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Abstract

Organochlorines (OCs) are common environmental pollutants that have been linked to cancer. This work aims to assess the role of OCs as a risk factor for breast cancer and to evaluate the cellular changes induced by exposure to such environmental contaminants. The study included 70 cancer patients subjected to thorough history taking and routine investigations. Samples from tumor and normal adjacent tissue were taken to measure OCs’ levels and to perform molecular analysis (some oncogenic and apoptotic markers) by flow cytometry. There were significantly higher concentrations of methoxychlor, dichloro-diphenyl-trichloroethane (DDT), hexa-chlorobenzene (HCB), and chlordane in tumor tissue samples compared to the surrounding normal tissue. There was a positive statistically significant correlation between G2m and dichloro-diphenyl-dichloroethane, DDT, and methoxychlor. There was also a negative correlation between propidium iodide (PI) and heptachlor as well as between PI, B-cell lymphoma 2, and methoxychlor. Annexin showed a negative correlation with HCB and methoxychlor. In conclusion, the higher level of organochlorine pesticides in the tissue specimens of breast cancer and the resultant molecular dysfunction highlight a possible association. Further research is warranted to elucidate the other possible mechanisms involved in the process of carcinogenesis.

Keywords

Pesticides breast cancer oncogenic and apoptotic markers

Introduction

Pesticides are chemicals widely used throughout the world. Organochlorine (OCs) are highly toxic due to their persistence, stability, and long half-lives (30–47 years). They can bioaccumulate and biomagnify in food chains. They also travel over long distances and can be detected even in areas where they have never been used.^1,2 They were prohibited from use throughout the world for more than 20 years ago. However, many of the banned pesticides are still sold or manufactured for export to developing countries such as Egypt.³

Some Egyptian studies documented the presence of OCs in vegetables and fruits, water, milk, and its products despite that the Egyptian Ministry of Agriculture banned them in the 1980s^{4
,
5} Furthermore, detection of OCs' residues in the ecosystem at Manzala Lake and in aquatic organisms is considered to be a serious threat to human health.³

Organochlorines are known to be stored in adipose tissues as they are lipophilic, resulting in extremely high concentrations in humans posing a public health hazard.¹ There is speculation that they may increase cancer risk of hematopoietic system, prostate, breast, pancreas, and liver.⁶

Breast cancer, in particular, is a major public health problem as one out of nine women will develop the disease in her lifetime and it is responsible for about 22% of the total cancer mortality.⁷ The increasing incidence of breast cancer could only be partially explained by improvements in genetic studies and screening programs.⁸

It is supposed that environmental factors may play a crucial role in the pathogenesis of the disease. Long-term exposure to environmental pollutants especially those having the estrogenic effect might make a significant contribution to the process of carcinogenesis particularly hormone-related tumors such as breast cancer. This issue is controversial and is still being researched intensively.^{6
,
9}

The aim of this work was to assess the potential role of organochlorines as a risk factor for breast cancer and to evaluate the cellular changes induced by exposure to such environmental contaminants.

Patients and methods

Study design

The present work is a comparative cross-sectional study that was conducted on breast cancer patients attending the Oncology Center, Mansoura University, Dakahlia Governorate, Egypt, in the period between April 2013 and July 2014.

Inclusion criteria

Patients who agreed to participate in the study were non-smokers. They did not receive either hormonal therapy, chemo-or radiotherapy before the surgery.

Exclusion criteria

Patients with positive family history of breast cancer or had previous surgery and came for recurrence or metastasis were excluded from the study.

Ethical consideration

A written informed consent was obtained from all studied patients participating in the study besides approval from the Ethical Committee of Mansoura University-Faculty of Medicine regarding all the procedures conducted in the present work.

Methods

All patients underwent thorough history taking including sociodemographic data, full detailed patient medical history (present, past, and family history), residence, and marital status.

