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. 2018 May 17;39(8):1037–1044. doi: 10.1093/carcin/bgy064

Aspirin abrogates impairment of mammary gland differentiation induced by early in life second-hand smoke in mice

Julia Santucci-Pereira 1,✉, Thomas J Pogash 1, Aman Patel 1, Navroop Hundal 1, Maria Barton 1,2, Anna Camoirano 3, Rosanna T Micale 3, Sebastiano La Maestra 3, Roumen Balansky 3,4, Silvio De Flora 3, Jose Russo 1
PMCID: PMC6067120  PMID: 29788174

Abstract

Epidemiological studies show that there is limited evidence that tobacco smoking causes breast cancer in humans. In rodents, many tobacco smoke chemicals cause mammary gland tumors. This study evaluated the mammary gland differentiation in mice exposed to environmental cigarette smoke (ECS), using 3R4F Kentucky reference cigarettes, starting after birth and continuing daily for 10 weeks (total particulate exposure 95 mg/m3; CO 610 ppm). We also analyzed the effects of oral administration of non-steroidal anti-inflammatory drugs (NSAIDs), aspirin (1600 mg/kg) or naproxen (320 mg/kg), on mammary gland differentiation, either in unexposed or ECS-exposed mice. The ECS exposure caused delay of mammary glands development. We speculate that this delay may result from aryl hydrocarbon receptor (AHR) signaling activation, which has an antiestrogenic effect and crosstalk to the estrogen metabolism pathway. Similarly, naproxen impaired gland differentiation in unexposed and ECS-exposed mice, while aspirin hindered its development only in unexposed mice. The lack of differentiation induced by the NSAIDs could be explained by their antiestrogenic effect through inhibition of aldo-keto reductases. In ECS-exposed animals, aspirin induced intense lobular formation, which could indicate that aspirin is counteracting the AHR signaling induced by ECS. Based on the differentiation induced by aspirin in ECS-exposed animals, we postulate that these mice would be less susceptible to mammary carcinogenesis. Our results suggest that exposure to smoke at an early age impairs the development of the mammary gland, thus resulting in a longer period of susceptibility and increased risk of breast cancer. However, addition of aspirin can abrogate this effect.

This study demonstrated for the first time that second-hand smoke at early age impairs mammary gland differentiation in mice, which could increase susceptibility to carcinogenesis. NSAIDs have similar effects; however, in animals exposed to cigarette smoke, aspirin abrogates its effect.

Introduction

Breast cancer is the most common cancer in women, with an estimated number of new cases of 63 410 in situ lesions and 252 710 invasive cases in the USA in 2017 (1). In the 40 countries of the four United Nations-defined areas of Europe, there were 464 000 new cases in 2012 (2). Besides genetic factors, environmental and behavioral risks play an important role in breast cancer epidemiology. One of them is tobacco use, which is a causative agent for this type of cancer (1,3,4). The analysis of more than 150 epidemiological studies of tobacco smoking and breast cancer consistently showed a small positive association. Hence, a re-evaluation by International Agency for Research on Cancer (IARC) (5) concluded that there is limited evidence that tobacco smoking causes breast cancer in humans. This conclusion is strengthened by the fact that many chemicals in tobacco smoke cause mammary gland tumors in animals, and these carcinogens are stored in breast adipose tissue in women (5). Among them, polycyclic aromatic hydrocarbons (PAH) are potent mammary carcinogens in rodents, but their effect on breast cancer development in women is not clear. Current data indicate that PAH–DNA adduct formation may influence breast cancer development, although the association does not appear to be dose dependent and may have a threshold effect (6). The formation of DNA adducts in liver and mammary epithelial cells of rats treated with 7,12-dimethylbenz(a)anthracene (DMBA) and their inhibition by chemopreventive agents correlated with the yield of mammary tumors in rats (7).

