Interactions between benzophenone-3 and dietary fat in mammary tumorigenesis

Benzophenone-3 is a putative endocrine disrupting chemical and common active ingredient in sunscreens and personal care products. The potential of endocrine disrupting chemicals to act as agonists or antagonists in critical hormonally regulated processes, such as mammary gland development and mammary tumorigenesis, demands evaluation of their potential in promoting breast cancer. We previously demonstrated promotion of mammary tumorigenesis by a diet high in saturated animal fat. This study examines the activity of benzophenone-3 in a dietary context to provide insight into its potential role in promoting breast cancer, and how diet might inuence this.


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Background Ovarian hormones are strongly implicated in the etiology of breast cancer [1,2], and are particularly important for the development of the breast during puberty and young adulthood [3]. Putative endocrine disrupting chemicals (EDCs), particularly estrogenic chemicals, have emerged as suspects in environmental promotion of breast cancer [4]. Environmental EDCs have the potential to act as agonists or antagonists in critical hormonally regulated processes, such as mammary gland development and mammary tumorigenesis. This warrants evaluation of their potential in promoting breast cancer. We demonstrated enhancement of mammary tumorigenesis by a diet high in saturated animal fat (HFD) [5,6,7,8]. Thus, examination of the activity of EDCs in a dietary context may provide additional insight into the potential role of EDCs in promoting breast cancer.
Benzophenone-3 (BP-3; oxybenzone) is a putative EDC and a common active ingredient in sunscreens and other personal care products [9]. Beyond direct exposure by these products, BP-3 is present in household dust [10], in sh lipids [11], and, notably, in the aqueous environment [9]. More recently, BP-3 was demonstrated to have pathological effects on coral [12]. Although BP-3 has a very short half-life, its presence is widespread in human urine [9], in as much as 98% of the general U.S. population [13]. A recent preliminary study found plasma concentrations greater than 0.5 ng/mL among a small human cohort using heavy topical applications of commercial sunscreens. This level, achieved after only one d exposure, exceeds Food and Drug Administration guidance for chemicals of "threshold of toxicological concern" [14].
BP-3 has known estrogenic and anti-estrogenic properties [9]. We have shown that both estrogen [15,16] and HFD [17,5] can modulate proliferative, in ammatory and angiogenic activity in the mammary gland. Thus, it is logical to test their individual and combined effects on mammary tumorigenesis. The present study examined the interaction of BP-3 with HFD on mammary tumorigenesis in BALB/c mice, using the Trp53-null transplantation model [18]. A level of BP-3 exposure was used that yielded levels in murine urine similar to that observed in humans subjected to heavy topical exposure of BP-3-containing commercial sunscreen [19]. We found that BP-3 had complex effects that were dependent upon dietary regimen and tumor histopathology. BP-3 was protective in regard to epithelial tumor promotion in mice fed a low fat diet (LFD) and was promotional for epithelial tumorigenesis in mice fed HFD restricted to adulthood. At the same time, BP-3 increased tumor cell proliferation, decreased tumor cell apoptosis, and increased tumor vascularity in a manner dependent on speci c dietary regimen and tumor histopathology. Increased mammary tumor-free survival was not always concordant with a decrease in properties associated with tumor progression. Notably, although BP-3 seemed protective for tumorigenesis in mice fed LFD, the spindle cell tumors that arose in these mice showed increased proliferation and decreased apoptosis. Collectively, these ndings suggest that BP-3 exposure may have adverse consequences in mammary tumorigenesis.