Preoperative investigations

Preoperative histopathological examination of breast mass by fine needle aspiration cytology (FNAC) or true cut biopsy. Then, postoperative histopathological examination of breast mass, lymph nodes, estrogen receptors, progesterone receptors, and human epidermal receptor 2.

Radiological examination by sono-mammography.

Surgery and collection of tissue samples (10 g each)

Modified radical mastectomy or wide local excision with at least 1 cm safety margin was done. During the surgical procedure, test samples were taken from the fresh malignant breast tissues. Control samples were also obtained from adjacent histologically healthy tissue (6 cm away from the tumor). All samples were divided into two halves – one for OCs detection and the other specimens were transported to the flow cytometry laboratory in Mansoura Children Hospital for molecular investigations.

Extraction and detection of organochlorines

All samples were stored at −20 °C until analysis. Then, samples were extracted and analyzed by gas chromatography for detection of organochlorine pesticides and estimation of their concentrations in breast tissue samples.

Analytical procedure:

– All solvents and reagents were pesticide analytical grade free of interfering residues. Reference chemical standards (13 organochlorines) were purchased from Ehrenstorfer (Ausberg, Germany). The purities of the standard pesticides were 97.4–99%.

– Pesticides analyzed in this study were hexachlorocyclohexane (HCH): α-HCH, β-HCH, hexa-chlorobenzene (HCB), the cyclodienes including aldrin, endrin, heptachlor, heptachlor epoxide, γ-isomer of HCH (γ-HCH, lindane), and γ-chlordane. Also, dichloro-diphenyl-trichloroethane (DDT) including DDT isomer (p,p′-DDT: 1,1,1-trichloro-2,2-bis-p-chlorophenyl-ethane), DDT metabolites: p,p′-p,p′-DDD (1,1-dichloro-2,2-bis-p-diphenyl-dichloroethane) and DDE (1,1-dichloro-2,2-bis-diphenyl-dichloroethylene), and methoxychlor.

– The tissue samples were extracted according to Petreas et al.¹⁰ One-fifth of the extract was taken to determine the lipid content gravimetrically. The samples were cleaned up by florisil column chromatography to separate pesticide residues by gas chromatography (Agilent Technologies, Inc., 6890 series) equipped with 63 Nickel Electron Capture Detector (63Ni) and auto-sampler injection.

– Gas chromatographic conditions were as follows: silica capillary column PAS-5 (30 m × 0.32 mm inner diameter × 0.25 μm film thickness), injector temperature 280 °C; detector 300 °C; column 160 °C, initial time 2 min hold 10 °C min⁻¹ to 280 °C for 10 min, the carrier gas was nitrogen. Peak areas were used as the basis for quantification. Residue levels are expressed relative to extracted lipid (ng/g lipid).

– The limit of detection was 0.5 ng/g of lipid for α, β, and γ-HCH, 0.25 ng/g lipid for HCB, aldrin, heptachlor, heptachlor epoxide, γ-chlordane, and p,p′-DDE while it was 0.99 ng/g lipid for endrin, p,p′ -DDD, p,p′ -DDT, and methoxychlor.

Molecular investigations by flow cytometry

Tissue samples were washed three times with normal saline solution and the surrounded fats were trimmed carefully.

Flow cytometer (Becton Dickinson, Sunnyvale, California, USA) was used for cell-cycle analysis. Preparation of single cell suspension and evaluation of apoptosis and DNA cell-cycle parameters were done.^{11
,
12} Then, samples were run in the flow cytometer. Data analysis was performed using DNA analysis program MODFIT (Verity Software House, Inc., Topsham, Maine, USA, version: 2.0 power Mac with 131072 KB registration no: 42000960827-16193213). This software measured DNA ploidy (diploid and aneuploid cycle %).

