Phthalate exposure and prostate cancer in a population-based nested case-control study

Journal Pre-proof Phthalate exposure and prostate cancer in a population-based nested case-control study Shu-Chun Chuang, Hui-Chi Chen, Chien-Wen Sun, Yuh-An Chen, Yin-Han Wang, Chun-Ju Chiang, Chu-Chih Chen, Shu-Li Wang, Chien-Jen Chen, Chao A. Hsiung PII:

S0013-9351(19)30699-1

DOI:

https://doi.org/10.1016/j.envres.2019.108902

Reference:

YENRS 108902

To appear in:

Environmental Research

Received Date: 26 June 2019 Revised Date:

5 November 2019

Accepted Date: 5 November 2019

Please cite this article as: Chuang, S.-C., Chen, H.-C., Sun, C.-W., Chen, Y.-A., Wang, Y.-H., Chiang, C.-J., Chen, C.-C., Wang, S.-L., Chen, C.-J., Hsiung, C.A., Phthalate exposure and prostate cancer in a population-based nested case-control study, Environmental Research (2019), doi: https:// doi.org/10.1016/j.envres.2019.108902. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

Phthalate exposure and prostate cancer in a population-based nested case-control study Shu-Chun Chuang, PhD1,, Hui-Chi Chen, PhD 2, Chien-Wen Sun, MSc3, Yuh-An Chen, MSc1, Yin-Han Wang, MSc1, Chun-Ju Chiang, PhD 4,5, Chu-Chih Chen, PhD 1, Shu-Li Wang, PhD 3,6,7*, Chien-Jen Chen, ScD, PhD 8, Chao A. Hsiung, PhD 1,* 1. Institute of Population Health Sciences, National Health Research Institutes, Zhunan, Miaoli, Taiwan 2. Department of Gynecology and Obstetrics, College of Medicine, National Taiwan University, Taipei, Taiwan 3. National Institute of Environmental Health Sciences, National Health Research Institutes, Zhunan, Miaoli, Taiwan 4. Graduate Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taipei, Taiwan 5. Taiwan Cancer Registry, Taipei, Taiwan 6. School of Public Health, National Defense Medical Center, Taipei, Taiwan 7. Department of Safety, Health and Environmental Engineering, National United University, Miaoli , Taiwan 8. Genomics Research Center, Academia Sinica, Taipei, Taiwan

*To whom correspondence should be addressed Shu-Li Wang, PhD and Chao Agnes Hsiung, PhD National Health Research Institutes 35 Keyan Road Zhunan, Miaoli County 35053 Taiwan Email: [email protected] and [email protected] Phone: +886 37-246-166 ext. 36509 and 36100 Fax: +886 37-586-467 Running title: Phthalate exposure and prostate cancer occurrence. Disclosure: The authors have declared no conflicts of interest.

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Abstract Background: Phthalic acid esters are established as endocrine disruptors. The study aimed to evaluate the association between urinary phthalate metabolites and prostate cancer occurrence. Methods: The study was based on the Taiwan Community-Based Cancer Screening Program, which was set up in 1991-1992 and followed periodically. By 2010, 80 incident prostate cancer cases were identified in the 12,020 men. For each case, 2 controls were randomly selected, matched by age (±3 years), urine collection date (±3 months), and residential township. Frequently used phthalate metabolites from the urine samples were quantified by liquid chromatography/electrospray ionization tandem mass spectrometry. Logistic regression was conducted to assess the association between the exposure levels and prostate cancer occurrence. Results: Exposure to di(2-ethylhexyl), butyl-benzyl and di-isobutyl phthalates (DEHP, BBzP, DiBP) was positively associated with prostate cancer in men with waist circumference (WC) ≥90 cm but not in the leans. Odds ratio for the DEHP metabolite summary score (upper tertile compared to the rest) and prostate cancer were 7.76 (95% CI=1.95-30.9, p<0.01) for WC≥90 cm. Conclusions: DEHP, BBzP, and DiBP exposure were associated with prostate cancer occurrence in abdominally obese men. The main limitation remains the lack of mechanistic experiments and comparable toxicological data. Keywords: phthalate; prostate cancer; obesity; environmental epidemiology; endocrine disruptor.

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1. Introduction Phthalic acid esters are plasticizers used worldwide in a variety of consumer and personal care products. Human are exposed to phthalates from various routes, e.g. ingestion, inhalation, and dermal exposures (1, 2). For example, human may expose to phthalates through ingestion food stored in plastic bags or containers or by dermal absorptions of lotions or perfumes (2-4). Phthalates are antiandrogens (5, 6), environmental estrogens (7, 8) and obesogens (9). Phthalates have been suggested to be associated with endocrine-related cancer risks; however, the available evidence in humans is still limited for conclusion. In addition, phthalate exposures occur in mixtures; thus, knowledge of the interactions among these compounds and their properties must be improved (8). In male rats, in utero phthalate exposure induced abnormal testis development, which was similar to human testicular dysgenesis syndrome, including insufficient spermatogenesis, cryptorchidism, hypospadias, and testicular germ cell tumors (10). In adult men, phthalate exposure has been linked to poor semen quality (11-13), lower circulating reproductive hormone (5, 14) and thyroid hormones (15, 16). The carcinogenic effect of long-term exposure to phthalates has been demonstrated in rodent species (17); however, the evidence from the human epidemiological observational studies has been inconclusive. Lifetime exposure to di(2-ethylhexyl) phthalate (DEHP) induced testicular cancer in rats and the DEHP-induced testicular cancer developed earlier than liver cancer (18). Recent studies used urinary phthalate metabolites to estimate the association between phthalate exposure and breast cancer occurrences. A case-control study in Northern Mexico women (19) revealed a positive association between mono-ethyl phthalate (MEP) and breast cancer occurrence, 3

