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This cross-sectional study was conducted among women living in Isfahan Province, Iran. Isfahan Province, with a population of 5,120,850 according to the 2016 census, is the third-most crowded province in Iran after Tehran and Mashhad Provinces.
Paired samples (n = 106) of malignant and normal tissues (adjacent to tumor tissues) were obtained from women with breast cancer (n = 53).
A researcher designed a questionnaire consisting of information on gender, age, body mass index (BMI), physical activity, breastfeeding duration, educational status, breast cancer and other family history of cancers, hormone therapy history, reasons to say yes to hormone therapy, age at first pregnancy, age at menarche, age at menopause, employment, status, deodorant, fast food and cosmetic consumption, and smoking. Based on the diagnosis, other information regarding the tumor, such as estrogen receptor/progesterone receptor/human epidermal receptor 2 (ER/PR/HER2) factors, were also obtained.
Breast tissues (malignant and normal) samples were obtained from Hojati and Khanavadeh Hospitals, and the procedure was subject to approval from the ethics committee of the Isfahan University of Medical Sciences. Participants signed the consent documents after the study procedure was explained by an environmental health researcher.
Samples were transported to the Poursina-Hakim Laboratory to be diagnosed and separated into normal and malignant tissues by a pathologist. The samples were kept in formalin at 4 °C until extraction.
All chemicals, solvents, and analytes, including MePB, EtPB, PrPB, and BuPB (with purity > 99%), were purchased from Sigma-Aldrich. Parabens stock solutions were prepared by dissolving 10 mg of each paraben in 1 mL of methanol, and then stored at 4 °C. The standard series were prepared in 10–100 ng/mL range to draw the calibration curves.
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Several previous methods[26-30] were assessed in designing the method adopted here. In this design, first, 1 to 5 g of breast tissue sample was grinded, followed by addition of 5 mL methanol. Next, the mixture was exposed to ultrasonic waves for 15 min to lyse the breast cells, and parabens were released from the breast tissue. The mixture was then centrifuged at 5,000 rpm for 5 min. The obtained supernatant was diluted by deionized water to 50 mL. Subsequently, 10 mL of the diluted solution was filtered through a 0.45 μm filter. Finally, based on the dispersive liquid-liquid micro-extraction (DLLME) method, 500 μL acetone and 30 μL chlorobenzene were rapidly injected into a 5 mL sample solution placed in a 10 mL conical centrifuge tube to form a cloudy suspension, which was then centrifuged at 5,000 rpm for 5 min[28].
Addition of these solvents to the tissue supernatant and the subsequent centrifugation allowed low-density insoluble compounds, such as lipids, to be dissolved with the assistance of acetone in the aqueous phase in the Falcon tube. In addition, owing to its high density and insolubility, chlorobenzene contributes in the separation of insoluble compounds, such as paraben, in the droplet on the bottom of the Falcon tube. This droplet was collected, dried by a mild flow of nitrogen, and derivatized with 10 μL N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA). The yield substance was analyzed through gas chromatography (GC) with an MS detector (Agilent 19091S-433MS column with 0.25 mm thickness, 30.0 m length, and 0.32 mm diameter)[31].
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Matrix (breast tissue) effects were evaluated using a spike recovery approach, and the results of the obtained calibration curve was modified for malignant and normal tissues by applying the relative recovery (RR%) obtained from Equation (1)[30]. For this reason, during sample preparation, the standards (130 ng/g) were spiked with sample, and matrix effects were evaluated by RR%.
$$ {\text{RR\%}} = \frac{{{n_{found}} - {n_{real}}}}{{{n_{added}}}} \times 100 $$ (1) Where, nfound is the amount of paraben detected in the real sample spiked with a known amount of paraben (130 ng/g). nreal is the amount of paraben detected in the non-spiked real sample and nadded is the amount of paraben added to the real sample[30].
To decrease background paraben contamination, all glassware were washed with deionized water and then placed at 250 °C for 24 h, all instrument operators avoided using products containing parabens to reduce sample contamination, and all solvent stocks were checked daily for contamination control[32]. In addition, a calibration standard (at a concentration range of 10–50 ng/mL) and a pure solvent (methanol) were injected after every 20 samples to check for changes in instrumental sensitivity and carry-over of parabens between samples, respectively[13].
Method validity was examined by analytical factors such as regression equation, linearity, limit of detection (LOD), limit of quantification (LOQ), retention time, and relative recovery (RR%). Correlation coefficients are equal or higher than 0.9976. The RR of these analytes was 88% for MePB and 133% for EtPB. The LOD was 0.28 ng/g for PrPB and 0.85 ng/g for BuPB. The LOQ was 0.88 ng/g for BuPB and 1.98 ng/g for MePB. Typical chromatograms of MePB, EtPB, PrPB, and BuPB are presented in Figure 1.