Mice
BALB/c Trp53+/breeding mice were obtained from Dr. D. Joseph Jerry (University of Massachusetts, Amherst MA), and Trp53-null mice were generated as described [18]. The female Trp53-null tissue donor mice were maintained on chow diet before mammary gland collection at eight weeks of age. Wild-type recipient female BALB/c mice were purchased from Charles River (Portage, MI) at 3 weeks of age.
To assess BP-3 stimulation of mammary epithelial proliferation in pubertal wild-type mammary glands, 3week-old female BALB/c mice were placed on LFD and HFD and, after one week on diet, those that had initiated estrous cycling were ovariectomized (OVX). Recovery was allowed for 3 weeks after OVX to permit complete terminal end bud regression before treatments [20]. To assess BP-3 stimulation of mammary epithelial proliferation in adult wild-type mammary glands, 10-week-old female BALB/c mice were placed on LFD and HFD and, after 3 weeks on diet, were OVX. Recovery was allowed for one week after OVX before treatments. For the long term uniform dosage experiment presented in Fig. 1A, mice were fed diets with and without BP-3 (70 mg/kg body weight (BW)) (Spectrum Chemical, New Brunswick, NJ), and then were injected daily for 5 d with saline control or 17-b-estradiol (E2) (1 µγ/injection) (Sigma, St. Louis, MO). For the acute dose-response experiment presented in Fig. 1B, 3-week-old female BALB/c mice were placed on HFD, and after one week were OVX. Recovery was allowed for 3 weeks after OVX before BP-3 and E2 treatments. Mice were injected daily for 5 d with saline control or E2 (1µ g/injection) and/or BP-3 by oral gavage in vegetable oil (70 mg/kg BW, 7 mg/kg BW, or 0.7 mg/kg BW).
For tumorigenesis experiments, female Trp53-null transplanted mice were randomly assigned into six diet groups (see Diets). Food and water were provided ad libitum. Mice were housed in a standard laboratory housing environment with a 12:12 h light-dark cycle, at 20 to 24 °C with 40 to 50% relative humidity. All mice were sacri ced at estrus. 5-bromo-2′-deoxyuridine (BrdU) (70 µγ/g body weight; Sigma-Aldrich, St. Louis, MO) was administered via intraperitoneal injection 2 h prior to sacri ce for analysis of cellular proliferation. All animal experimentation was conducted in accord with accepted standards of humane animal care under guidelines approved by the All University Committee on Animal Use and Care at Michigan State University (AUF #07/17-128-00).
Trp53 -null mouse model Fragments of donor mammary epithelium were collected from female BALB/c Trp53-null mice at 8 weeks of age, and transplanted into the cleared inguinal mammary fat pads of 3-week-old female wild type BALB/c mice as previously described [21,22]. To minimize donor bias from secondary genetic alterations, mammary duct fragments from 4 donor mice were transplanted to recipient mice in each diet group in equal distribution. Body weights and food consumption were monitored weekly. Animals were palpated for tumor development twice a week starting at 13 weeks of age. Tumors were harvested at 1 cm in diameter. Portions of tumors and mammary glands were formalin-xed, para n embedded for H&E and immunohistochemistry. Mice were monitored for 500 d, and at termination of the studies, mammary glands were formalin-xed and processed as whole mounts to evaluate transplantation success rate.

Diets
Low fat diet (D11012202; 10% kcal fat) and high fat diet (D11012204; 60% kcal fat) were purchased from Research Diets (New Brunswick, NJ). See Table S1 for detailed composition of the diets. For the continuous LFD group, the diet was initiated after transplantation at 3 weeks of age and maintained throughout the studies. For the HFD-LFD and LFD-HFD groups, mice were initially fed one diet from 3 weeks until 10 weeks of age, and then switched to the other diet thereafter. For diets containing BP-3, BP-3 (Spectrum Chemical, New Brunswick, NJ) was compounded into the diets at 0.75 g/kg chow for pubertal animals (3 to 10 weeks of age) and 1.5 g/kg chow for adult animals; the difference in BP-3 between the diets for pubertal and adult animals was intended to compensate for the differences in food consumption and body weight with age.