particular among premenopausal women. Another study in Alaska Native women (20) also found a positive association between mono-(2-ethylhexyl) phthalate (MEHP) and increased breast cancer incidence. However, a third study predominantly in white women (21) suggested inverse association between several phthalate metabolites and breast cancer risk and the subsequent survival. Besides paternal exposure to DBP was found to be associated with female offspring’ BCA risk in New Zealand (22). Thereby it is essential to investigate male exposure to phthalates and its relation to prostate cancer with obesity considered. Prostate cancer is an age-related, androgen-dependent tumor. The only established risk factors for prostate cancer are age, African-American ethnicity, and a family history of prostate cancer (23, 24). There is some evidence that obese men are associated with higher risk of aggressive, high-grade prostate cancer (25-27); and testosterone is positively associated with low-grade prostate cancer but negatively associated with high-grade cancer (28). In addition, the experimental study provided evidence that MEHP may impact on lipolysis, glucose uptake/glycolysis, and mitochondrial respiration/biogenesis in fat cells (29). Thus, the impact of phthalates on prostate cancer risk may be different in obese and lean men. To our knowledge, no study has evaluated the association between phthalate exposures and prostate cancer risks. A small study conducted in 2008 suggested a median DEHP exposure of 33.9 µg/kg body weight/day in Taiwan (30), compared to 10.3µg/kg body weight/day in Germany in 2003 (31). Similarly, a remarkably high median urinary mono-(2-ethylhexyl) phthalate (MEHP) levels was observed in the 2008 study (15.9µg/L, (30)), compared to 4.6 µg/L in a German sample in 2003 (32) and 2.20 µg/L in a US sample collected in 2007-2008 (33). The high dose and long-term phthalates exposure has raised health concerns in Taiwan. 4

This study evaluated the association between urinary phthalate metabolite concentrations and prostate cancer occurrence and explored the potential effect modification of obesity in a prospective cohort study. 2. Methods 2.1 Study Cohort The Community-Based Cancer Screening Program was established between 1991 and 1992 in Taiwan. The cohort was described in detail previously (34). In brief, 12,024 men who lived in seven townships in Taiwan and were 30-65 years old at recruitment participated in the study. Information on sociodemographic characteristics, diet, cigarette smoking, alcohol use, betel-nut chewing, personal medical and surgical history, and any family history of hepatocellular carcinoma or liver cirrhosis were included in the lifestyle questionnaires. Each subject provided written informed consent for the interview, blood and urine sample collection, and serologic and biochemical assays. The current study was approved by the Institutional Review Board of the National Health Research Institutes (NHRI) and by the National Taiwan University College of Public Health. 2.2 Cases and control selection Incident prostate cancer cases were identified through computerized linkages with the Taiwan Cancer Registry in 2015. The Taiwan Cancer Registry was set up in 1979. In Taiwan, all hospitals with 50 or more beds must report all newly diagnosed malignant cancers to the Cancer Registry. All participants were followed from recruitment (1991-1992) until cancer development, death, or the end of the follow-up period (1991-2010).

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Among the 12,020 men, 49 had prevalent cancer before recruitment and 591 did not provide samples. During the 20 year follow-up, 123 incident prostate cancer cases were identified (ICD-O-3: C61). Forty-three cases were further excluded due to a lack of urine samples (Supplementary Table 1). For each incident case, two controls were selected by cumulative sampling and matched by age (± 3 years), residence, and date of urine sample collection (± 3 months). Four controls were further excluded because their urine samples were insufficient for the assay. The final dataset for analysis included 80 incident prostate cancer cases and 156 controls. 2.3 Urinary phthalate metabolite concentrations Spot urine samples were collected at recruitment (1991-1992), frozen at -30°C, and not been thawed until analysis. Urinary concentrations of 11 phthalate metabolites were examined using solid phase extraction coupled with liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) at the NHRI analytical organic toxicology laboratory, per the previously published methodology (35). The metabolites were mono-methyl phthalate (MMP), mono-ethyl phthalate (MEP), mono-isobutyl phthalate (MiBP), mono-n-butyl phthalate (MnBP), mono-benzyl phthalate (MBzP), mono-isononyl

phthalate

(MiNP),

mono-(2-ethyl-5-hydroxyhexyl) phthalate

(MEOHP),

mono-(2-ethylhexyl)

phthalate

(MEHHP),

mono-(2-ethyl-5-carboxypentyl)

phthalate

(MEHP),

mono-(2-ethyl-5-oxohexyl) phthalate

(MECPP),

and

mono-(2-carboxymethylhexyl) phthalate (MCMHP). Blank samples with water (Sigma-Aldrich, Switzerland) were simultaneously included in every experiment. Measurements were performed in the laboratory at the National Institute of Environmental Health Sciences, National Health Research Institutes, Taiwan, at which an annual external quality assurance by German External Quality Assessment Scheme for Biological Monitoring is conducted. All the laboratory work was done by the same 6

technical personnel in blind fashion. The laboratory results, questionnaire data, and the disease status were then merged and analyzed by another person. The inter- and intra-batch coefficients of variance were between 8% for MEOHP and 27% for MEHP and between 3% for MEOHP and 26% for MEHP, respectively (Supplementary Table 2). For measurements below the limit of detection (LOD), we assigned a default value of LOD divided by 2. The MiNP detection rate was low in our study samples (7%); thus this metabolite was eliminated from further analysis. Urinary creatinine concentration (mg/dL) was measured using an ADVIA 1800 Clinical Chemistry System (Siemens, Erlangen, Germany) at the Union Clinical Laboratory (UCL, Taipei, Taiwan). We used the creatinine-adjusted method (µg per g creatinine) (36) to standardize the renal function and urine dilution. In addition to examining creatinine-adjusted concentrations, we combined molar sums for DEHP. The molar sum was calculated by dividing each metabolite concentration by its molar mass, summing the individual metabolite concentrations, and correcting

for the

creatinine concentrations.