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Data were analyzed in SPSS (IBM SPSS Statistics 25). The paired sample t-test was used to compare the means of paraben concentration, and Spearman correlation coefficients were applied to determine the correlation between parabens in adjacent normal-malignant tissues. The association between paraben concentrations with traditional risk factors of breast cancer was assessed using the generalized estimating equations (GEE) model. All tests were conducted at a 5% error level. Total paraben concentration was calculated using Equation (2):
$$ \sum \text{PB}\text{s} = \text{MePB} + \text{EtPB} + \text{PrPB} + \text{BuPB} $$ (2) Estrogen equivalency (EEQ) factor of parabens was calculated using Equation (3)[33].
$$\begin{aligned} \text{EEQ}\;(\text{parabens}) =& \; (\text{MePB} \times 1) + (\text{PrPB} \times 83.3) \\ & + (\text{BuPB} \times 250)\\ \end{aligned}$$ (3) In Equations (2) and (3), the concentration of each paraben was expressed as ng/g and mol/g, respectively.
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The socio demographic characteristics of the subjects are shown in Table 1. Most subjects were aged 40–49 years (42.3%), followed by 30–39 years (19.2%). The obtained data indicated that 36.5%, 32.7%, 25.0%, and 5.8% of women were considered obese, normal, overweight, and underweight, respectively. Only 17.3% of the subjects were with menarche at age < 12 years and 61.5% were at age > 12 years. Based on the obtained results, 5.8% and 25.0% of women were considered as postmenopausal and premenopausal, respectively. Most participants were unemployed (83.0%); thus, they were more willing to complete and return the questionnaires than the participants who were employed. The most frequent highest education was < high school (53.8%) (Table 1).
Table 1. The socio-demographic and behavioral characteristics of the study population (N = 53)
Demographic variables Grouping variables N (%) Age (years) ≤ 30 4 (7.7) 30−39 10 (19.2) 40−49 22 (42.3) 50−59 8 (15.4) ≥ 60 8 (15.4) Body mass index (BMI) ≤ 18.5 (underweight) 3 (5.8) 18.5−24.9 (normal weight) 17 (32.7) 25−29.9 (over weight) 13 (25.0) ≥ 30 (obesity) 19 (36.5) Physical activity Regular 7 (13.5) Irregular 29 (55.8) Passive 16 (30.7) Breast feeding duration Without children 8 (15.1) < 6 months 6 (11.3) 6−24 months 39 (73.6) Educational status < High school 28 (53.8) High school 11 (21.2) > High school 13 (25.0) Degree of family history
of breast cancerNone 29 (54.7) First or second degree 24 (45.3) Degree of family history
of other cancerNone 35 (66.0) First or second degree 18 (34.0) Hormone therapy history Yes 23 (41.8) No 32 (58.2) Reasons to say yes to hormone therapy Birth control 20 (87.0) Infertility 3 (13.0) Age at first pregnancy (years) < 30 43 (87.8) 30 3 (6.1) > 30 3 (6.1) Employment status Employed 9 (17.0) Unemployed 44 (83.0) Age at menarche (years) < 12 9 (17.3) 12 11 (21.2) > 12 32 (61.5) Age at menopause
(years)Non-menopausal 36 (69.2) < 55 13 (25.0) > 55 3 (5.8) -
In both tissues, 100% of samples contained MePB, EtPB, PrPB, and BuPB. According to the results obtained from paired sample t-test, no significant difference in all the measured paraben metabolites (MePB, EtPB, PrPB, and BuPB) was observed between these two tissues (P > 0.05) (Table 2). In both tissues, the order of paraben concentration was as follows: MePB > EtPB > PrPB > BuPB.
Table 2. Comparison of the mean and median of paraben metabolite (ng/g) distribution between malignant and normal tissues
Paraben metabolites (ng/g wet w.) Tissue Mean Median SD IQR Min Max t df P-value MePB Malignant 178.47 148.36 107.10 110 22.36 529.23 0.798 52 0.429 Normal 164.86 135.49 114.92 169 7.72 547.42 EtPB Malignant 136.69 131.37 84.90 132 11.77 369.98 −0.581 52 0.564 Normal 144.11 112.83 120.01 126 11.68 547.42 PrPB Malignant 99.89 84.48 63.87 76 23.50 266.19 0.202 52 0.840 Normal 97.44 68.82 95.72 91 5.89 422.18 BuPB Malignant 87.88 61.93 80.38 112 7.56 310.28 −1.019 52 0.313 Normal 107.53 45.44 138.82 122 4.76 769.06 ∑PBs Malignant 497.02 507.31 242.29 356 99.35 1114.70 −0.252 52 0.802 Normal 507.81 432.20 325.43 411 30.05 1522.65 In this study, the EEQ or estrogenic body burden of parabens for malignant and normal tissues were 7.97 and 6.15 mol/g, respectively. The results indicated that the estrogenic effect of parabens in malignant tissues was 1.3 (7.97:6.15) times higher than that in normal tissues.