Determination of CD95 in tissue was performed.^{13
,
14}

Determination of Bcl2 was done.¹⁵

Cell death was evaluated by determination of propidium iodide (PI) “oncotic marker” and annexin V (apoptotic marker) using flow cytometry: dot plots were generated and divided into four quadrants (UL: upper left; UR: upper right; LL: lower left; LR: lower right). The LL quadrant shows cells negative for both annexin V and PI (living non-apoptotic cells). The UL quadrant shows dead cells (positive for PI, but negative to annexin V), whereas the UR quadrant shows late apoptosis: cells positive for both annexin V and PI. The LR quadrant shows living, early apoptotic cells positive for annexin V, but negative for PI.¹⁶

Statistical analysis

Data entry and statistical analyses were performed using SPSS (statistical package of social sciences) version 20.0 (SPSS Inc., Chicago, Illinois, USA). Distribution of data was first tested by one sample K-S test. Parametric data was expressed in mean and standard deviation. Non-parametric data was expressed in median, minimum, and maximum. Independent t-test was used to compare means for continuous parametric variables, while Mann–Whitney U test was done for comparison of non-parametric continuous variables between two groups. For comparison of qualitative data among groups, χ ² square test was performed. Spearman’s correlation coefficient was used to illustrate the strength of relationship between non-parametric variables. p value < 0.05 was considered as statistically significant.

Results

Seventy female patients fulfilled the inclusion criteria and were finally enrolled in the study. Their age ranged from 29 to 58 years (mean ± SD was 54.8 ± 12.4). The main characteristics of the studied patients as regards age, residence, occupation, marital status, and the hormonal analysis were illustrated in Table 1.

Table 1.

Sociodemographic data and hormonal analysis of the studied breast cancer cases (n = 70).

Parameter	Statistical data
Age	Mean ± SD: 54.8 ± 12.4 Min–max: 29–58
	N (%)
Marital status
Married	69 (98.6)
Single	1 (1.4)
Residence
Rural	60 (85.7)
Urban	10 (14.3)
Occupation
Housewives	55 (78.6%)
Employees	15 (21.4%)
Hormonal analysis
PR
Positive	45 (64.3)
Negative	25 (35.7)
ER
Positive	42 (60)
Negative	28 (40)
HER2
Negative	51 (72.9)
Positive	19 (27.1)

PR: progesterone receptors; ER: estrogen receptors; HER2: human epidermal receptors 2; n: number.

Table 2 shows the concentrations of organochlorine pesticides in addition to the odds ratio (OR) of their levels in the tested tissue samples. There were higher concentrations of methoxychlor, DDT, HCB, and chlordane in tumor tissue samples compared to the surrounding normal tissue in the studied cases and the differences were statistically significant (p < 0.05).

Table 2.

Levels of organochlorine pesticides in tissue samples (n = 140).

Organochlorine pesticides	Tumor n = 70		Normal n = 70		p ^a Value	p ^b Value OR (95% CI)
Organochlorine pesticides	Median (min−max)	N (%)	Median (min−max)	N (%)	p ^a Value	p ^b Value OR (95% CI)
HCB	<LOD (0–15.09)	14 (20)	<LOD (0–7.6)	2 (2.9)	0.002^c	0.001^c 8.5 (1.85–38.9)
α-HCB	<LOD (0–0.83)	2 (2.9)	<LOD (0–1.78)	1 (1.4)	0.5	1.00 2.02 (0.18–22.9)
γ-HCB	<LOD (0–12.23)	7 (10)	<LOD (0–2.04)	2 (2.9)	0.07	0.08 3.7 (0.75–18.86)
β-HCH	<LOD (0–56.01)	12 (17.1)	<LOD (0–36.01)	5 (7.1)	0.07	0.07 2.69 (0.89–8.09)
Heptachlor	<LOD (0–0.85)	3 (4.3)	<LOD (0–0)	0 (0)	0.08	0.08 0.48 (0.41–0.58)
Aldrin	<LOD (0–7.09)	5 (7.1)	<LOD (0–3.71)	1 (1.4)	0.09	0.09 5.3 (0.60–46.6)
Heptachlor epoxide	<LOD (0–0)	0 (0)	<LOD (0–0)	0 (0)	—	—
Chlordane	<LOD (0–12.77)	13 (18.6)	<LOD (0–7.7)	3 (4.3)	0.009^c	0.008^c 5.09 (1.38–18.7)
Andrin	<LOD (0–0)	0 (0)	<LOD (0–0)	0 (0)	—	—
Methoxychlor	<LOD (0–44.06)	21 (30)	<LOD (0–19.4)	6 (8.6)	0.001^c	0.001^c 4.5 (1.7–12.18)
DDE	<LOD (0–2.5)	3 (4.3)	<LOD (0–0)	0 (0)	0.08	0.08 0.48 (0.41 –0.58)
DDD	<LOD (0–10.77)	10 (14.3)	<LOD (0–8.2)	6 (8.6)	0.26	0.28 1.77 (0.60–5.19)
DDT	<LOD (0–17.7)	17 (24.3)	<LOD (0–10.74)	7 (10)	0.03^c	0.02^c 2.88 (1.9–13.3)