BP-3 dosage
We sought a BP-3 dosage below a level found to have carcinogenic effects in mice. A prior study subjecting female B6C3F1 mice to 2 years of BP-3 ingestion at 150 mg/kg BW/d found weakly carcinogenic effects [25]. Our dosage of 0.75 g/kg chow for pubertal animals and 1.5 g/kg chow for adult animals yielded BP-3 consumption of approximately 70 mg/kg BW/d; BP-3 consumption was similar between LFD and HFD, and between pubertal and adult animals. Excretion in mice is divided between urine and feces [26]. To examine BP-3 urine excretion, BALB/c mice were fed LFD or HFD from 3 to 7 weeks of age, and then subjected to a single dose of BP-3 by oral gavage (1.3 mg/100 µΛ vegetable oil; equivalent to 70 mg/kg BW for an average body weight of 18.2 g). Determination of BP-3 levels in urine was performed by the Division of Laboratory Sciences of the National Center for Environmental Health, Center for Disease Control and Prevention (Atlanta, GA). Greater than 90% of BP-3 excretion occurred by 8 h post BP-3 treatment ( Figure S1) with no signi cant difference between diets. In longer-term experiments with an extended period of BP-3 ingestion (Fig. 1A), excretion in the urine voided at sacri ce for mice treated with BP-3 plus E2 ranged between 1.0 and 6.1 mg/kg BW with no signi cant differences between diet or life stage. We compared these values to observations in a small human cohort that was exposed to heavy topical application of sunscreen for 5 d in succession followed by 5 d of urine collection without BP-3 exposure [19]. The total urine excretion in that population averaged approximately 2.3 mg/kg BW, assuming an average European adult body weight of 70.8 kg [27]. Considering the rapid excretion in mice, it is likely that our dosage generates urine excretion on the same order as that observed in heavily exposed humans.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) 5 µm tumor sections were depara nized and rehydrated. TUNEL analysis was performed using the TdT-FragEL DNA Fragmentation Detection Kit (EMD Millipore, Billerica, MA) following the manufacturer's directions. At least 1000 cells per tumor were analyzed.

Metabolic parameters
Plasma glucose and insulin levels were measured from samples collected at sacri ce from non-fasting tumor-bearing animals, as previously described [5]. Plasma glucose levels were determined by OneTouch UltraMini (LifeScan, Inc., Milpitas, CA) and the insulin levels were determined with the rat/mouse insulin ELISA kit (EMD Millipore), according to the manufacturer's instructions.

Statistical analysis
Data were analyzed by two-way analysis of variance (ANOVA) and post-hoc t-test, when applicable. The Mantel-Cox test to analyze signi cance in Kaplan-Meier temporal tumor development, exact Fisher test to analyze proportions of spindle versus epithelial tumors, non-parametric Mann-Whitney test to analyze tumor latency, 2-way ANOVA, and two-tailed t-test are from the GraphPad PRIZM 7.03 software package (San Diego, CA). Results are shown as means ± standard error of the mean (SEM) or ± standard deviation, as noted in gure legends. Differences were considered signi cant at p < 0.05 using statistical approaches as noted in gure legends.

Results
BP-3 enhances estrogen-stimulated mammary gland proliferation in pubertal mice fed HFD Since BP-3 showed estrogenic activity as a proliferative stimulus to MCF-7 breast cancer cells [29,30], we sought to see if BP-3 could stimulate in vivo mammary epithelial proliferation. To that end, both pubertal and adult BALB/c mice were placed on LFD or HFD with and without BP-3 (see Materials and Methods), OVX, allowed time for recovery and clearance of endogenous hormones, and then treated with E2 or control for 5 d. While no BP-3 effects were seen in the adult mice (data not shown), the pubertal mice fed HFD plus BP-3 showed higher proliferation in response to E2 than did mice fed HFD alone ( Figure 1A). No BP-3 effects were observed in the absence of E2, and no BP-3 effects were observed in mice fed LFD.
As the initial experiment involved relatively long-term treatment with BP-3 and acute exposure to E2, we examined the effects of BP-3 in an acute exposure regimen at our standard dose, as well as 0.1 and 0.01 doses, with and without co-treatment with E2 in OVX pubertal BALB/c mice fed HFD. While BP-3 by itself showed no effects at any dose (data not shown), BP-3 augmented the proliferative response to E2 in both ducts and duct ends at the standard dose and in duct ends at the 0.1 dose ( Figure 1B).