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The correlation coefficients of all parabens (MePB, EtPB, PrPB, and BuPB) were compared between malignant and normal tissues by Spearman correlation coefficients (Figure 2). The individual paired samples revealed positive correlations between malignant and normal tissues for all paraben concentrations. According to the Spearman correlation coefficients, all parabens were correlated with each other at P < 0.01, except for BuPB (P = 0.05).
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Table 3 shows the association between paraben concentrations with breast cancer risk factors, as analyzed using the GEE model. Analysis of the effects of ER/PR/HER2 on paraben exposure revealed a significant association between the dose-response pattern of MePB [OR = 98.34 (11.43, 185.2), P = 0.027] for both ER+ and PR+ women and the dose-response pattern of MePB [OR = 164.3 (CI: 112.3, 216.3), P < 0.001] for HER2+ women, compared with those women for the receptors- negative (Table 3).
Table 3. Association between parabens concentrations with breast cancer risk factors, analyzed using the generalized estimating equations (GEE) model
Risk factors of breast cancer MePB EtPB PrPB BuPB ∑PBs B (CI 95%) P B (CI 95%) P B (CI 95%) P B (CI 95%) P B (CI 95%) P ER
(Ref. = negative.)Positive 98.34
(11.43, 185.2)0.027 11.32
(−83.24, 105.8)0.814 −47.89
(−186.1, 90.32)0.497 −21.49
(−192.8, 149.8)0.806 16.97
(−372.8, 406.8)0.932 PR
(Ref. = negative.)Positive 98.34
(11.43, 185.2)0.027 11.32
(−83.24, 105.8)0.814 −47.89
(−186.1, 90.32)0.497 −21.49
(−192.8, 149.8)0.806 16.97
(−372.8, 406.8)0.932 HER2
(Ref. = negative.)Positive 164.3
(112.3, 216.3)< 0.001 −17.49
(−69.16, 34.17)0.507 −66.74
(−140.6, 7.12)0.077 −23.18
(−111.8, 65.49)0.608 46.21
(−144.8, 237.2)0.635 Tumor location in breast
(Ref. = outer)Inter 22.07
(−53.59, 97.74)0.567 17.33
(−41.17, 75.83)0.561 −5.66
(−51.52, 40.20)0.809 1.08
(−51.26, 53.42)0.968 50.19
(−121.4, 221.8)0.567 Upper −27.76
(−92.17, 36.63)0.398 55.33
(−63.88, 174.5)0.363 −10.48
(−109.9, 88.97)0.836 −10.42
(−109.6, 88.78)0.837 23.35
(−257.0, 303.7)0.870 Education status
(Ref. ≥ high school)< High school −3.52
(−48.95, 41.90)0.879 −43.19
(−120.5, 34.13)0.274 −11.82
(−45.12, 21.48)0.487 −11.52
(−61.80, 38.75)0.653 −106.1
(−284.6, 72.6)0.245 High school 20.80
(−7.20, 48.81)0.146 −50.27
(−135.5, 34.9)0.248 40.43
(−28.65, 109.5)0.251 19.59
(−66.27, 105.4)0.655 −9.26
(−244.3, 225.8)0.938 Activity
(Ref. = passive)Regular −72.58
(−149.7, 4.53)0.065 40.10
(−79.03, 159.2)0.509 9.57
(−75.21, 94.36)0.825 −32.30
(−88.76, 24.16)0.262 −52.31
(−290.7, 186.1)0.667 Irregular 5.44
(−72.57, 83.46)0.891 46.32
(2.59, 90.05)0.538 −3.15
(−44.34, 38.04)0.881 19.18
(−40.19, 78.57)0.527 34.04
(−104.2, 172.2)0.629 Breast feeding duration
(Ref. = without children)< 6 month −41.85
(−124.5, 40.82)0.321 −69.52
(−170.2, 31.16)0.176 −83.02
(−144.8, −21.2)0.008 −67.48
(−148.4, 13.47)0.102 −225.3
(−463.5, 12.93)0.064 > 6 month −32.38
(−85.26, 20.80)0.230 −57.89
(−145.9, 30.19)0.198 −62.68
(−123.5, −1.85)0.043 −44.80
(−115.2, 25.64)0.213 −164.2
(−336.2, 7.75)0.061 Age at menarche
