study 5 (174 tumors in 66 mice), pooled ETS-exposed mice from Study 5* (41 tumors in 27 mice), and pooled control mice from Study 5 (81 tumors in 84 mice). Tumor-diameter means (±1 SE) in ETS-exposed mice from Studies 5 and 5* (both ~1.0 ± 0.1 mm) are each significantly less than that of pooled control animals from Study 5 (1.4 ± 0.1 mm).
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Discussion
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Data on ETS-induced lung tumors in A/J mice obtained by Witschi et al. (7,3539), summarized in Table I
, indicate that respiratory ETS exposure clearly can increase combined malignant and benign lung tumors in multiple experiments involving male and female A/J mice, and thus provide convincing evidence that ETS is a positive mouse carcinogen. Tumorigenic potencies estimated from these A/J mouse bioassay data predict a corresponding range of increased human risk (0.160.51%) that overlaps the range (0.270.53%) implied by casecontrol studies now widely considered to demonstrate significantly increased risks of lung cancer in nonsmoking women living with smoking versus nonsmoking spouses (Table II
). The A/J mouse bioassay data thus appear to corroborate and predict human epidemiological data on lung tumor risks posed by ETS exposure.
The ETS risk range predicted by A/J mouse bioassay data and by ETS casecontrol data may be compared to a lung cancer risk posed by residential ETS exposure estimated from epidemiological data on the relative risk (RR) of lung cancer in US active smokers versus nonsmokers (RR
11 at ~20 cigarettes/day for 67% of a lifetime; Materials and methods). Assuming a mean historical mainstream TSP content of ~20 mg/cigarette (71), and a lifetime TWA ETS concentration of (0.0349 mg/m3) x (7 m3/day) x (N = 1.5)
0.37 mg/day for typical households (Materials and methods), which corresponds is equivalent to about 0.018 cigarette/day. The corresponding estimate of increased lifetime risk to never-smokers from a lifetime of typical US residential ETS exposure levels may therefore be estimated as R0(RR1) = 0.85% x (0.018/20)(3/2)(11 1) = 0.011%. This risk estimate is about 10 to 50-fold less than the range of those cited above based on human casecontrol data or A/J mouse bioassay data, which is consistent with the hypothesis that ETS is more potent than mainstream tobacco smoke as a lung carcinogen. This hypothesis is supported by observations that cigarette sidestream-smoke condensate is more carcinogenic in skin painting studies than full-smoke condensate (72).
Results obtained concerning lung tumor size and the effect of different exposure durations affect the question of ETS's tumorigenic mechanism of action. In A/J mice exposed to a significantly tumorigenic ETS concentration, lung tumors were found to be significantly smaller than those in corresponding control mice that were not exposed to ETS. Moreover, although lung tumors were significantly increased in mice exposed to ~130 mg/m3 of ETS either for 5 or for 9 months, the mice exposed for the longer duration had significantly fewer tumors/animal. These findings are consistent with the hypothesis that ETS does not promote growth of spontaneous neoplastic foci in A/J mice, but rather may suppress it. Such suppression might be due to selective ETS-induction of growth-suppression and/or apoptosis in spontaneous as well as ETS-induced premalignant focal cells. It has recently been shown, e.g. that tobacco smoke not only induces apoptosis in human cell in vitro, but also in hair follicle cells in the skin of mice and in the bronchiolar epithelium of rats (7375). Tumor-diameter results obtained in the present study are thus consistent with the hypothesis that ETS-induced lung tumor risk in A/J mice occurs by a predominantly genotoxic mechanism of action, which may be suppressed partially by sustained high-level ETS exposure. Epidemiological data also indicate a pattern among smokers who quit whereby lung cancer risk appears to increase during the first 14 years after smoking cessation (42,7678), which pattern may be due partly or entirely to a greater likelihood of quitting following the onset of symptoms of undiagnosed lung cancer, although we know of no epidemiological study that has addressed this issue directly.
The four major types of human lung cancer are squamous cell carcinoma, small cell carcinoma (oat cell cancer), adenocarcinoma and undifferentiated large-cell carcinoma. The biggest risk factor for lung cancer is active smoking and ~90% of all lung cancers are found in past or current smokers (42). Originally, most lung cancers found in smokers were squamous cell carcinomas. They often were referred to as bronchogenic carcinoma, i.e. tumors originating most likely from the cells lining the conducting airways. Mouse lung tumors do not resemble this type of human lung cancer (34,79). However, during the last two decades there has been a noticeable shift in the types of human lung cancers. The relative frequency of squamous cell cancers has decreased, whereas that of adenocarcinomas, often of peripheral origin, is clearly increasing (80). This has been attributed to the `changing' cigarette (81,82). The design of effective filters removed much of the tar from inhaled tobacco smoke. The tar fraction contains most of the polycyclic aromatic hydrocarbons, including numerous carcinogens known to produce squamous cell lung cancer in animals (3). However, filters also retain some nicotine. As the use of filtered cigarettes has become predominant, smokers have inhaled more deeply and have retained smoke longer in the deep lung in order to satisfy nicotine craving (83). This altered pattern is likely responsible for the shift in US lung cancer histology patterns away from squamous cell carcinomas towards adenocarcinomas of the type produced in experimental animals.