BP-3 reduced tumorigenesis in mice fed LFD and promoted tumorigenesis in mice fed LFD-HFD
Having observed growth promoting effects, we examined whether BP-3 treatment would promote tumorigenesis in the Trp53-null mammary transplant model in BALB/c mice. We previously reported that HFD exposure at either puberty or adulthood promoted mammary tumorigenesis in this model [7]. Since our examination of BP-3 stimulated growth in the mammary gland only found effects at puberty with mice fed HFD, we examined the effects of BP-3 on tumorigenesis with mice fed LFD, pubertally-restricted HFD (HFD-LFD), and adulthood-restricted HFD (LFD-HFD). As in our earlier studies, most tumors were epithelial in composition (Figure 2A), while some were poorly differentiated spindle cell carcinomas ( Figure 2B). While an adult-restricted HFD (LFD-HFD) increased the proportion of spindle cell tumors compared to LFD, the proportion of epithelial versus spindle cell tumors was increased by BP-3 treatment in mice fed LFD-HFD ( Figure 2C).
Kaplan-Meier analysis revealed that BP-3 reduced tumorigenesis of epithelial tumors in mice fed LFD ( Figure 3A). On the other hand, consistent with the increased proportion of epithelial tumors, BP-3 was promotional for epithelial tumorigenesis in mice fed LFD-HFD ( Figure 3C), while reducing spindle cell tumorigenesis ( Figure 3D). No signi cant effects were observed with Kaplan-Meier analysis for either spindle cell tumors in mice fed LFD ( Figure 3B), or for both epithelial and spindle cell tumors from mice fed HFD-LFD ( Figure 3E and F).
BP-3 treatment increased latency of both epithelial and spindle cell tumors in mice fed LFD ( Figure 4A and B). No signi cant effects on latency were found for BP-3 treatment on other diets. ANOVA found signi cance for dietary effects in both epithelial and spindle cell tumors (Supplementary Table S2). This is consistent with mice fed LFD-HFD tending to have shorter tumor latencies than mice fed either LFD or HFD-LFD, although this trend is not signi cant by Mann-Whitney U test.

Tumor characteristics
Most of the epithelial tumors were ER-PR-, ranging from 89 to 100% among the dietary regimens and BP-3 treatments, and did not vary signi cantly by histological type, diet, or BP-3 treatment. Similarly, most spindle cell tumors were ER-PR-, ranging from 88 to 100% among the dietary regimens and BP-3 treatment (Supplementary Table S3).
Since unregulated proliferation and resistance to apoptosis are hallmarks of cancer, we measured tumor cell proliferation and apoptosis by quantifying BrdU incorporation and TUNEL, respectively. Epithelial tumors arising in mice fed LFD-HFD and HFD-LFD showed greater proliferation with BP-3 treatment ( Figure 5A) and spindle cell tumors arising in mice fed LFD and LFD-HFD showed greater proliferation with BP-3 treatment ( Figure 5B). Both epithelial and spindle cell tumors arising in mice fed HFD-LFD showed reduced proliferation compared to LFD-fed mice. ANOVA (Supplementary Table S2) found that overall dietary effects on proliferation of epithelial tumors were not signi cant, while BP-3 effects were signi cant and showed a signi cant interaction with diet. ANOVA found both signi cant dietary and BP-3 effects on proliferation of spindle cell tumors, but no interaction between these treatments. BP-3 did not signi cantly alter apoptosis, except for spindle cell tumors arising in mice fed LFD ( Figure 6). Apoptosis in spindle cell tumors from LFD + BP-3 mice was reduced by half compared to those from LFD mice ( Figure 6B). We also observed a reduction of apoptosis in epithelial tumors arising in mice fed HFD-LFD ( Figure 6A) and in spindle cell tumors arising in mice fed LFD-HFD compared to LFD ( Figure 6B). It is noteworthy that the spindle cell tumors arising in mice fed LFD + BP-3 showed both higher proliferation and lower apoptosis, but longer latency. ANOVA (Supplementary Table S2) found a signi cant dietary effect on apoptosis in epithelial tumors, but no signi cance to the effects of diet and BP-3 in spindle cell tumors. However, the interaction between diet and BP-3 was signi cant for spindle cell tumors, consistent with BP-3 having a signi cant effect only on tumors arising in mice fed LFD.
Having observed increased proliferation with BP-3 treatment in epithelial tumors arising in mice fed LFD-HFD and HFD-LFD and in spindle cell tumors arising in mice fed LFD and LFD-HFD, we examined whether this would be re ected in the number of epithelial proliferative lesions observed in the mammary glands of 26-week old mice, prior to the appearance of palpable tumors, as well as in the proliferation of normal mammary tissue. BP-3 treatment only increased the number of lesions in mice fed HFD-LFD ( Figure 7A), while proliferation was increased by BP-3 treatment in all dietary groups ( Figure 7B). No association was observed between BP-3 treatment effects on normal cellular proliferation and tumorigenesis, or between BP-3 treatment effects on epithelial proliferative lesions and tumorigenesis. Mice fed HFD-LFD also showed increased lesions compared to mice fed LFD ( Figure 7A). ANOVA (Supplementary Table S2) found both signi cant dietary and BP-3 effects on the number of lesions, although these effects show no signi cant interaction. This is consistent with BP-3 showing a trend toward increased lesions in all dietary groups, and the HFD-LFD group showing a signi cantly higher number of lesions compared to the LFD and LFD-HFD groups. ANOVA only found a signi cant effect for BP-3 treatment on proliferation in mammary glands of 26-week old mice.
Because we found that HFD promoted angiogenesis among epithelial tumors arising in both the Trp53null transplantation model [7] and in the 7,12-dimethylbenz[a]anthracene (DMBA) model [5], we examined the intra-tumoral vascularization of tumors with and without BP-3 treatment using the endothelial cell marker, CD31. Epithelial tumors arising in mice fed LFD-HFD showed increased vascularization in response to BP-3 treatment ( Figure 8A). Spindle cell tumors as a group showed increased vascularization compared to epithelial tumors ( Figure 8A and B; Supplementary Figure S2), as previously reported [7]. ANOVA (Supplementary Table S2) found signi cant BP-3 effects for both epithelial and spindle cell tumors, consistent with small but statistically insigni cant increases in vascularization of LFD and HFD-LFD epithelial tumors, as well as small but statistically insigni cant decreases in vascularization of spindle cell tumors with BP-3 treatment.