(Ref > 12 years)< 12 years −45.37
(−126.5, 35.83)0.273 7.152
(−62.80, 77.10)0.841 −37.11
(−74.07, −0.150)0.05 2.75
(−66.44, 71.95)0.938 −100.3
(−271.3, 70.55)0.250 = 12 years −20.42
(−107.1, 66.27)0.644 −69.95
(−120.1, −18.32)0.008 −44.79
(−83.14, −6.43)0.022 −2.74
(−56.66, 51.16)0.920 −132.9
(−276.8, 10.90)0.070 Age at first pregnancy
(Ref ≥ 30)< 30 years −5.88
(−40.6, 28.86)0.740 32.91
(−13.59, 79.43)0.165 −1.96
(−73.08, 69.15)0.957 53.17
(10.27, 96.07)0.015 77.29
(−65.43, 220.1)0.288 = 30 years 92.09
(−18.15, 202.3)0.102 166.2
(110.4, 222.1)< 0.001 13.84
(−57.23, 84.91)0.703 136.40
(66.89, 205.9)0.001 329.5
(77.48, 581.6)0.010 Age at menopause
(Ref ≥ 55 years)None menopause −19.68
(−134.3, 94.94)0.736 41.54
(−43.83, 126.9)0.340 36.24
(10.21, 62.28)0.006 3.95
(−55.39, 63.29)0.896 63.49
(−174.3, 301.3)0.601 ≤ 55 years −15.75
(−134.7, 103.2)0.795 45.89
(−35.02, 144.86)0.231 32.41
(3.52, 61.30)0.028 39.95
(−29.40, 109.3)0.259 81.61
(−159.3, 322.5)0.507 Illicit drug consumption
(Ref. = none)Yes 22.73
(−80.18, 125, 6)0.665 −71.79
(−131.8, −11.77)0.119 −12.33
(−73.30, 48.63)0.692 −32.39
(−89.06, 24.28)0.263 −86.75
(−342.4, 168.9)0.506 Smoking consumption
(Ref. = none)Yes −5.09
(−69.40, 59.22)0.877 −33.04
(−88.97, 22.88)0.247 −22.55
(−60.65, 15.55)0.246 −49.03
(−92.21, −5.85)0.026 −93.58
(−242.4, 55.32)0.218 Fast food consumption
(Ref. = none)Yes 3.505
(−46.31, 53.32)0.890 25.35
(−21.71, 72.42)0.291 −11.08
(−50.06, 27.89)0.577 −8.42
(−58.33, 41.48)0.741 −9.27
(−138.8, 120.2)0.888 Cosmetic consumption
(Ref. = none)Yes 30.53
(−23.64, 84.72)0.269 20.14
(−36.94, 77.22)0.489 −25.85
(−61.20, 9.48)0.152 −27.08
(−72.90, 18.70)0.246 5.58
(−133.1, 144.2)0.937 Deodorant consumption
(Ref. = none)Yes 18.34
(−34.70, 71.38)0.498 0.786
(−50.65, 52.23)0.976 −42.22
(−75.32, −9.11)0.062 −17.94
(−65.96, 30.08)0.464 −30.29
(−164.1, 103.6)0.657 History of breast cancer
(Ref. = first degree)None −95.89
(−157.9, −33.88)0.002 −61.04
(−132.5, 10.40)0.094 −4.96
(−42.83, 32.90)0.797 −15.52
(−71.56, 40.51)0.587 −164.3
(−308.5, −20.13)0.026 Second −47.79
(−128.9, 33.34)0.248 −73.83
(−150.3, 2.68)0.059 −31.49
(−70.60, 7.61)0.114 −11.35
(−76.79, 54.07)0.734 −147.3
(−333.9, 39.23)0.122 History of other cancers
(Ref. = first degree)None −23.22
(−96.87, 50.42)0.537 −41.29
(−131.6, 49.05)0.370 9.59
(−35.64, 54.84)0.678 −43.60
(−112.9, 25.73)0.218 −76.07
(−258.0, 105.8)0.413 Second −7.12
(−112.1, 97.76)0.894 −37.74
(−141.5, 66.04)0.476 3.52
(−64.99, 72.04)0.920 −59.91
(−132.6, 12.80)0.106 −75.43
(−326.3, 175.4)0.556 Hormone therapy
(Ref. = none)Yes −49.80
(−95.81, −3.79)0.054 −6.94
(−59.26, 45.37)0.795 −22.26
(−57.64, 13.10)0.217 −13.14
(−59.91, 33.61)0.582 −83.84
(−213.6, 45.96)0.206 Reasons to say yes to hormone therapy
(Ref. = infertility)Birth control −61.39
(−127.9, 5.12)0.070 −155.6
(−317.9, 6.59)0.060 −73.60
(−168.7, 21.57)0.130 −44.81
(−148.2, 58.61)0.396 −320.4
(−635.9, −4.81)0.047 Age
(Re ≥ 60)< 30 −40.27
(−126.9, 46.43)0.363 −101.6
(−141.6, −61.51)< 0.001 −38.97
(−106.4, 28.53)0.258 −98.75
(−144.5, −52.97)< 0.001 −250.2
(−403.3, −97.2)0.001 30−39 29.32
(−55.39, 114.1)0.498 49.22