n: Number; HCB: hexa-chlorobenzene; HCH: hexachlorocyclohexane; DDE: dichloro-diphenyl-dichloroethylene; DDD: dichloro-diphenyl-dichloroethane, DDT: dichloro-diphenyl-trichloroethane; CI: Confidence interval; LOD: Limit of detection; min: minimum; Max: maximum; χ² : Chi square test.

^aMann–Whitney U test.

^b χ ² test, detection levels in tissue samples are expressed as ng/gm/lipid (total number is not absolute).

^c p Value < 0.05 is statistically significant.

Data of molecular analysis of tissue samples are shown in Table 3. There were higher levels of PI, bcl2, CD95, apoptosis, STm, G2m, annexin (necrosis, early and late apoptosis) in tumor tissue compared to normal samples which were statistically significant (p < 0.05). On the other hand, PIM2, G01, and annexin (viable) were highly detected in normal tissue compared to tumor tissue samples (p < 0.05).

Table 3.

Molecular analysis in malignant versus normal breast tissue samples (n = 140).

Markers	Statistics	Tumor tissue (n = 70)	Normal tissue (n = 70)	p ^a value
PIM1	Mean ± SD	68.27 (19.94)	46.5 (18.44)	<0.001^b
PIM2^a	Median (min–max)	14.6 (1.9–39.9)	35.3 (8.5–75.5)	<0.001^b
PIM3^a	Median (min–max)	4.3 (0.48–59.7)	6.0 (0.8–62.0)	0.2
PIM4^a	Median (min–max)	1.42 (0.15–48.7)	2.12 (0–8.5)	0.6
Bcl-2	Mean ± SD	60.86 (15.41)	38.13 (15.98)	<0.001^b
CD95	Mean ± SD	69.4 (13.95)	38.76 (12.97)	<0.001^b
Apoptosis	Mean ± SD	44.18 (19.97)	3.00 (1.35)	<0.001^b
G01	Mean ± SD	62.5 (23.46)	90.75 (1.97)	<0.001^b
STm	Mean ± SD	17.31 (8.75)	5.67 (2.48)	<0.001^b
G2m	Mean ± SD	7.96 (4.28)	2.8 (1.64)	<0.001^b
Annexin necrosis^a	Median (min–max)	0.12 (0–5.6)	0.01 (0–15.0)	<0.001^b
Annexin viable^a	Median (min–max)	52.6 (38.7–96.9)	88.7 (50.11–98.2)	<0.001^b
Annexin early apoptosis^a	Median (min–max)	16.8 (1.74–59.5)	6.3 (1.03–58.5)	<0.001^b
Annexin late apoptosis^a	Median (min–max)	6.54 (0.45–44.4)	1.45 (0.16–12.3)	<0.001^b

N: Number; SD: standard deviation.

^aMann-Whitney U test or independent t-test.

^b p Value < 0.05 is statistically significant.