The sum

of DEHP includes

creatinine-adjusted molar sum of its five metabolites: MEHP, MEHHP, MEOHP, MECPP, and MCMHP, and is expressed as MEHP, molar weight 278 (21). 2.4 Statistical analyses The characteristics for the cases and the controls were compared using chi-square test for categorical and Kruskal-Wallis test for continuous data. Since most cancer cases were diagnosed more than 10 years after recruitment, tertiles of each creatinine-adjusted metabolite or summary scores were categorized based on total sample distributions. Conditional logistic regression was used to estimate odds ratios (OR) and 95% 7

confidence intervals (CI) for prostate cancer, with the lowest tertile serving as the reference category. The models were adjusted for education (illiterate, primary school, middle school, and high school or above) and waist circumference. Further adjusted for smoking, drinking, and betel nut chewing status did not change the results. Tests for trend were performed by modeling the median values of each category as a continuous variable. Because prostate cancers in obese patients are more likely to be high-grade and androgen-independent (25-27), we also examined the modification effect by waist circumference (≥90 vs. <90 cm and ≥92 vs. <92 cm) and body mass index (BMI, ≥25 vs. <25 and ≥27 vs. <27 cm). Abdominal obesity was defined as waist circumference ≥90 cm in Taiwanese men. The other cutoffs were chosen for sensitivity analyses. In the subgroup analyses, we split the matched sets and conducted unconditional logistic regression and adjusted for the matched variables (age at urine collection and residence) and education (37). Due to the small sample size in the subgroup analysis, Firth logistic regression was used to confirm the observed associations (38). Because creatinine is associated with age, sex, race. BMI, and fat-free mass, the observed phthalate-prostate cancer association could be because of creatinine-prostate cancer association (39). We repeated analyses treating creatinine as a covariate of urinary phthalate concentrations in addition to examining creatinine-adjusted concentrations. Interactions between urinary phthalate metabolites and waist circumference or BMI were tested by introducing an interaction term of metabolite (tertile) and waist circumference or BMI category. Sensitivity analyses were also performed by excluding cases who were followed for <2, <5, or <10 years and by using metabolite concentration cut-offs at every 10th percentile. The daily intake of phthalates was estimated from the metabolite concentrations in the urine based on the formula provided by the Mage et al., 2008 (40). All analyses 8

were performed using SAS 9.4. All tests were two sided and statistical significance was assessed at the α level of 0.05. 3. Results Table 1 shows the characteristics for the cases and the controls. The mean age at recruitment was 58 years and the average age at cancer diagnosis was 71 years old. Seventy-eight percent cases were followed for more than 10 years before their cancer was diagnosed. Overall, the controls had higher education levels; but smoking, drinking, betel-nut chewing, body mass index, and waist circumference did not differ. Table 2 presents the LOD, detection rate, geometric means and standard error of the urinary level and creatinine-adjusted metabolites by case and control status. The correlation coefficients between metabolites are shown in the Supplementary Table 3. The distribution of creatinine-adjusted metabolites by waist circumference (≥90 and <90 cm) in the controls is presented in the Supplementary Table 4. No major difference was observed, except for that MEP concentrations were higher in the abdominally obese participants (median=18.65 µg/g creatinine) than that in the normal participants (median=10.83 µg/g creatinine, p=0.03). Overall, no association between urinary phthalate metabolites and prostate cancer risk was observed (Table 3 and Supplementary Table 5 and 6). However, seven of the 10 measured metabolites were positively associated with prostate cancer in the abdominally obese subjects at α<0.05: MnBP, MBzP, MiBP, MEHHP, MEOHP, MECPP, and MCMHP (Table 4). At α<0.005, MECPP still associated with higher prostate cancer risks (OR3rd vs. 1st tertile=19.4, 95% CI=2.85-132, p=0.0025). Summary scores for DEHP confirmed this observation (OR3rd vs. 1st tertile=21.0, 95% CI=3.02-146, p=0.0021). Due to the small sample size, the Firth logistic regression was conducted and obtained similar 9

results (Supplementary Table 7). The associations were further confirmed in the model by treating creatinine as a covariate of the original urinary phthalate metabolites (supplementary Table 8). The estimated median DEHP intake in the highest tertile group was 8.00 µg/kg BW/day. Sensitivity analyses suggested similar associations (Supplementary Table 9). 4. Discussion In this study, we report for the first time an association between urinary phthalate metabolites and prostate cancer occurrence in abdominally obese subjects. It is also the first time to use prospective cohort design to study prostate cancer in relation to phthalate. This is important because phthalates exposure is associated with therapeutic procedures and easily altered through life-style changes. In our samples collected during 1991 and 1992, the 95th percentile of urinary MEHP concentrations in our control sample was 122.95 µg/L, which was much higher than that of a German sample collected in 1991 (43.8 µg/L) (32) and a US sample collected during 1999-2010 (male 25.3 µg/L) (33). The 75th percentile of the urinary MEHP concentration in our sample was 23.55 µg/L (Table 2). Our results may be unique to populations with high exposure levels. After Bonferroni correction, MECPP was still related to prostate cancer in the abdominal obese participants. As MECPP is the stronger biomarker of DEHP exposure (41), this might suggest that exposure to DEHP may be associated with higher prostate cancer occurrence in abdominally obese subjects. Several other DEHP metabolites confirmed this observation, including MEHP, MEHHP, and MCMHP. Sensitivity analyses using different cutoffs provided similar associations. These associations were also checked using the Firth logistic regression (Supplementary Table 7 and 9). 10