(−49.35, 147.8)0.328 13.96
(−45.28, 73.20)0.644 5.13
(−79.44, 89.72)0.905 131.6
(−83.21, 346.4)0.230 40−49 5.83
(−53.18, 64.84)0.846 −2.60
(−48.77, 43.57)0.912 10.65
(−31.46, 52.77)0.620 −21.31
(−80.46, 37.84)0.480 15.66
(−86.98, 118.3)0.765 50−59 −1.71
(−73.98, 70.56)0.963 1.99
(−74.68, 78.66)0.959 −20.53
(−64.46, 23.39)0.360 −61.84
(−119.1, −4.54)0.064 −51.99
(−195.7, 91.81)0.479 BMI
(Ref. = obesity)Under weight 46.05
(−30.49, 122.5)0.238 102.3
(−189.5, 394.3)0.492 32.55
(8.28, 56.82)0.009 −24.75
(−80.86, 31.36)0.387 134.8
(−260.8, 530.5)0.504 Normal weight −14.81
(−76.87, 47.24)0.640 −28.83
(−83.48, 25.80)0.301 28.96
(−13.61, 71.54)0.182 24.70
(−34.14, 83.55)0.411 6.19
(−150.5, 162.9)0.938 Over weight −6.41
(−71.68, 58.85)0.847 −5.66
(−61.10, 49.76)0.841 31.12
(−19.34, 81.59)0.227 30.27
(−31.58, 92.13)0.337 32.15
(−134.5, 198.8)0.705 Breastfeeding duration showed a significant negative association with the response-dose of PrPB [OR = −83.02 (CI: −144.8, −21.2), P = 0.008] and PrPB [OR = −62.68 (CI: −123.5, −1.85), P = 0.043] in women with breastfeeding duration of < 6 months and > 6 months, compared with those in women with no children, respectively (Table 3).
These results indicated that the response-dose of EtPB [OR = −69.95 (CI: −120.1, −18.32), P = 0.008] and PrPB [OR = −44.79 (CI: −83.14, −6.43), P = 0.022] decreased by 69 and 44-fold among women with age at menarche of 12 years, compared to that in women with age at menarche of > 12 years, respectively (Table 3). We also observed that age at first pregnancy had a significant association with increased doses of EtPB [OR = 166.2 (CI: 110.4, 222.1), P < 0.001], BuPB [OR = 136.4 (CI: 66.89, 205.9), P = 0.001], and ∑PBs [OR = 329.5 (CI: 77.48, 581.6), P = 0.01] in women with age at first pregnancy of 30 years, compared with the reference. Moreover, the dose response of BuPB [OR = 53.17 (CI: 10.27, 96.07), P = 0.015] for age at first pregnancy of < 30 years was significant (Table 3).
A 36-fold increase was recorded in PrPB dose [OR = 36.24 (CI: 10.21, 62.28), P = 0.006] among women with no menopause compared with the reference, and a 32-fold increase was recorded in PrPB dose [OR = 32.41 (CI: 32.52, 61.30), P = 0.028] among women with menopause at < 55 years compared with the reference (Table 3).
The 95-fold decrease in MePB dose [OR = −95.89 (CI: −157.9, −33.88), P = 0.002] and the 164-fold decrease in ∑PBs dose [OR = −164.3 (CI: −308.5, −20.13), P = 0.026] were significant in women with no family history of breast cancer compared to those in the reference (first-degree relatives with breast cancer) (Table 3).
Compared with women using hormone therapy for infertility treatment, ∑PBs dose [OR= −320.4 (CI = −635.9, −4.81), P = 0.047] was 320-fold lower among women using hormone therapy for birth control (Table 3).
Stratification of data by age revealed a significant association with the response-dose of EtPB [OR = −101.6 (CI: −141.6, −61.51) P < 0.001], BuPB [OR = −98.75 (CI: −144.5, −52.97), P < 0.001], and ∑PBs [OR = −250.2 (CI: −403.3, −97.2), P = 0.001] among women aged < 30 years compared with women aged ≥ 60 years (Table 3).