Table 4 illustrates the correlation between different molecular analytical data and OC pesticides detected in tumor tissue samples. There was a weak positive statistically significant correlation between G2m and DDD, DDT and methoxychlor (p ≤ 0.05). Also, there was a negative correlation between PIM1 and heptachlor. Annexin necrotic showed a negative correlation with HCB. Methoxychlor showed a negative correlation as regards annexin (early apoptosis), PIM1, and Bcl-2.

Table 4.

Correlation between molecular analytical data and Organochlorine pesticides levels in tumor tissue samples (N = 70).^a

Organochlorine pesticides	Annexin necrotic r (p)	Annexin viable r (p)	Annexin early apoptosis r (p)	Annexin late apoptosis r (p)	G2m r (p)	G01 r (p)	STm r (p)	PIM1 r (p)	PIM2 r (p)	Bc1-2 r (p)	CD95 r (p)	Apoptosis r (p)
HCB	−0.25 (0.03^b)	−0.02 (0.8)	0.09 (0.4)	−0.19 (0.09)	0.01 (0.9)	0.14 (0.23)	0.19 (0.1)	−0.13 (0.24)	−0.12 (0.28)	−0.0.03 (0.76)	0.1 (0.37)	−0.003 (0.9)
α-HCH	−0.01 (0.8)	0.15 (0.19)	0.09 (0.43)	0.07 (0.52)	0.02 (0.83)	0.09 (0.4)	−0.12 (0.3)	0.07 (0.54)	0.02 (0.8)	0.12 (0.30)	0.10 (0.37)	−0.08 (0.48)
γ-HCH	0.05 (0.6)	−0.03 (0.7)	0.01 (0.9)	0.02 (0.8)	0.1 (0.3)	0.12 (0.3)	−0.005 (0.9)	−0.10 (0.3)	0.13 (0.25)	−0.01 (0.89)	0.03 (0.7)	−0.006 (0.9)
β-HCH	0.04 (0.6)	−0.11 (0.3)	−0.01 (0.9)	0.03 (0.7)	0.1 (0.2)	−0.12 (0.3)	−0.19 (0.1)	0.002 (0.9)	−0.09 (0.4)	−0.03 (0.77)	0.08 (0.4)	0.13 (0.2)
Heptachlor	0.003 (0.9)	−0.12 (0.2)	0.2 (0.09)	−0.008 (0.9)	0.2 (0.1)	0.05 (0.6)	0.06 (0.6)	−0.25 (0.02)^b	0.18 (0.1)	−0.07 (0.54)	−0.01 (0.8)	0.03 (0.7)
Aldrin	−0.13 (0.25)	−0.07 (0.5)	0.08 (0.4)	−0.03 (0.7)	0.02 (0.8)	0.04 (0.7)	−0.03 (0.7)	0.16 (0.17)	−0.04 (0.73)	0.04 (0.7)	0.01 (0.9)	−0.07 (0.5)
Chlordane	−0.02 (0.8)	−0.12 (0.2)	−0.03 (0.7)	0.04 (0.7)	0.13 (0.26)	−0.05 (0.6)	−0.12 (0.3)	−0.14 (0.22)	0.08 (0.49)	0.04 (0.7)	0.04 (0.6)	0.07 (0.5)
pp-DDE	0.08 (0.4)	−0.004 (0.9)	0.04 (0.7)	0.07 (0.5)	0.21 (0.07)	0.06 (0.6)	0.04 (0.7)	−0.016 (0.15)	0.1 (0.35)	−0.16 (0.15)	0.15 (0.2)	0.07 (0.5)
pp-DDD	0.1 (0.3)	−0.1 (0.3)	−0.08 (0.4)	0.09 (0.4)	0.3 (0.02)^b	−0.1 (50.2)	−0.15 (0.2)	−0.0.07 (0.5)	0.02 (0.8)	−0.1 (0.4)	0.03 (0.7)	0.07 (0.5)
pp-DDT	−0.02 (0.8)	−0.1 (0.3)	0.01 (0.8)	−0.06 (0.5)	0.24 (0.04)^b	0.02 (0.8)	−0.03 (0.7)	−0.1 (0.3)	0.12 (0.27)	−0.07 (0.5)	0.14 (0.2)	0.02 (0.8)
Methoxychlor	−0.02 (0.8)	−0.04 (0.6)	−0.23 (0.05^b)	−0.11 (0.3)	0.4 (0.001^b)	0.17 (0.15)	0.03 (0.7)	−0.25 (0.03^b)	0.2 (0.07)	−0.22 (0.05^b)	0.1 (0.3)	−0.05 (0.6)