There is evidence regarding the association between active smoking, involving inhalation of mainstream cigarette smoke and breast cancer (4). Second-hand smoke (or passive smoking) involves involuntary exposures to environmental cigarette smoke (ECS), which is a mixture of sidestream cigarette smoke and mainstream cigarette smoke in a ratio of about 8:1. Second-hand smoke causes sudden infant syndrome, respiratory and ear infections, and asthma attacks in infants and children, and coronary heart diseases, stroke and lung cancer in adult nonsmokers, being on the whole responsible for more than 41 000 deaths in adults and 400 deaths in infants each year in the USA (8). The association of second-hand smoke with breast cancer is less clear. There are reports indicating that cumulative exposures to high levels of ECS may increase breast cancer risk among postmenopausal women who themselves have never smoked tobacco products (9). Moreover, women who had smoked and were exposed to passive smoke had a significant increased risk of breast cancer (10). Current evidence confirms that young women who smoke or who have regular long-term exposure to second-hand smoke have an increased risk of developing premenopausal breast cancer (11).

The potentially causal role for smoking and breast cancer is more evident when exposed to smoke initiates at an early age (12). This is likely to be ascribed to the fact that the breast undergoes a complete series of changes from intrauterine life to senescence. In humans, the main development occurs during puberty, when there is lobule formation, which is only completed by the end of the first full-term pregnancy (13). With the approach of puberty, the rudimentary mammary gland begins to show growth activity both in the glandular and stroma tissues. The glandular increase is due to the growth and division of small bundles of primary and secondary ducts. The ducts grow, divide and form club-shaped terminal end buds (TEBs), which can differentiate into alveolar buds (ABs). ABs can cluster around a terminal duct (TD), forming the lobule type 1 (Lob1). Lobule formation in the female breast occurs within 1–2 years after onset of the first menstrual period (13) and the mammary gland can continue to develop with new structures until approximately 35 years old. Additional sprouting of ABs can occur originating lobules type 2 (Lob2) and 3 (Lob3). Similarly, in mice there is little growth of the mammary gland until puberty. At puberty, TEBs, present at the end of the ductal structures, elongate farther penetrating into the fat pad and branching (14). These TEBs bifurcate and secondary branches sprout forming several ducts, and alveolar structures (14). The development of the breast is an orchestrated process triggered and controlled by hormones. Noxious events at prepubertal or pubertal periods can alter the normal process having deleterious consequences on this organ also later in life. As a rule, mammary carcinomas arise from undifferentiated structures of the gland, TEBs and the number of adenocarcinomas is proportional to the number of these structures in the mammary gland (15). The more differentiated a given structure is, the more benign and organized is the lesion that develops upon its exposure to a carcinogen (15,16).

These premises prompted us to carry out an experimental study in which we evaluated the effect on mammary gland differentiation resulting from exposure of mice to ECS, starting soon after birth and continuing daily for 10 weeks. This period covers the immediate post-natal time, during which the sudden transition from the maternal-mediated respiration of the fetus to the autonomous respiration of the newborn causes paraphysiological genomic and postgenomic alterations, as demonstrated in mouse lung (17), followed by the lactation period until completion of weaning and then adolescence, which in mice covers the period from 23 to 48 days (18), and then continues during early adulthood.

A second objective of the present study was to evaluate the modulation of mammary gland differentiation consequent to the oral administration of non-steroidal anti-inflammatory drugs (NSAIDs) to post-weaning mice, either unexposed or exposed to ECS. Due to the prominent role of inflammation in the development of cancer in general and in particular of smoke-associated cancers (19), NSAIDs provide a promising approach to cancer prevention. Within the broad family of NSAIDs, we used aspirin (acetylsalicylic acid) and naproxen, both of which are dual inhibitors of cyclooxygenase (COX) activities, including COX-1, a constitutive isoform and COX-2, the inducible, proinflammatory isoform (20). In the same animal model used in this study, both aspirin and naproxen exerted protective effects towards ECS-induced early nucleotide alterations in both mouse genders and towards lung tumors in female mice (21).

The results obtained show that exposure of newborn mice through adulthood to ECS causes alterations of mammary glands development, decreasing their ramification, potentially increasing their susceptibility to mammary tumor formation. NSAIDs also affect the differentiation both in sham-exposed mice and ECS-exposed mice. Striking differences were observed in ECS-exposed animals that received aspirin. In fact, this group presented accentuated differentiation of the mammary gland, indicating that aspirin induces further differentiation of the mammary gland abrogating the effect of cigarette smoke.