Aside from androgens, ERα and ERβ are also expressed in human prostate glands (42-44). Testosterone would be aromatized to estradiol within adipocytes and prostate cells, thus may lead to elevated serum and intracellular estradiol levels in obese men (45). Androgen is hypothesized to promote cancer and complete the malignant transformation (42, 46), while estrogen induces prostate cancer cell growth (47). Molecular interplay between these sex hormones in prostate glands may increase tumor proliferation, migration, and reduce tumor apoptosis and transit to androgen independence, thus result in more aggressive prostate cancer (25). Exposure to phthalates may exacerbate endocrine imbalance and drive prostate cancer progression in obese patients In addition, a study reported increased urinary phthalate metabolites 3-6 months after weight loss in obese subjects (48). The study suggested that phthalates may accumulate in fatty tissue and be released during weight loss (48). In vitro studies have shown that phthalate exposure can induce DNA damage, alter apoptosis, affecting DNA methylation, and increase cell proliferation and invasiveness of tumor cell lines (49-51). A recent study showed early-life exposure to DEHP pose increased prostate cancer risk via epigenetics changes of PSCA hypo-methylation in rats (52). A new evidence suggested that Peroxisome proliferator-activated receptor-γ (PPAR-γ) may be oncogenic in prostate cancer development and progression (53). An in vitro experiment found that repeated exposure to MEHP and its metabolites may induce a pro-inflammatory state in differentiated adipocytes partially mediated by PPARγ (50). In human, PPAR-γ is important in adipocyte differentiation, the inflammatory response, and glucose utilization, and fatty acids are endogenous PPAR-γ ligands (50, 54, 55). PPARs can be activated by phthalates (56, 57). DEHP may cause prostate cancer via activation of PPAR-γ in obese patients. 11

DEHP, DBP and their metabolites are weak aryl hydrocarbon receptor (AhR) agonists (58). AhR is constitutively active in advanced prostate cancer cells (59), and prostate cancer cell signaling facilitates their invasion (60). Furthermore, AhR may disrupt fat metabolism and contribute to obesity (61). Activation of AhR may be another mechanism to explain the association between phthalates and prostate cancer in obesity. Another study suggested that MEHP and MBzP may act as a tumor promoter through their associations with longer leukocyte telomere length (62). In a prospective study, longer leukocyte telomere length was associated with prostate cancer, particularly with aggressive cancer in men with a family history of the disease (63). Thereby phthalates may be accumulated in fatty tissues, activate PPARs, disrupt cholesterol metabolism, interrupt androgen and estrogen synthesis, and leading to increased prostate cancer occurrence. Our study had several limitations. The small sample size increases the possibility of chance findings due to multiple comparisons. Given our sample size, the power to detect an OR<2 was only 69%. However, the p-values for MECPP and the summary scores for DEHP was small (p<0.005). Further analyses using Firth logistic regression, a procedure to provide a solution to small sample size in logistic regression (38), suggested similar associations. DEHP and its metabolites, and MBzP were consistently positively associated in the abdominally obese subjects. Using different cut-offs confirmed the observed associations. Second, spot urine may not accurately represent an individual’s exposure during follow-up years. It has been reported that 75% of administered DEHP is excreted in the urine after two days (41, 64-66). Nevertheless, urinary phthalate metabolites appeared moderately reproducibility over years, suggesting single measurement of urinary phthalate metabolites might be able to indicate a steady state of exposure (67). The total concentrations of phthalate 12

metabolites remained unchanged in urine samples stored at -70°C for up to 1 year (68). Although it is possible that these metabolites might undergo some degree of degradation in samples frozen at -30°C for over 20 years, such degradation is very limited (69) and usually attenuates the relative risks and reduces statistical power. In the 1990s, however, people may have been unaware of phthalate exposure. The exposure sources and patterns may also have been changed. For example, people might use plastic bag for hot food packaging more frequently and applied less personal care products than they do nowadays. Because the samples were taken long before the prostate cancer developed and analyzed at the same lab/round using standardized protocol, and the lab technician did not know the case/control status of the study, the misclassification of phthalate exposure may be non-differential with respect to prostate cancer incidence and may result in bias toward the null. Third, we were not able to rule out of a possibility of residual confounding from other unmeasured environmental exposures. Nevertheless, we tried to manage this limitation by matching the residential area and adjusted this variable in subgroup analyses. Fourth, because the creatinine-adjusted concentrations may introduce association between creatinine and disease (39), we repeated the analyses treating creatinine as a covariate and examine the association independent of creatinine. DEHP and its metabolites, as well as MBzP and MiBP, were still associated with prostate cancer in abdominal obese men. Finally, clinical information for the prostate cancer cases was only available after 2010 and thereafter. It would be thought-provoking to further investigate whether phthalate exposure is associated with high-grade prostate cancer. On the other hand, the incidence rate of prostate cancer was low in Taiwan. In 2012, the age-standardized rate was 40.2/100,000 in Taiwan (70), while it was 81.3/100,000 in the USA, 72.9/100,000 in UK, and 111.0/100,000 in Norway (71), for example. In Taiwan, all newly diagnosed cancers from hospitals with 13

more than 50 beds have to report to the Taiwan Cancer Registry since 1979. In 2010, the completeness of the Taiwan Cancer Registry was around 97%. As a result, the case finding in our study should be relatively completed. Secondly, we measured the human metabolites of phthalates. There should be minimum interaction between the plastic vials and the biological content. The mechanisms how phthalate affect prostate cancer risk, particularly in obese men, remain unknown. 5. Conclusion In conclusion, our study suggests an association between phthalate exposure and prostate cancer risk in obese men. The results need to be validated in larger cohorts and in different ethnic groups. The mechanism behind this observation warrants further investigation.

Acknowledgements: The work was supported by the National Health Research Institutes of Taiwan (EH-105-SP-01). The authors would like to thank Dr. Hsing-Jien Kung (Institute of Molecular and Genomic Medicine, National Health Research Institutes) and Dr. Kai-Hsiung Chang (Institute of Cellular and System Medicine, National Health Research Institutes) for their comments and advice.