Statistical analyses revealed that PrPB dose [OR = 32.55 (CI: 8.28, 56.82), P = 0.009] increased by 32-fold among underweight women compared with that among obese women (Table 3).
doi: 10.3967/bes2019.112
Paraben Content in Adjacent Normal-malignant Breast Tissues from Women with Breast Cancer
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Abstract:
Objective Accumulation of estrogenic compounds and other carcinogens in normal breast tissues contributes to unpredictable breast cancer incidence during adolescence and throughout life. To assess the role of parabens in this phenomenon, the paraben content of adjacent normal-malignant breast tissues is measured in women with breast cancer living in Isfahan Province, Iran. Methods Adjacent normal-malignant breast tissue samples were obtained from 53 subjects. The parabens including methyl-paraben (MePB), ethyl-paraben (EtPB), propyl-paraben (PrPB), and butyl-paraben (BuPB) were extracted from the sample supernatant and then subjected to gas chromatography analysis. Results Some risk factors for breast cancer were stimulated by parabens in adjacent malignant-normal breast tissues among young and middle-aged women with breast cancer. We observed a significant association for dose-response pattern of MePB [OR = 98.34 (11.43–185.2), P = 0.027] for both ER+ and PR+ women and MePB [OR = 164.3 (CI: 112.3–216.3), P < 0.001] for HER2+ women than women with negative receptors. The risk of 95-fold increase in MePB dose and 164-fold increase in ∑PBs dose were significant for women with hereditary breast cancer in first-degree relatives. Conclusions These results may promote future epidemiology studies and strategies to improve women’s lifestyle and consume paraben-free products. -
Key words:
- Paraben /
- Breast cancer /
- Malignant tissues /
- Normal tissues /
- Demography /
- Risk factors
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Table 1. The socio-demographic and behavioral characteristics of the study population (N = 53)
Demographic variables Grouping variables N (%) Age (years) ≤ 30 4 (7.7) 30−39 10 (19.2) 40−49 22 (42.3) 50−59 8 (15.4) ≥ 60 8 (15.4) Body mass index (BMI) ≤ 18.5 (underweight) 3 (5.8) 18.5−24.9 (normal weight) 17 (32.7) 25−29.9 (over weight) 13 (25.0) ≥ 30 (obesity) 19 (36.5) Physical activity Regular 7 (13.5) Irregular 29 (55.8) Passive 16 (30.7) Breast feeding duration Without children 8 (15.1) < 6 months 6 (11.3) 6−24 months 39 (73.6) Educational status < High school 28 (53.8) High school 11 (21.2) > High school 13 (25.0) Degree of family history
of breast cancerNone 29 (54.7) First or second degree 24 (45.3) Degree of family history
of other cancerNone 35 (66.0) First or second degree 18 (34.0) Hormone therapy history Yes 23 (41.8) No 32 (58.2) Reasons to say yes to hormone therapy Birth control 20 (87.0) Infertility 3 (13.0) Age at first pregnancy (years) < 30 43 (87.8) 30 3 (6.1) > 30 3 (6.1) Employment status Employed 9 (17.0) Unemployed 44 (83.0) Age at menarche (years) < 12 9 (17.3) 12 11 (21.2) > 12 32 (61.5) Age at menopause
(years)Non-menopausal 36 (69.2) < 55 13 (25.0) > 55 3 (5.8) Table 2. Comparison of the mean and median of paraben metabolite (ng/g) distribution between malignant and normal tissues