Materials and methods

Mice

Male and female A/J mice were purchased from Harlan Laboratories (San Pietro al Natisone, Udine, Italy) and used for breeding. The mice were housed in MakrolonTM cages on sawdust bedding and maintained on standard rodent chow (Teklad 9607, Harlan Laboratories) and tap water ad libitum. The animal room temperature was 23 ± 2°C, with a relative humidity of 55% and a 12-h day-night cycle. Treatment of mice was conducted at the University of Genoa, Italy. Housing and treatment of mice were in accordance with NIH, European (2010/63/UE Directive) and institutional guidelines.

Experimental groups

The mice used in this study and in a parallel one evaluating lung carcinogenesis (21) were divided into six groups of newborn mice. For the present experiment, each group contained five female mice. As shown in Figure 1, the groups were as follows: (A) Sham, mice kept in filtered air; (B) ECS, mice exposed for 10 weeks to ECS, starting within 12 h after birth; (C) Aspirin, mice receiving aspirin with the diet after weaning, at 4 weeks of age; (D) ECS + Aspirin, mice exposed to ECS for 10 weeks and receiving a diet supplemented with aspirin after weaning; (E) Naproxen, mice receiving naproxen with the diet after weaning; (F) ECS+Naproxen, mice exposed to ECS for 10 weeks and receiving a diet supplemented with naproxen after weaning.

Figure 1.

Figure 1.

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Experimental design showing the time line of exposure of the six groups. In group (A) Sham, mice were kept in filtered air during the whole length of the experiment (white bar). Group (B) ECS, mice were exposed for 10 weeks to ECS using 3R4F Kentucky reference cigarettes, starting within 12 h after birth (dark-gray bar). Mice in group (C) Aspirin were exposed to filtered air for 10 weeks, receiving aspirin with the diet after weaning at 4 weeks of age. In group (D) ECS+Aspirin, mice were exposed to ECS for 10 weeks and received a diet supplemented with aspirin after weaning. Group (E) Naproxen, mice were exposed to filtered air, receiving naproxen with the diet after weaning. And in group (F) ECS+Naproxen, mice were exposed to ECS and received a diet supplemented with naproxen after weaning.

Exposure to ECS

The mice were exposed whole body to ECS, using 3R4F Kentucky reference cigarettes (College of Agriculture, The Reference Cigarette Program, University of Kentucky, Lexington, KY), having a declared content of 9.4 mg tar and 0.7 mg nicotine and delivering 12 mg CO each. Five cigarettes were burnt at one time using a smoking machine (model TE-10C, Teague Enterprises, Davis, CA), in which mainstream cigarette smoke (11%) and sidestream cigarette smoke (89%) are mixed to generate ECS. Cigarettes were smoked using the Federal Trade Commission (FTC) method of puffing a volume of 35 cm3 for 2 s once a minute. Two rounds of exposure were performed daily by burning a total of 120 cigarettes per day. The total particulate matter in the exposure chambers was on an average 95 mg/m3 and CO was 610 ppm.

Treatment with NSAIDs

Aspirin and naproxen were purchased from Sigma-Aldrich (Milan, Italy). The two NSAIDs were incorporated in Teklad 9607 diet (Harlan Laboratories) at the concentrations of 1600 and 320 mg/kg, respectively. These doses were selected based on the results of a subchronic toxicity study with varying concentrations of NSAIDs. They corresponded to the 80% of the maximum tested dose that did not produce any apparent adverse effects in mice (21).

Mammary gland collection and morphological analyses

At the age of 10 weeks, all mice were euthanized by CO2 asphyxiation. The pelts containing the abdominal mammary gland were collected, fixed in 10% neutral-buffered formalin and shipped to Fox Chase Cancer Center, Philadelphia, PA.