This shift to adenocarcinomas makes A/J mouse lung tumors is highly relevant for the study of human adenocarcinomas. The lung tumors observed in studies by Witschi et al. (7,3539) are in many respects similar to human adenocarcinoma. They originate from the same cells, the type II alveolar epithelial cells and Clara cells. They also share many of the molecular changes found in cell cycle and cellcell communicating genes, have striking similarities in certain enzyme activities and other biochemical processes, and share other genetic and epigenetic traits (34,79). A/J mouse lung tumors are thus a highly relevant model for human pulmonary adenocarcinomas, including those that may be induced by ETS. In this regard, it is noteworthy that few casecontrol studies have had the power to detect ETS-related increases in specific lung tumor types. One that did was a multicenter casecontrol study on lung cancer in lifetime female never smokers with a smoking versus nonsmoking spouse, in which the only histologically distinct tumor type found to be significantly increased was pulmonary adenocarcinoma (adjusted OR = 1.28, P < 0.05) (11).
In conclusion, an analysis of bioassay data indicates that ETS is a clearly positive lung carcinogen in A/J mice. Corresponding estimates of the carcinogenic potency of ETS based on these bioassay data were shown to imply levels of increased lung cancer risk for typically exposed US nonsmokers that overlap the range of risks indicated by casecontrol studies that also are consistent with the hypothesis that ETS is a human lung carcinogen. Finally, an analysis of the size of lung tumors in A/J mice subjected to different ETS exposure conditions was shown to be most consistent with the hypothesis that these tumors occur by a predominantly genotoxic mechanism of action, which may be suppressed partially by sustained high-level ETS exposure.
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Appendix
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For analytic convenience, empirical binary-response probabilities pi (e.g. at corresponding doses di) often are modeled by fitting a linear (or general linear) function that predicts logistically transformed ordinate values,
i = log(pi/(1 pi)), using minimum-
2 or numerical maximum likelihood (ML) methods for parameter optimization (8487), even when a biological or mechanistic basis for this transformation is not evident. The somewhat more biologically based multistage model, pi = R(di) (see Eq. 1), generally has been fit to cancer-bioassay data using constrained numerical ML methods, assuming a binomial likelihood function involving the numbers xi of animals with tumor, the numbers ni of animals exposed and examined, and corresponding predicted response probabilities R(di) at bioassay doses di, as previously described (43,88,89).
The advantages of ML estimates (namely, consistency/unbiasedness, normality, efficiency) are only asymptotic properties, and as such do not necessarily (or typically) pertain to nonlinear (e.g. logit or multistage) models fit to relatively few and/or non-normally distributed data (66,90). In contrast, parametric bootstrap methods may be used to estimate values and confidence bounds of regression parameters for models involving assumed non-Gaussian errors, parameter constraints, nonlinear parameter transformations, and/or small sample sizes (9193). When asymptotic ML estimation versus Monte Carlo (ML-parametric bootstrap) simulation were compared as methods to fit a simple (non-negatively constrained linear-quadratic) multistage model to bioassay data involving realistically small sample sizes (e.g. three dose groups, 50 animals/group), some asymptotic ML results obtained were found to differ dramatically from more accurate results obtained by Monte Carlo simulation (89).
In view of limitations on ML estimates of parameters and confidence limits based on finite samples, a best estimate (
1) of multistage potency (q1) was calculated in the present study by numerical, iteratively reweighted minimization of the multistage chi-square objective function,
 | (A1) |
of observed data (di, ni, xi) and multistage parameters (qi) to be optimized, where error in xi is assumed to be ~Binomial (ni,
(di)), and where
(di) is the multistage function R(di) (see Eq. 1) conditional on estimated parameters
i. This procedure yields estimates having asymptotic advantages similar to ML estimates (66,94), but which are obtained by optimizing the
2 goodness-of-fit criterion traditionally used for multistage fits (43), modified to include a continuity-correction factor. Tumor multiplicity was fit similarly by minimizing
 | (A2) |
with error in xi assumed to be ~Poisson(ni P(di)), where
(di) = -ln(1-
(di)). Numerical optimizations were done using an exhaustive-search approach that fits linear or nonlinear objective functions involving non-negatively constrained polynomial coefficients (95,96). This approach was implemented using nested applications of the Mathematica 4.0® Catch, FixedPoint, and FindMinimum functions (69), applied to
2 defined by Eqs (A1A2).
Monte Carlo simulation was used to approximate a parametric-bootstrap 95% one-tailed upper confidence bound (
*1) (92), corresponding to each
1 estimate obtained as explained above. Each Monte Carlo
*1 estimate was calculated as the mean of 10 repeated estimates each based on 200 sets of dose-response data simulated based upon corresponding assumed error distributions noted above, using independent sample-value vectors obtained by systematic Latin-hypercube sampling (97). Monte Carlo estimation error associated with each
*1 estimate thus obtained was characterized by its relative standard error (100%
*1/101/2).
 |
Notes
|
---|
3 To whom correspondence should be addressed Email: bogen{at}LLNL.gov. 
 |
Acknowledgments
|
---|
The authors thank Imelda Espiritu and Dale Uyeminami for help in performing A/J mouse experiments and Dr R.R.Maronpot for histopathological evaluation of the tumor material. This work was supported in part by National Institute of Environmental Health Sciences (NIEHS) grants ES07908, ES07499 and ES05707; it reflects views of the authors and not necessarily those of the NIEHS, National Institutes of Health, or Lawrence Livermore National Laboratory.
 |
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Received January 19, 2001;
revised October 18, 2001;
accepted December 12, 2001.