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induce adiposity, metabolic syndrome and prostate disease, via aberrant ER-alpha and GPER signalling. Mol Cell Endocrinol. 2012;351(2):269-78. 46. Bosland MC. The role of steroid hormones in prostate carcinogenesis. J Natl Cancer Inst Monogr. 2000;(27):39-66. 47. Friedman AE. The Estradiol-Dihydrotestosterone model of prostate cancer. Theor Biol Med Model. 2005;2:10. 48. Dirtu AC, Geens T, Dirinck E, Malarvannan G, Neels H, Van GL, et al. Phthalate metabolites in obese individuals undergoing weight loss: Urinary levels and estimation of the phthalates daily intake. Environ Int. 2013;59:344-53. 49. Caldwell JC. DEHP: genotoxicity and potential carcinogenic mechanisms-a review. Mutat Res. 2012;751(2):82-157. 50. Manteiga S, Lee K. Monoethylhexyl Phthalate Elicits an Inflammatory Response in Adipocytes Characterized by Alterations in Lipid and Cytokine Pathways. Environ Health Perspect. 2017;125(4):615-22. 51. Singh S, Li SS. Epigenetic effects of environmental chemicals bisphenol A and phthalates. Int J Mol Sci. 2012;13(8):10143-53. 52. Xia B, Wang Y, Wang X, Wu J, Song Q, Sun Z, et al. In utero and lactational exposure of DEHP increases the susceptibility of prostate carcinogenesis in male offspring through PSCA hypomethylation. Toxicol Lett. 2018;292:78-84. 53. Elix C, Pal SK, Jones JO. The role of peroxisome proliferator-activated receptor gamma in prostate cancer. Asian J Androl. 2018;20(3):238-43. 54. Lemberger T, Desvergne B, Wahli W. Peroxisome proliferator-activated receptors: a nuclear receptor signaling pathway in lipid physiology. Annu Rev Cell Dev Biol. 1996;12:335-63. 55. Desvergne B, Feige JN, Casals-Casas C. PPAR-mediated activity of phthalates: A link to the obesity epidemic? Mol Cell Endocrinol. 2009;304(1-2):43-8. 56. Lapinskas PJ, Brown S, Leesnitzer LM, Blanchard S, Swanson C, Cattley RC, et al. Role of PPARalpha in mediating the effects of phthalates and metabolites in the liver. Toxicology. 2005;207(1):149-63. 57. Issemann I, Green S. Activation of a member of the steroid hormone receptor 19

superfamily by peroxisome proliferators. Nature. 1990;347(6294):645-50. 58. Kruger T, Long M, Bonefeld-Jorgensen EC. Plastic components affect the activation of the aryl hydrocarbon and the androgen receptor. Toxicology. 2008;246(2-3):112-23. 59. Richmond O, Ghotbaddini M, Allen C, Walker A, Zahir S, Powell JB. The aryl hydrocarbon receptor is constitutively active in advanced prostate cancer cells. PLoS One. 2014;9(4):e95058. 60. Ide H, Lu Y, Yu J, Noguchi T, Kanayama M, Muto S, et al. Aryl hydrocarbon receptor signaling involved in the invasiveness of LNCaP cells. Hum Cell. 2017;30(2):133-9. 61. Kerley-Hamilton JS, Trask HW, Ridley CJ, Dufour E, Ringelberg CS, Nurinova N, et al. Obesity is mediated by differential aryl hydrocarbon receptor signaling in mice fed a Western diet. Environ Health Perspect. 2012;120(9):1252-9. 62. Scinicariello F, Feroe AG, Attanasio R. Urinary Phthalates and Leukocyte Telomere Length: An Analysis of NHANES 1999-2002. EBio Medicine. 2016;6:96-102. 63. Julin B, Shui I, Heaphy CM, Joshu CE, Meeker AK, Giovannucci E, et al. Circulating leukocyte telomere length and risk of overall and aggressive prostate cancer. Br J Cancer. 2015;112(4):769-76. 64. Koch HM, Bolt HM, Preuss R, Angerer J. New metabolites of di(2-ethylhexyl)phthalate (DEHP) in human urine and serum after single oral doses of deuterium-labelled DEHP. Arch Toxicol. 2005;79(7):367-76. 65. Frederiksen H, Kranich SK, Jorgensen N, Taboureau O, Petersen JH, Andersson AM. Temporal variability in urinary phthalate metabolite excretion based on spot, morning, and 24-h urine samples: considerations for epidemiological studies. Environ Sci Technol. 2013;47(2):958-67. 66. Johns LE, Cooper GS, Galizia A, Meeker JD. Exposure assessment issues in epidemiology studies of phthalates. Environ Int. 2015;85:27-39. 67. Townsend MK, Franke AA, Li X, Hu FB, Eliassen AH. Within-person reproducibility of urinary bisphenol A and phthalate metabolites over a 1 to 3 year period among women in the Nurses' Health Studies: a prospective cohort study. Environ Health. 2013;12(1):80. 68. Samandar E, Silva MJ, Reidy JA, Needham LL, Calafat AM. Temporal stability of eight phthalate metabolites and their glucuronide conjugates in human urine. Environ Res. 2009;109(5):641-6 20

69. Schwedler G, Seiwert M, Fiddicke U, Issleb S, Holzer J, Nendza J, et al. Human biomonitoring pilot study DEMOCOPHES in Germany: Contribution to a harmonized European approach. Int J Hyg Environ Health. 2017;220(4):686-96. 70. Chiang CJ, Lo WC, Yang YW, You SL, Chen CJ, Lai MS. Incidence and survival of adult cancer patients in Taiwan, 2002-2012. J Formos Med Assoc. 2016;115(12):1076-88. 71. Cancer Incidence in Five Continents, CI5plus: IARC CancerBase No. 9 [Internet]. [Internet]. International Agency for Research on Cancer. 2018. Available from: Available from: http://ci5.iarc.fr.