Effects on metabolic parameters
Animal weight was followed over the time course of tumorigenesis and BP-3 exposure had no signi cant impact on body weight (Supplementary Figure S3). BALB/c mice were previously reported to be obesityresistant when fed HFD [5,6,7,8]. In the present study, we observed considerable weight gain among mice fed LFD-HFD. The mice fed LFD-HFD and LFD-HFD + BP-3 were signi cantly heavier than those fed LFD and LFD + BP-3 by 11 weeks of age, with at least 20% weight gain by 16 weeks of age in (Supplementary Figure S3). While certainly not obesity-resistant in these experiments, the weight gain of mice fed LFD-HFD is not correlated with elevated non-fasting plasma glucose levels (Supplementary Figure S4A), although non-fasting plasma insulin levels were elevated in mice fed LFD-HFD (Supplementary Figure   S4B). BP-3 modestly reduced glucose levels in mice fed LFD-HFD and HFD-LFD ( Figure S4A), and modestly increased insulin levels in mice fed HFD-LFD ( Figure S4B). ANOVA (Supplementary Table S2) found signi cant BP-3 effects on glucose levels, consistent with the modest reductions found across dietary groups. ANOVA (Supplementary Table S2) also found signi cant dietary effects on insulin levels, consistent with modest elevation of insulin across dietary groups. Kaplan-Meier analysis revealed that the weight gain in mice fed LFD-HFD did not signi cantly alter epithelial tumorigenicity, whether with or without BP-3 treatment (Supplementary Figure S5A and B). Segregation of spindle cell tumors between weight groups do not provide an adequate sample size for meaningful analysis. We speculate that this change in weight gain from prior experiments may be the result of housing changes in our mouse facility.
Weight gain in mice has been attributed to a shift from sub-thermoneutral to thermoneutral conditions [31].