Paraben metabolites (ng/g wet w.) Tissue Mean Median SD IQR Min Max t df P-value MePB Malignant 178.47 148.36 107.10 110 22.36 529.23 0.798 52 0.429 Normal 164.86 135.49 114.92 169 7.72 547.42 EtPB Malignant 136.69 131.37 84.90 132 11.77 369.98 −0.581 52 0.564 Normal 144.11 112.83 120.01 126 11.68 547.42 PrPB Malignant 99.89 84.48 63.87 76 23.50 266.19 0.202 52 0.840 Normal 97.44 68.82 95.72 91 5.89 422.18 BuPB Malignant 87.88 61.93 80.38 112 7.56 310.28 −1.019 52 0.313 Normal 107.53 45.44 138.82 122 4.76 769.06 ∑PBs Malignant 497.02 507.31 242.29 356 99.35 1114.70 −0.252 52 0.802 Normal 507.81 432.20 325.43 411 30.05 1522.65 Table 3. Association between parabens concentrations with breast cancer risk factors, analyzed using the generalized estimating equations (GEE) model
Risk factors of breast cancer MePB EtPB PrPB BuPB ∑PBs B (CI 95%) P B (CI 95%) P B (CI 95%) P B (CI 95%) P B (CI 95%) P ER
(Ref. = negative.)Positive 98.34
(11.43, 185.2)0.027 11.32
(−83.24, 105.8)0.814 −47.89
(−186.1, 90.32)0.497 −21.49
(−192.8, 149.8)0.806 16.97
(−372.8, 406.8)0.932 PR
(Ref. = negative.)Positive 98.34
(11.43, 185.2)0.027 11.32
(−83.24, 105.8)0.814 −47.89
(−186.1, 90.32)0.497 −21.49
(−192.8, 149.8)0.806 16.97
(−372.8, 406.8)0.932 HER2
(Ref. = negative.)Positive 164.3
(112.3, 216.3)< 0.001 −17.49
(−69.16, 34.17)0.507 −66.74
(−140.6, 7.12)0.077 −23.18
(−111.8, 65.49)0.608 46.21
(−144.8, 237.2)0.635 Tumor location in breast
(Ref. = outer)Inter 22.07
(−53.59, 97.74)0.567 17.33
(−41.17, 75.83)0.561 −5.66
(−51.52, 40.20)0.809 1.08
(−51.26, 53.42)0.968 50.19
(−121.4, 221.8)0.567 Upper −27.76
(−92.17, 36.63)0.398 55.33
(−63.88, 174.5)0.363 −10.48
(−109.9, 88.97)0.836 −10.42
(−109.6, 88.78)0.837 23.35
(−257.0, 303.7)0.870 Education status
(Ref. ≥ high school)< High school −3.52
(−48.95, 41.90)0.879 −43.19
(−120.5, 34.13)0.274 −11.82
(−45.12, 21.48)0.487 −11.52
(−61.80, 38.75)0.653 −106.1
(−284.6, 72.6)0.245 High school 20.80
(−7.20, 48.81)0.146 −50.27
(−135.5, 34.9)0.248 40.43
(−28.65, 109.5)0.251 19.59
(−66.27, 105.4)0.655 −9.26
(−244.3, 225.8)0.938 Activity
(Ref. = passive)Regular −72.58
(−149.7, 4.53)0.065 40.10
(−79.03, 159.2)0.509 9.57
(−75.21, 94.36)0.825 −32.30
(−88.76, 24.16)0.262 −52.31
(−290.7, 186.1)0.667 Irregular 5.44
(−72.57, 83.46)0.891 46.32
(2.59, 90.05)0.538 −3.15
(−44.34, 38.04)0.881 19.18
(−40.19, 78.57)0.527 34.04
(−104.2, 172.2)0.629 Breast feeding duration
(Ref. = without children)< 6 month −41.85
(−124.5, 40.82)0.321 −69.52
(−170.2, 31.16)0.176 −83.02
(−144.8, −21.2)0.008 −67.48
(−148.4, 13.47)0.102 −225.3
(−463.5, 12.93)0.064 > 6 month −32.38
(−85.26, 20.80)0.230 −57.89
(−145.9, 30.19)0.198 −62.68
(−123.5, −1.85)0.043 −44.80
(−115.2, 25.64)0.213 −164.2
(−336.2, 7.75)0.061 Age at menarche
(Ref > 12 years)< 12 years −45.37
(−126.5, 35.83)0.273 7.152
(−62.80, 77.10)0.841 −37.11
(−74.07, −0.150)0.05 2.75
(−66.44, 71.95)0.938 −100.3
(−271.3, 70.55)0.250 = 12 years −20.42
(−107.1, 66.27)0.644 −69.95
(−120.1, −18.32)0.008 −44.79
(−83.14, −6.43)0.022 −2.74
(−56.66, 51.16)0.920 −132.9
(−276.8, 10.90)0.070 Age at first pregnancy
(Ref ≥ 30)< 30 years −5.88
(−40.6, 28.86)0.740 32.91
(−13.59, 79.43)0.165 −1.96
(−73.08, 69.15)0.957 53.17
(10.27, 96.07)0.015 77.29
(−65.43, 220.1)0.288 = 30 years 92.09
(−18.15, 202.3)0.102 166.2
(110.4, 222.1)< 0.001 13.84
(−57.23, 84.91)0.703 136.40
(66.89, 205.9)0.001 329.5
(77.48, 581.6)0.010 Age at menopause
(Ref ≥ 55 years)None menopause −19.68