21

Table 1. Study population characteristics

Age at recruitment, mean±SD Age at diagnosis, mean±SD Residence Penghu islets Hsinchu Chiayi Pingtung Taipei county Education Illiterate Primary school Middle school High school or above Smoking status No Yes Unknown Drinking status No Yes Unknown Betel-nut chewing status No Yes Unknown BMI <25 25-30 ≥30 Waist circumference <90 cm ≥90 cm

Control N=156 % 57.53±6.00

Case N=80 % 57.74±6.02 71.21±6.19

49 21 31 37 18

31.4 13.5 19.9 23.7 11.5

25 11 16 19 9

31.3 13.8 20.0 23.8 11.3

Matched

26 86 16 28

16.7 55.1 10.3 17.9

13 47 15 5

16.3 58.8 18.8 6.3

0.04

61 94 1

39.1 60.3

38 42 0

47.5 52.5

0.38

121 34 1

77.6 21.8

66 14 0

82.5 17.5

0.56

140 15 1

89.7 9.6

72 8 0

90.0 10.0

0.77

93 56 7

59.6 35.9 4.5

53 24 3

66.3 30.0 3.8

0.61

107 49

68.6 31.4

56 24

70.0 30.0

0.82

22

p Matched

Table 2. Urinary phthalate metabolites by case and control status, Taiwan, 1991-1992 Control Cases Molar Detection Geometric LOD Geometric mass Rate (%) p5 p25 median p75 p95 p95 p5 p25 Median p75 means means 1 MMP 180 0.12 95.76 8.29 0.06 5.18 9.67 21.27 54.54 8.33 1.23 4.24 7.95 18.68 44.00 MMP2 9.96 0.29 4.33 9.67 19.10 38.09 7.47 1.17 3.51 7.33 15.11 51.76 1 MEP 194 0.12 87.29 10.29 0.06 5.46 14.65 34.06 168.80 5.56 0.06 2.29 10.07 25.35 107.40 MEP2 16.14 0.11 5.66 12.32 26.90 178.75 11.30 0.06 2.24 7.51 26.71 70.06 MnBP1 221 0.12 99.15 34.53 2.06 18.23 39.63 77.94 263.56 43.97 8.06 20.76 47.83 98.14 207.54 MnBP2 34.77 5.94 18.29 35.09 63.67 149.99 37.34 9.36 20.61 40.20 59.28 147.25 MBzP1 255 0.12 79.24 1.82 0.06 0.92 2.49 7.40 36.74 2.16 0.06 0.94 3.27 7.52 54.55 MBzP2 3.51 0.08 0.87 2.05 4.77 23.99 3.88 0.07 0.89 2.82 5.46 35.90 MiBP1 222 0.24 87.29 3.64 0.12 1.90 4.40 10.90 33.94 3.71 0.12 1.85 4.42 12.61 38.03 MiBP2 5.36 0.16 1.90 3.76 8.57 35.71 4.50 0.18 1.83 3.55 8.94 20.02 MiNP1 419 1.2 6.78 0.63 0.60 0.60 0.60 0.60 1.03 0.64 0.60 0.60 0.60 0.60 0.77 MiNP2 1.26 0.23 0.35 0.52 0.97 2.24 1.41 0.20 0.36 0.46 0.89 1.81 MEHP1 278 0.24 90.25 6.29 0.12 2.49 8.46 23.35 122.95 7.57 0.73 3.82 8.56 18.25 59.91 2 MEHP 9.15 0.22 2.45 7.96 16.88 85.74 7.66 0.88 3.05 7.05 13.24 39.31 MEHHP1 294 0.12 100.00 28.22 3.42 13.28 28.93 52.27 360.67 28.93 6.52 15.82 26.53 43.42 199.01 MEHHP2 26.72 7.70 15.56 22.24 38.19 176.41 24.57 9.74 13.79 22.79 37.61 98.17 MEOHP1 292 0.24 98.73 15.98 1.80 7.76 18.20 34.67 161.80 17.56 3.95 10.34 17.82 29.58 88.00 MEOHP2 16.14 3.61 8.25 13.93 24.60 83.68 14.91 5.02 8.49 14.30 21.74 49.10 MECPP1 307 0.048 100.00 40.22 5.44 17.69 36.45 78.44 320.96 44.26 9.88 20.55 38.40 75.17 340.05 2 MECPP 38.08 11.50 20.13 32.35 59.23 230.04 37.58 12.83 21.92 34.00 52.71 155.79 MCMHP1 307 0.048 99.15 10.68 1.58 4.83 10.25 20.93 77.30 11.76 2.57 5.83 12.28 19.80 113.53 MCMHP2 10.43 3.36 5.53 8.37 14.49 61.44 10.61 3.46 5.75 8.55 15.84 62.18 Total DEHP3 297.21 27.62 52.26 83.31 143.71 587.90 204.01 38.03 52.18 82.76 136.04 385.25 1. Urinary metabolite level (µg/L) 2. Creatinine-adjusted metabolites (µg/g creatinine) 3. Creatinine-adjusted molar sum of DEHP metabolites: MEHP, MEHHP, MEOHP, MECPP, and MCMHP, and is expressed as MEHP, molar weight 278. Metabolite