Discussion
In this study, we showed that long term BP-3 exposure affects the course of Trp53-null initiated tumorigenesis in a manner dependent upon both the temporal inclusion of HFD in the feeding regimen of mice and the histopathology of the tumor. BP-3 exposure to mice fed an adult-restricted HFD (LFD-HFD) promoted the incidence of epithelial tumors with increased proliferation and increased vascularization, while decreasing the incidence of spindle cell tumors. Spindle cell tumors occurring in mice fed LFD-HFD with BP-3 exposure also had increased proliferation. In contrast, BP-3 exposure to mice fed a lifelong LFD decreased the incidence of epithelial tumors, and increased the latency of both epithelial and spindle cell tumors. Interestingly, while BP-3 increased the latency of spindle cell tumors in mice fed LFD, those tumors that did occur showed both increased proliferation and a two-fold decrease in apoptosis. Thus, although the latency of spindle cell tumors increased, these tumors displayed two characteristics of higher-grade tumors.
BP-3 exposure to mice fed a pubertally restricted HFD (HFD-LFD) produced no signi cant change in the incidence of either epithelial or spindle cell tumors compared to mice not treated with BP-3. It should be noted, however, that the brief exposure to HFD between three and ten weeks of age was su cient to eliminate all protective effects of BP-3 found in mice fed LFD, suggesting puberty as a sensitive window of susceptibility to the effects of BP-3 with HFD. In these studies, our initial examination of BP-3 effects on estrogen-stimulated proliferation in wild type mammary glands only found enhanced proliferation in pubertal mice. This is consistent with the notion of puberty as a particularly sensitive window to environmental toxicants.
In our previous studies, we found that HFD promoted mammary tumorigenesis in both the DMBA-induced tumorigenesis model [5,6,8] and in the current Trp53-null transplantation model [7]. A pubertal window of susceptibility was observed in these studies and the current study of BP-3 exposure reinforces the nding that puberty is a very sensitive time window for adverse exposures. Regarding BP-3, it will be valuable to eventually explore pubertal versus adult exposure to BP-3 on a constant diet regimen.
The observation of BP-3 promotion of epithelial tumors in mice fed LFD-HFD, while seemingly protecting against spindle cell tumors, raises the question of whether this is an issue of differential response of tumors with differing histopathologies or BP-3 blocking the progression of epithelial tumors to a spindle cell histopathology under conditions of adult HFD. If the latter scenario were the case, one would expect increased latency of spindle cell tumors with BP-3 treatment. This is not the case. How might an LFD-HFD diet regimen favor epithelial tumors over spindle cell tumors? Possible mechanisms might include alteration of the target cell population, alteration of the immune milieu, and alteration of the spectrum of available growth factors. None of these are mutually exclusive.
Examination of the mammary glands of 26-week old mice for BP-3 effects on the occurrence of lesions and proliferation were discordant with results from tumors. While BP-3 effects on tumor promotion were most dramatic for epithelial tumors occurring in mice fed a HFD in adulthood (i.e., LFD-HFD), BP-3 was only seen to enhance the number of pre-neoplastic lesions in animals that received a pubertal HFD (i.e., HFD-LFD). This is consistent with our observation that BP-3 only contributed to proliferation in intact mammary glands of mice fed a pubertal HFD. Perhaps, the increase in lesions in HFD-LFD mice re ects this enhanced proliferation during puberty. On the other hand, all mammary glands of 26-week old mice, irrespective of diet, showed enhanced proliferation. This is super cially at odds with our experiments in intact animals that found no proliferative effects in adult mammary glands, irrespective of diet. Perhaps, this discordance re ects the difference between the relatively brief 5-d exposure in our preliminary experiments with intact wild-type mammary glands compared to the lengthy exposure in the mice that comprised the tumorigenesis experiments. Our metabolic studies in BP-3 treated tumor-bearing mice, where BP-3 exposure is long term, suggest modestly increased insulin levels that could plausibly enhance proliferation [32,33,34,35]. Another possibility is that differences from wild type tissue re ect altered growth regulation in Trp53-null epithelium. Neither rationale is su cient to explain epithelial proliferation being increased by BP-3 under all diets at 26 weeks of age, while epithelial tumorigenesis as assessed by Kaplan-Meier analysis being promoted by BP-3 only in mice fed an adult HFD. Clearly, tumorigenesis outcomes integrate not only effects intrinsic to the epithelium, but also effects impacting immune status and extrinsic growth factors, to name just two examples. Both are certainly ripe areas for further study.
Unlike several earlier studies that found rather minimal BP-3 activity in vivo in rodents with doses higher than those in the current study: 1500 mg/kg BW/d [29]; 1000 mg/kg BW/d [36]; 150 mg/kg BW/d [25], our tumorigenesis experiments showed signi cant effects at 70 mg/kg BW/d. This dosage elicits levels of BP-3 excretion in urine similar to that observed in humans with heavy topical application of BP-3containing sunscreen [19]. Furthermore, our initial experiments in wild type BALB/c mice found signi cant effects on mammary epithelial proliferation at only 7 mg/kg BW/d. Our observations suggest caution in the use of BP-3-containing sunscreens.