(−134.3, 94.94)0.736 41.54
(−43.83, 126.9)0.340 36.24
(10.21, 62.28)0.006 3.95
(−55.39, 63.29)0.896 63.49
(−174.3, 301.3)0.601 ≤ 55 years −15.75
(−134.7, 103.2)0.795 45.89
(−35.02, 144.86)0.231 32.41
(3.52, 61.30)0.028 39.95
(−29.40, 109.3)0.259 81.61
(−159.3, 322.5)0.507 Illicit drug consumption
(Ref. = none)Yes 22.73
(−80.18, 125, 6)0.665 −71.79
(−131.8, −11.77)0.119 −12.33
(−73.30, 48.63)0.692 −32.39
(−89.06, 24.28)0.263 −86.75
(−342.4, 168.9)0.506 Smoking consumption
(Ref. = none)Yes −5.09
(−69.40, 59.22)0.877 −33.04
(−88.97, 22.88)0.247 −22.55
(−60.65, 15.55)0.246 −49.03
(−92.21, −5.85)0.026 −93.58
(−242.4, 55.32)0.218 Fast food consumption
(Ref. = none)Yes 3.505
(−46.31, 53.32)0.890 25.35
(−21.71, 72.42)0.291 −11.08
(−50.06, 27.89)0.577 −8.42
(−58.33, 41.48)0.741 −9.27
(−138.8, 120.2)0.888 Cosmetic consumption
(Ref. = none)Yes 30.53
(−23.64, 84.72)0.269 20.14
(−36.94, 77.22)0.489 −25.85
(−61.20, 9.48)0.152 −27.08
(−72.90, 18.70)0.246 5.58
(−133.1, 144.2)0.937 Deodorant consumption
(Ref. = none)Yes 18.34
(−34.70, 71.38)0.498 0.786
(−50.65, 52.23)0.976 −42.22
(−75.32, −9.11)0.062 −17.94
(−65.96, 30.08)0.464 −30.29
(−164.1, 103.6)0.657 History of breast cancer
(Ref. = first degree)None −95.89
(−157.9, −33.88)0.002 −61.04
(−132.5, 10.40)0.094 −4.96
(−42.83, 32.90)0.797 −15.52
(−71.56, 40.51)0.587 −164.3
(−308.5, −20.13)0.026 Second −47.79
(−128.9, 33.34)0.248 −73.83
(−150.3, 2.68)0.059 −31.49
(−70.60, 7.61)0.114 −11.35
(−76.79, 54.07)0.734 −147.3
(−333.9, 39.23)0.122 History of other cancers
(Ref. = first degree)None −23.22
(−96.87, 50.42)0.537 −41.29
(−131.6, 49.05)0.370 9.59
(−35.64, 54.84)0.678 −43.60
(−112.9, 25.73)0.218 −76.07
(−258.0, 105.8)0.413 Second −7.12
(−112.1, 97.76)0.894 −37.74
(−141.5, 66.04)0.476 3.52
(−64.99, 72.04)0.920 −59.91
(−132.6, 12.80)0.106 −75.43
(−326.3, 175.4)0.556 Hormone therapy
(Ref. = none)Yes −49.80
(−95.81, −3.79)0.054 −6.94
(−59.26, 45.37)0.795 −22.26
(−57.64, 13.10)0.217 −13.14
(−59.91, 33.61)0.582 −83.84
(−213.6, 45.96)0.206 Reasons to say yes to hormone therapy
(Ref. = infertility)Birth control −61.39
(−127.9, 5.12)0.070 −155.6
(−317.9, 6.59)0.060 −73.60
(−168.7, 21.57)0.130 −44.81
(−148.2, 58.61)0.396 −320.4
(−635.9, −4.81)0.047 Age
(Re ≥ 60)< 30 −40.27
(−126.9, 46.43)0.363 −101.6
(−141.6, −61.51)< 0.001 −38.97
(−106.4, 28.53)0.258 −98.75
(−144.5, −52.97)< 0.001 −250.2
(−403.3, −97.2)0.001 30−39 29.32
(−55.39, 114.1)0.498 49.22
(−49.35, 147.8)0.328 13.96
(−45.28, 73.20)0.644 5.13
(−79.44, 89.72)0.905 131.6
(−83.21, 346.4)0.230 40−49 5.83
(−53.18, 64.84)0.846 −2.60
(−48.77, 43.57)0.912 10.65
(−31.46, 52.77)0.620 −21.31
(−80.46, 37.84)0.480 15.66
(−86.98, 118.3)0.765 50−59 −1.71
(−73.98, 70.56)0.963 1.99
(−74.68, 78.66)0.959 −20.53
(−64.46, 23.39)0.360 −61.84
(−119.1, −4.54)0.064 −51.99
(−195.7, 91.81)0.479 BMI
(Ref. = obesity)Under weight 46.05
(−30.49, 122.5)0.238 102.3
(−189.5, 394.3)0.492 32.55
(8.28, 56.82)0.009 −24.75
(−80.86, 31.36)0.387 134.8
(−260.8, 530.5)0.504 Normal weight −14.81
(−76.87, 47.24)0.640 −28.83
(−83.48, 25.80)0.301 28.96
(−13.61, 71.54)0.182 24.70
(−34.14, 83.55)0.411 6.19
(−150.5, 162.9)0.938 Over weight −6.41
(−71.68, 58.85)0.847 −5.66
(−61.10, 49.76)0.841 31.12
(−19.34, 81.59)0.227 30.27
(−31.58, 92.13)0.337 32.15
(−134.5, 198.8)0.705 -
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