23

Table 3. Association between urinary phthalate metabolites and prostate cancer risk µg/g creatinine1 Median1 Controls Cases OR2 95% CI OR3 DEHP4 <58.85 42.86 52 26 1.00 1.00 ≥58.85 - <105.63 83.02 52 27 1.01 (0.51, 2.02) 1.14 ≥105.63 182.95 52 27 1.05 (0.53, 2.09) 1.26 5 Continuous 0.99 (0.80, 1.19) 0.99 Q3 vs. Q1+Q2 1.05 (0.58, 1.89) 1.18 MEHP <3.63 1.82 53 35 1.00 1.00 ≥3.63 - <12.67 7.45 48 31 1.21 (0.59, 2.46) 1.30 ≥12.67 21.10 55 24 0.91 (0.43, 1.91) 0.99 Continuous5 1.03 (0.90, 1.17) 1.03 Q3 vs. Q1+Q2 0.76 (0.42, 1.36) 0.84 MEHHP <17.11 11.95 48 30 1.00 1.00 ≥17.11 - <29.31 22.35 54 25 0.73 (0.37, 1.44) 0.83 ≥29.31 49.99 54 25 0.81 (0.42, 1.57) 0.86 Continuous5 0.95 (0.78, 1.15) 0.96 Q3 vs. Q1+Q2 0.89 (0.49, 1.60) 0.95 MEOHP <9.91 6.85 52 26 1.00 1.00 ≥9.91 - <19.02 14.05 51 28 1.11 (0.56, 2.18) 1.32 ≥19.02 32.75 53 26 1.09 (0.55, 2.16) 1.17 Continuous5 1.00 (0.83, 1.20) 1.01 Q3 vs. Q1+Q2 0.97 (0.55, 1.73) 1.00 MECPP <23.91 17.66 53 25 1.00 1.00 ≥23.91 - <44.66 32.69 50 29 1.31 (0.65, 2.64) 1.31 ≥44.66 75.80 53 26 1.08 (0.53, 2.20) 1.20 5 Continuous 0.99 (0.82, 1.21) 1.01 Q3 vs. Q1+Q2 0.95 (0.53, 1.68) 1.02 MCMHP <6.41 4.71 50 28 1.00 1.00 ≥6.41 - <11.35 8.40 54 25 0.85 (0.44, 1.64) 0.82 ≥11.35 20.04 52 27 1.00 (0.50, 2.02) 1.06 5 1.00 (0.84, 1.19) 1.02 Continuous Q3 vs. Q1+Q2 1.05 (0.59, 1.87) 1.19 MBzP <1.29 0.20 48 30 1.00 1.00 ≥1.29 - <3.74 2.19 59 20 0.55 (0.28, 1.10) 0.59 ≥3.74 7.89 49 30 1.11 (0.50, 2.05) 1.06 1.02 (0.91, 1.14) 1.04 Continuous5 Q3 vs. Q1+Q2 1.33 (0.73, 2.42) 1.42 MiBP <2.51 1.11 49 29 1.00 1.00 ≥2.51 - <6.35 3.70 55 24 0.77 (0.37, 1.57) 0.71 ≥6.35 12.47 52 27 0.85 (0.43, 1.67) 0.88 Continuous5 0.97 (0.84, 1.11) 0.99 Q3 vs. Q1+Q2 1.00 (0.56, 1.78) 1.05 MnBP <24.26 14.91 54 24 1.00 1.00 ≥24.26 - <52.45 36.87 48 31 1.46 (0.76, 2.79) 1.35 ≥52.45 81.07 54 25 1.05 (0.51, 2.18) 1.13 Continuous5 1.10 (0.90, 1.33) 1.14 Q3 vs. Q1+Q2 0.85 (0.47, 1.53) 0.93 MMP <5.10 2.71 49 29 1.00 1.00 ≥5.10 - <13.57 8.20 50 29 1.00 (0.52, 1.90) 1.09 ≥13.57 23.22 57 22 0.57 (0.27, 1.21) 0.61 5 Continuous 0.95 (0.81, 1.11) 0.98 Q3 vs. Q1+Q2 0.59 (0.31, 1.15) 0.58 MEP <5.96 2.03 43 35 1.00 1.00 ≥5.96 - <17.09 10.54 57 22 0.43 (0.20, 0.91) 0.36 ≥17.09 47.40 56 23 0.42 (0.20, 0.91) 0.45 5 Continuous 0.85 (0.76, 0.96) 0.86 Q3 vs. Q1+Q2 0.69 (0.36, 1.32) 0.78 1. Tertiles and medians were rounded to the second decimal. 2. Conditional logistic regression without adjustment 3. Conditional logistic regression adjusted for education and circumference. 4. Creatinine-adjusted molar sum of DEHP metabolites and is expressed as MEHP, molar weight 278. 5. Assessed by the base 2 logarithm of the creatinine adjusted level.

24

95% CI

(0.55, (0.61, (0.81, (0.64,

2.36) 2.60) 1.21) 2.18)

(0.63, (0.46, (0.90, (0.46,

2.70) 2.14) 1.18) 1.54)

(0.41, (0.43, (0.79, (0.52,

1.69) 1.73) 1.17) 1.73)

(0.63, (0.57, (0.84, (0.55,

2.74) 2.41) 1.23) 1.81)

(0.62, (0.56, (0.83, (0.56,

2.77) 2.56) 1.23) 1.85)

(0.42, (0.51, (0.85, (0.65,

1.63) 2.20) 1.22) 2.18)

(0.28, (0.51, (0.92, (0.76,

1.23) 2.23) 1.16) 2.66)

(0.33, (0.44, (0.86, (0.59,

1.52) 1.77) 1.14) 1.88)

(0.68, (0.53, (0.92, (0.50,

2.71) 2.41) 1.40) 1.72)

(0.55, (0.28, (0.83, (0.29,

2.14) 1.35) 1.15) 1.17)

(0.16, (0.20, (0.76, (0.39,

0.78) 1.02) 0.97) 1.56)