The morphology of the mice right abdominal mammary gland was evaluated in whole-mounted tissue. The mammary glands were removed from the pelt and defatted in acetone for at least 48 h. The glands of 2 out of 30 animals, one from Group B (ECS) and one from Group E (naproxen), were damaged during the process, therefore they were excluded from the analysis. The tissues were hydrated and stained with a solution of 0.025% toluidine blue. The glands were then fixed in 4% ammonium molybdate, dehydrated in successive solutions and submitted to a secondary dissection to remove excess connective tissue. Then the glands were mounted on slides and cover slipped using Permount mounting media. Pictures of the whole mammary glands were taken using an Olympus SZX10 stereo microscope attached to QIClick camera (QImaging, Canada) using MetaMorph software. The epithelial structures of the glands were classified as either TEB, TDs, AB, lobules type 1 (Lob1) or lobules type 2 (Lob2) based on their morphological characteristics. To determine the number of each epithelial structure, every whole mount preparation was blinded and analyzed by two trained and independent people. All the structures presented that could be clearly identified were counted using ImageJ Software (22).

Statistical analysis

We modeled count data (TEB, TD, AB, Lob1, Lob2) using Poisson regression models with Generalized Estimating Equations with robust standard errors to account for within-mouse effects. We included ECS, Aspirin, Naproxen and interactions between ECS with Aspirin and Naproxen as predictors in the model. Pair-wise comparisons were performed using linear combinations of the relevant parameter estimates from the multivariate model. When models would not fit due to treatment conditions with all zero outcomes (TEB, Lob2), we used Wilcoxon tests for the pair-wise comparisons.

Results

We evaluated the mammary gland differentiation through whole mount preparations of mice belonging to the six groups depicted in Figure 1 (Sham, ECS, Aspirin, ECS + Aspirin, Naproxen, ECS + Naproxen). Examples of whole mounts prepared from one mice/group are shown in Figure 2. The number of structures was analyzed and Figure 3 reports box-plots with the number of TEB, TD, AB, Lob1 and Lob2, respectively. Table 1 shows the relative ratio of the results obtained by comparing the six experimental groups for the analyzed epithelial structures.

Figure 2.

Figure 2.

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Representative pictures of mammary gland whole mounts.

Figure 3.

Figure 3.

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Box plot of the number of TEBs, TDs, ABs, lobules type 1 (Lob 1) and lobules type 1 (Lob 2) found in the right abdominal mammary gland of each group.

Table 1.

Table 1.

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Ratio and P-values (P) for all comparisons among the six experimental groups per morphological structure

Effects of ECS, aspirin and naproxen alone on mammary gland differentiation

The mice exposed to ECS did not have significant changes on the number of the least differentiated structure, TEB or on the most differentiated structures (Lob1) when compared to unexposed mice. However, ECS-exposed mice showed approximately 40% decrease on the number of TD and 60% decrease of AB. Similar effect was observed in animals that received aspirin after weaning, with about 50% reduction in TD and 65% in AB. Mice that received naproxen, presented similar results to aspirin, decreasing TD and AB, but also statistically significantly decreased the number of TEB. Early exposure to ECS, aspirin or naproxen impairs the normal mammary gland differentiation.

Effects of ECS combined with aspirin or naproxen on mammary gland differentiation

Although the number of TEB did not have significant changes in animals exposed to ECS alone, ECS when in combination with aspirin or naproxen significantly decreased TEB. In the combination groups, TEB were inexistent in most of the mammary glands evaluated (Figure 3).

The number of TD significantly decreased in all the evaluated groups when compared with the sham mice. The most accentuated reduction of TD was observed in the mice exposed to the combination of ECS and aspirin, which reduced this structure by 70%. The reduction of TD in this group was also statistically significant compared to aspirin alone, to ECS alone, or to the combination of ECS and naproxen. The reduction of TD in the ECS + aspirin group was consistent with the increase of more developed structures such as AB, Lob1 and Lob2 observed in this group.

Interestingly, the combination of ECS with aspirin increased the formation of ABs five times in comparison to the unexposed group. No other groups showed increase of this structure, indeed, the number of AB decreased in all the other groups when compared with animals not exposed either to ECS or to NSAIDs. The combined exposure to ECS and aspirin further stimulated the development of the mammary gland, showing high increase in lobules type 1 and even lobules type 2. Lob2 were not found in any other group, and are not expected to be observed in virgin animals that do not are exposed to hormonal stimulation.

On the whole, these data demonstrate that the combination of ECS with NSAIDs decreases the number of the most undifferentiated structures (TEB), but only the combination with aspirin induces further stimulation by increasing the number of differentiated structures (AB, Lob1 and Lob2).