Conclusions
These studies reveal signi cant effects on the course of Trp53-null mammary tumorigenesis induced by exposure to benzophenone-3 (BP-3; oxybenzone), a common active ingredient in sunscreens and other personal care products. Utilizing Kaplan-Meier analysis to measure lifetime "survival" of tumor-free mammary glands, we found that BP-3 elicits both promotional and protective effects on mammary tumorigenesis dependent upon dietary regimen and tumor histopathology. However, even in instances where this analysis shows an ostensibly protective effect, other parameters suggest the potential for greater risk. For example, while BP-3 treatment enhances tumor-free survival in mice fed LFD, the spindle cell tumors that do occur display a higher level of proliferation and a lower level of apoptosis, properties associated with a poorer prognosis in human cancers. We also found that pubertal exposure to HFD was su cient to counter the ostensibly protective effect of BP-3 in mice fed LFD. Thus, our studies contribute to an existing literature suggesting that puberty is a critical window of susceptibility for later mammary tumorigenesis.
Taken together, these ndings suggest that BP-3 exposure may have adverse consequences in mammary tumorigenesis. They point to a need for further studies of BP-3 in both animal models and humans as a potential risk factor in breast cancer. They also point to the more general need to evaluate endocrine disrupting chemicals in the context of varying diets. Future studies are needed to identify the mechanistic basis for BP-3 effects on mammary tumorigenesis and how dietary fat interacts with BP-3 to alter outcomes.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
These studies were supported by the Breast Cancer and the Environment Research Program Grant 5 U01 ES026119 from the National Institute of Environment Health Science (NIEHS) and the National Cancer Institute (NCI), NIH, DHHS. Its contents are solely the responsibility of the authors and do not necessarily represent the o cial views of NIEHS, NCI, or NIH.
Authors' contributions AK carried out the majority of the tumorigenesis experiments, histopathological analysis of the tumors, apoptosis assays, assessment of lesions, and assisted in gure preparation. OM assisted with histopathological analysis of the tumors, performed analysis of vascularization and receptor expression, and assisted in gure preparation. RH performed metabolic analyses and assisted in gure preparation.
MDA carried out urine excretion experiments, proliferation assays in normal mammary gland, and assisted in setting up tumorigenesis experiments. MAB performed proliferation assays in tumors and pretumor mammary glands. SZH conceived and designed the study with RCS and assisted in interpretation of the results. RCS conceived and designed the study with SZH, interpreted the results, and wrote the manuscript. All authors read and approved the nal manuscript.
37. Schlecht C, Klammer H, Jarry H, Wuttke W. Effects of estradiol, benzophenone-2 and benzophenone-3 on the expression pattern of the estrogen receptors (ER) alpha and beta, the estrogen receptor-related receptor 1 (ERR1) and the aryl hydrocarbon receptor (AhR) in adult ovariectomized rats. Toxicology.
2004;205:123-30. Figure 1 BP-3 enhances estrogen-stimulated mammary gland proliferation in pubertal mice fed HFD. (A) Pubertal OVX BALB/c mice were placed on LFD or HFD with and without BP-3, and then treated with E2 or control for 5 d. The pubertal mice fed HFD plus BP-3 showed higher proliferation in response to E2 than did mice fed HFD alone. (B) Pubertal OVX BALB/c mice BP-3 were placed on HFD and treated for 5 d with E2 (E) or E2 + BP-3 (1.0x, 0.1x, 0.01x 70mg/kg BW). BP-3 augmented the proliferative response to E2 in both ducts and duct ends at the standard dose and in duct ends at the 0.1 dose. The values presented are means +/-SEM. Signi cance of differences between samples was assessed using an unpaired two-tailed Student's t-test. *, p<0.05.