Table 4. Association between urinary phthalate metabolites and prostate cancer risk by abdominal obesity Waist <90 cm Waist ≥90 cm µg/g creatinine1 Median1 Control Case OR2 LL UL Control Case OR2 LL UL 3 DEHP <58.85 42.86 34 24 1.00 18 2 1.00 ≥58.85 - <105.63 83.02 34 19 0.71 (0.31, 1.62) 18 8 4.95 (0.75, 32.8) ≥105.63 182.95 39 13 0.49 (0.21, 1.15) 13 14 21.0 (3.02, 146) 0.73 (0.53, 1.00) 1.61 (1.06, 2.44) Continuous4 Q3 vs. Q1+Q2 0.57 (0.27, 1.23) 7.76 (1.95, 30.9) MEHP <3.63 1.82 35 20 1.00 18 5 1.00 ≥3.63 - <12.67 7.45 32 23 1.04 (0.45, 2.39) 16 8 2.61 (0.57, 11.9) 0.47 (0.19, 1.17) 15 11 4.75 (0.98, 23.2) ≥12.67 21.10 40 13 Continuous4 0.92 (0.78, 1.09) 1.33 (0.99, 1.77) Q3 vs. Q1+Q2 0.46 (0.20, 1.02) 2.61 (0.78, 8.74) MEHHP <17.11 11.95 33 28 1.00 15 2 1.00 ≥17.11 - <29.31 22.35 36 15 0.49 (0.22, 1.12) 18 10 6.19 (0.92, 41.6) ≥29.31 49.99 38 13 0.44 (0.19, 1.02) 16 12 9.01 (1.45, 56.0) 4 Continuous 0.69 (0.50, 0.95) 1.51 (1.03, 2.20) Q3 vs. Q1+Q2 0.60 (0.28, 1.29) 2.76 (0.86, 8.82) MEOHP <9.91 6.85 37 24 1.00 15 2 1.00 ≥9.91 - <19.02 14.05 32 16 0.85 (0.37, 1.96) 19 12 5.56 (0.92, 33.7) ≥19.02 32.75 38 16 0.66 (0.29, 1.50) 15 10 10.7 (1.53, 74.8) 4 0.84 (0.65, 1.08) 1.54 (1.03, 2.28) Continuous Q3 vs. Q1+Q2 0.71 (0.34, 1.48) 3.04 (0.88, 10.5) MECPP <23.91 17.66 33 23 1.00 20 2 1.00 ≥23.91 - <44.66 32.69 35 19 0.74 (0.33, 1.66) 15 10 8.58 (1.30, 56.6) 0.51 (0.22, 1.21) 14 12 19.4 (2.85, 132) ≥44.66 75.80 39 14 Continuous4 0.75 (0.55, 1.02) 1.67 (1.09, 2.55) 0.60 (0.28, 1.28) 5.07 (1.40, 18.4) Q3 vs. Q1+Q2 MCMHP <6.41 4.71 33 23 1.00 17 5 1.00 ≥6.41 - <11.35 8.40 35 17 0.69 (0.30, 1.60) 19 8 1.58 (0.36, 6.94) ≥11.35 20.04 39 16 0.56 (0.24, 1.30) 13 11 7.31 (1.38, 38.7) Continuous4 0.81 (0.63, 1.05) 1.69 (1.08, 2.64) Q3 vs. Q1+Q2 0.67 (0.32, 1.41) 5.54 (1.41, 21.8) MBzP <1.29 0.20 31 24 1.00 17 6 1.00 ≥1.29 - <3.74 2.19 41 15 0.47 (0.20, 1.08) 18 5 1.06 (0.23, 4.79) ≥3.74 7.89 35 17 0.69 (0.30, 1.57) 14 13 4.06 (1.03, 15.9) Continuous4 0.94 (0.82, 1.08) 1.27 (1.01, 1.59) Q3 vs. Q1+Q2 0.99 (0.47, 2.06) 3.95 (1.24, 12.6) MiBP <2.51 1.11 35 24 1.00 14 5 1.00 ≥2.51 - <6.35 3.70 34 17 0.64 (0.27, 1.53) 21 7 1.72 (0.33, 8.98) ≥6.35 12.47 38 15 0.47 (0.20, 1.12) 14 12 8.28 (1.37, 49.9) 4 Continuous 0.88 (0.74, 1.04) 1.48 (1.04, 2.12) Q3 vs. Q1+Q2 0.59 (0.28, 1.24) 5.65 (1.52, 21.1) MnBP <24.26 14.91 36 20 1.00 18 4 1.00 ≥24.26 - <52.45 36.87 34 22 1.07 (0.47, 2.44) 14 9 5.80 (1.06, 31.6) ≥52.45 81.07 37 14 0.61 (0.25, 1.45) 17 11 6.10 (1.14, 32.7) Continuous4 1.06 (0.83, 1.36) 1.35 (0.93, 1.95) Q3 vs. Q1+Q2 0.58 (0.27, 1.26) 2.16 (0.67, 6.94) MMP <5.10 2.71 35 22 1.00 14 7 1.00 ≥5.10 - <13.57 8.20 37 18 0.79 (0.35, 1.78) 13 11 2.23 (0.56, 8.78) ≥13.57 23.22 35 16 0.76 (0.33, 1.76) 22 6 0.62 (0.15, 2.51) Continuous4 0.98 (0.82, 1.17) 0.99 (0.71, 1.38) Q3 vs. Q1+Q2 0.85 (0.40, 1.81) 0.40 (0.12, 1.32) MEP <5.96 2.03 31 24 1.00 12 11 1.00 ≥5.96 - <17.09 10.54 46 17 0.41 (0.18, 0.93) 11 6 0.51 (0.12, 2.19) ≥17.09 47.40 30 16 0.54 (0.21, 1.35) 26 7 0.30 (0.07, 1.22) Continuous4 0.89 (0.79, 1.00) 0.84 (0.69, 1.03) 0.88 (0.39, 1.96) 0.41 (0.12, 1.40) Q3 vs. Q1+Q2 1. Tertiles were rounded to the second decimal. 2. Unconditional logistic regression adjusted for age at urine collection, residence, and education. 3. Creatinine-adjusted molar sum of DEHP metabolites: MEHP, MEHHP, MEOHP, MECPP, and MCMHP, and is expressed as MEHP, molar weight 278.

25

4.

Assessed by the base 2 logarithm of the creatinine adjusted level.

26




Phthalic acid esters are established as endocrine disruptors. Phthalates may accumulate in fatty tissue, inducing inflammatory responses in adipocytes. Thus, we hypothesized that obesity may modify the association between phthalates and prostate cancer risks.




We conducted a nested case-control study based on incident prostate cancer cases identified from a previous cohort (1991-2010) and the urine samples were collected at recruitment (1991-1992).




We found that DEHP, BBzP, and DiBP exposure were associated with prostate cancer occurrence in abdominally obese men (waist≥90 cm).