Discussion

To the best of our knowledge, this is the first study that evaluates the effects of cigarette smoke exposure on the development of the mammary gland. Other groups have studied the effects of individual or combinations of chemicals present in tobacco smoke, however, it is estimated that cigarette smoke contains over 7000 chemicals in its composition (12). The first finding of the present study is that the whole-body exposure of mice to second-hand cigarette smoke resulted in decreased differentiation of the mammary gland. These mice presented significantly fewer TDs and ABs in the mammary gland compared to animals not exposed to ECS. Undifferentiated structures are known to present higher rate of cell proliferation and lower rate of reparative activity, which makes them more susceptible to carcinogenic agents (16). Similar effect was observed in mice prepubertally exposed to cadmium, one component of the cigarettes (23). These mice presented lower degree of mammary gland development, suggesting that at prepubertal period cadmium has antiestrogenic effects (23).

The decreased differentiation induced by the cigarette smoke may tentatively be ascribed to the activation of aryl hydrocarbon receptor (AHR), which has an antiestrogenic effect and crosstalk to the estrogen metabolism pathway (Figure 4) (24–26). Cigarette smoke contains several chemicals, including PAHs that trigger the activation of the AHR pathway (12,27). This pathway is responsible for xenobiotic metabolism (26,28), and it is also involved in other cellular processes such as proliferation, cell growth and differentiation through its transcriptional factor role (28). AHR is found in a variety of organs, including breast and its activation is tissue-specific depending on co-regulators present in different cell types (26,28). It has been described that the AHR complex has the ability to bind directly to estrogen receptors producing its antiestrogenic effect (25,28). In addition, the AHR pathway activates phases I and II metabolic cytochrome P450 (CYP) enzymes, such as CYP1A1, CYP1A2 and CYP1B1 (26,28), which are part of the estrogen metabolism (Figure 4). Mostly in the liver, CYP1A1/2 catalyzes 17-β estradiol hydroxylation into 2-hydroxyestradiol, while in other tissues, including mammary, ovary and uterus, CYP1B1 predominantly catalyzes estradiol hydroxylation into 4-hydroxyestradiol (29,30). 4-Hydroxyestradiol is a strong estrogen agonists with similar potency to estradiol (31), but both 2-hydroxyestradiol and 4-hydroxyestradiol are rapidly inactivated by catechol O-methyltransferase (COMT) (29,30). The metabolites of estradiol can be further metabolized leading to the formation of quinones and semiquinones, which either stably bind to DNA or induce DNA adducts formation (29,30). The carcinogenicity of estrogens and PAHs are attributed in part to this DNA damage and adducts formation (29,30).

Figure 4.

Figure 4.

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Schematic representation of estrogen metabolism and AHR signaling pathways. Full arrows correspond to estrogen metabolism and dashed arrows correspond to AHR signaling pathway. 17β-HSD (17β-hydroxysteroid dehydrogenase) is inhibited by NSAIDs. NQO1 (NAD(P)H quinone oxidoreductase 1) and GST (glutathione S-transferases) are enzymes which activity is induced by Aspirin. Cyp1A1/A2 and Cyp1B1 are induced by AHR activation, however, the use of aspirin could decrease these enzymes.

The activation of the AHR pathway is a possible mechanism by which the cigarette smoke could inhibit mammary gland differentiation, decreasing endogenous estrogenic effect. Supporting this hypothesis, studies in which rodents were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent AHR ligand, demonstrated delay in mammary gland development (32,33). Moreover, Cyp1b1 is suggested to be essential for the antiestrogenic activity of TCDD mediated by AHR (30). Indeed, tobacco smoke induces Cyp1b1 (34,35) and 4-hydroxyestradiol expression within the mouse lung (35). In addition to the mechanisms discussed above, it has been demonstrated that dioxins can degrade nuclear estrogen receptor through the activation of proteasome (28,30). Important to notice is that other mechanisms may be involved, taking into account that ECS contains several chemicals and not only PAH.

Under our experimental conditions, exposure of mice to ECS for 10 weeks, starting soon after birth, mimics an exposure to passive smoke or second-hand smoke, during a period of time that in humans would cover the post-natal time, followed by puberty, adolescence and early adulthood. The ECS doses to which the mice were exposed are high but fall within possible human exposures. In fact, it has been reported that the respirable particulate matter in certain entertainment venues ranges from less than 15 mg/m3, where smoking is prohibited, up to 350 mg/m3 where smoking is allowed. In the home environment, peaks up to 300 mg/m3 have been detected, and inside vehicles concentrations are estimated to range from about 90 mg/m3, a value that is similar to the one used in our study, to well over 1000 mg/m3 (36).

Children are especially sensitive to the effects of exposure to second-hand smoke (36). As demonstrated in the respiratory tract, mice exposed early in life are more susceptible than adult mice to molecular, biochemical and cytogenetic alterations induced by ECS. Such an increased susceptibility has been ascribed to oxidative stress and to the resulting epigenetic alterations and DNA damage (37). At early stages, the mammary gland is also very susceptible because most of its development in humans and rodents happens during the approach of puberty (13,14).

Treatment with either aspirin or naproxen, starting after weaning, mimics an intervention after puberty in individuals exposed to second-hand smoke since birth. The doses of NSAIDs used in the present study did not produce any toxic effects in a preliminary subchronic toxicity test and did not cause any significant bleeding of the gastric mucosa (21). Although they were 3–4 times higher than those used in humans for therapeutic purposes, it should be taken into account that, due to metabolic reasons, mice eat as often as they can. Therefore, the intake of drugs by mice was highly fractionated during the day (21). Aspirin and other NSAIDs were proposed to be used as chemopreventives against breast cancer. Inverse association between aspirin use alone or aspirin and other NSAIDs and breast cancer risk relative to non-users have been observed (38). More pronounced inverse association was observed with aspirin users and those with hormone receptor-positive cancers (38). NSAIDs inhibit COX activity and thereby reduce prostaglandin synthesis. Additional anticancer effects are induction of apoptosis and inhibition of cell proliferation (39). Herein, we tested the effects of aspirin and naproxen in the differentiation of the mammary gland. When tested alone, the two tested NSAIDs had similar effects to ECS in the mammary gland, decreasing TEBs, TDs and alveolar structures. Naproxen had very similar effects in animals either exposed or not to ECS. However, notable was the differentiation induced by aspirin in the mammary gland of ECS-exposed mice.

The lack of differentiation induced by the NSAIDs could be in part explained by their antiestrogenic effect through inhibition of aldo-keto reductases (AKR). AKRs participate in the production of steroid hormones, such as testosterone, estradiol and also prostaglandins (Figure 4) (40). More specifically in the breast, AKR1C3 (aldo-keto reductase family 1 member C3), also known as 17β-hydroxysteroid dehydrogenase type 5 (17β-HSD), catalyzes the reduction of Δ4-androstene-3,17-dione to yield testosterone, which is converted to estradiol by aromatase, or directly reduces estrone to estradiol (Figure 4) (40). Over-expression of AKR1C3 is commonly found in breast and prostate cancers (41). Both naproxen and aspirin have shown to inhibit different isoforms of AKR1C (40). Naproxen has been demonstrated to be a strong inhibitor of AKR1C3, while aspirin is less potent (40). This inhibition of AKR1C3 can be diminishing the estradiol in the mammary gland of these mice. Although NSAIDs have been tested as potential preventive agent against breast cancer, the use of these drugs at this early life stage, before full mammary gland development, might be impairing the ability of this organ to reach a normal level of differentiation.

Unexpectedly, we observed great differentiation of the mammary gland in mice that received aspirin during ECS exposure. Although the reason for this development induction is not clear, we hypothesize that the combination of aspirin with cigarette smoke may cause a shift in the AHR and/or estrogen metabolism pathways that increases the availability of estradiol thus inducing lobule formation in these glands. Lewis et al. have shown that TCDD-exposed animals that received exogenous estrogen treatment had increased differentiation of the mammary gland with larger percentage of lobule formation, demonstrating that even glands that were impaired by this contaminant were not refractory to hormone stimulation (32).

Our first hypothesis is that the combination of ECS and aspirin could change the effect of the AHR pathway. Although AHR pathway is largely known for its antiestrogenic effect, there is evidence that it might have estrogenic effect depending on its activation (24,42). In hepatocytes, aspirin induces AHR gene expression (43). Another explanation for the increase of estradiol in the ECS + aspirin animals would be that aspirin is counteracting the AHR signaling. While AHR activation increases Cyp1A1/A2 and Cyp1B1, the use of aspirin could decrease these enzymes, allowing the accumulation of estradiol instead of its metabolites. Studies describing the effect of aspirin on Cyp1A had different results in a variety of models. Low-dose aspirin in male non-smoker volunteers had no effect on CYP1A2, CYP2D6 and CYP2E1 (44). In human hepatoma cells, aspirin had no inhibitory effect on TCDD-induced CYP activity (45). However, in MCF-7 breast cells in which CYP1A was induced by β-naphthoflavone, aspirin decreased the CYP1A1/A2 expression (46). To the best of our knowledge, the effect of aspirin on CYP1B1 has not yet been elucidated. Our data in combination with the above reported data suggest that ECS + aspirin exposure could result in accumulation of estradiol in the mammary gland of these animals due to aspirin inhibition of CYP1A1/A2, and/or accumulation of 4-hydroxyestradiol due to the ECS-induced increase of CYP1B1. We could not find any work describing the effect of aspirin on COMT, which if decreased, would favor the accumulation of 4-hydroxyestradiol.

In addition, aspirin has been showed to increase gene expression and activity of NAD(P)H quinone oxidoreductase 1 (NQO1) in hepatocytes (47) and in HT-29 human colon adenocarcinoma cells (48). NQO1 reduces the estradiol-3,4-quinone to 4-hydroxyestradiol (49). Estradiol-3,4-quinone forms depurinating adducts (29). The increase of NQO1 suggests accumulation 4-hydroxyestradiol and decrease of DNA adducts in these mice. Moreover, aspirin has also been demonstrated to increase the activity of glutathione S-transferases (GST) (48), phase II detoxification enzymes that conjugate the quinones with glutathione (29), not allowing them to form DNA adducts. Although we did not measure DNA adduct formation in the mammary gland of these animals, in the parallel pulmonary carcinogenesis study, DNA adducts were considerably increased in ECS-exposed mice, while aspirin and naproxen remarkably inhibited these nucleotide alterations (21). These findings are corroborated by Abbadessa et al. (46) that observed that salicylate, an aspirin metabolite, inhibited DMBA-induced DNA adduct formation in breast cancer cells. Collectively, these mechanisms could indicate that there is accumulation of 4-hydroxyestradiol in the mammary gland of the ECS-exposed mice that received aspirin, inducing lobule formation in their mammary gland. Naproxen in combination with ECS probably did not induce the same differentiation level due to its stronger potency to inhibit AKR1C3 than aspirin, decreasing the levels of estradiol.

Interestingly, in the parallel pulmonary carcinogenesis study, the NSAIDs attenuated the yield of lung adenomas, an effect that was statistically significant only in female mice (21). A selective inhibition of lung adenomas by both NSAIDs was also observed in female Swiss H mice exposed to mainstream cigarette smoke (50). These findings suggest a crosstalk between estrogens and smoke in pulmonary carcinogenesis, as also supported by metabolic studies (34,35).

Based on the differentiation induced by aspirin in ECS-exposed animals, we postulate that aspirin-treated mice would be less susceptible to mammary carcinogenesis. Our results suggest that exposure to smoke at an early age impairs the development of the mammary gland, thus resulting in a longer period of susceptibility and increased risk of breast cancer. However, the treatment with aspirin can abrogate this effect.

Funding

This work was supported by the National Cancer Institute (Grant P30-CA006927).

Acknowledgements

We thank Rachael McClenahan and Theresa Nguyen for their assistance on whole mount preparation, Dr. Elizabeth Handorf for statistical services and Dr. Yanrong Su for review of the manuscript.

Conflict of Interest Statement: None declared.

Abbreviations

AB

alveolar bud

AHR

aryl hydrocarbon receptor

COX

cyclooxygenase

ECS

environmental cigarette smoke

NSAID

non-steroidal anti-inflammatory drug

PAH

polycyclic aromatic hydrocarbon

TD

terminal duct

TEB

terminal end bud

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