N: number; HCB: hexa-chlorobenzene; HCH: hexachlorocyclohexane; DDE: dichloro-diphenyl-dichloroethylene; DDD: dichloro-diphenyl-dichloroethane; DDT: dichloro-diphenyl-trichloroethane.

^aHeptachlor epoxide and endrin were excluded because its median levels were nil in tumor tissue.

^b p Value < 0.05 is statistically significant.

Discussion

Organochlorines (OCs) are common environmental pollutants that have been linked to various health hazards including cancer. Studies concerning the relation between breast cancer and OCs are too few to provide a conclusion. Some reports did not confirm a possible correlation,¹⁷ while others were positive.¹⁸ Inconclusive results necessitate further investigation. Moreover, mechanisms that explain a potential association still in need to be elucidated.^{17
,
19}

To the best of our knowledge, this work is one of the few studies that assesses the correlation between levels of different OCs and some molecular markers detected in tissue specimens of human breast cancer.

The present findings showed that HCB, chlordane, methoxychlor, and DDT are significantly higher in malignant tissue samples than in the surrounding normal tissue specimens with higher risk of breast cancer in relation to these compounds (OR: 8.5, 5.09, 4.5, 2.88). Endrin and heptachlor epoxide are not detected in any of the tested samples. It is also found that 78.6% of the patients are housewives and 85.7% are residents of rural areas.

The concentration of the detected tissue OCs is lipid adjusted in order to give a better estimation of the burden of exposure as those compounds are lipophilic.²⁰ Serum OC concentrations are not used in the present work as they have some limitations in comparison to adipose breast tissue. Crinnion²¹ reported that serum OC concentrations are only good estimators of recent exposure. They could not indicate long-term exposure, which is affected by current exposure levels and lipid mobilization.

Nearly similar, Salerno et al.²² investigated the association between farming (a proxy for pesticide exposure) and cancer in the Vercelli suburban area (Italy). They reported that farmers showed higher odds for all cancers (OR = 1.459, p < 0.001) including breast cancer.

In line with the current work, it was found that β-hexachlorocyclohexane (β-HCH), HCB, heptachlor, and p,p′-DDE are positively associated with breast cancer risk. Further statistical adjustment for the selected covariates indicated that only β-HCH, heptachlor, and p,p′-DDE remained statistically significant.²³ In addition, Cohn et al.²⁴ indicate an association between breast cancer and DDT exposure as evidenced in the present study.

It was also reported that sera of breast cancer patients have a combination of aldrin, DDE, and DDD and the risk of breast cancer was moderately associated with the later. The authors suggested that organochlorine pesticide mixtures could play a possible role in breast cancer.²⁵

In contrast, other studies did not report an evidence to support a causal association between breast cancer and OCs.^{26
,
27} Comparison between the present work and other studies is difficult and discrepancies may be explained by differences in biological samples used to assess exposure, target populations, study designs, and varied historical exposure levels.

Noteworthy, the well-known correlation between prolonged exposure to estrogens and breast cancer suggests that environmental pollutants, namely, xeno-estrogens such as organochlorines, may play a critical role in the cellular and molecular changes occurring during breast carcinogenesis.²⁸

In the present work, there are higher levels of PIM1, bcl2, CD95, apoptosis, STm, G2m, annexin necrosis, and annexin (early and late apoptosis) in tumor tissue compared to normal tissue (p < 0.05). There is a significant correlation between annexin (necrotic) and HCB as well as between G2m and methoxychlor, DDD, and DDT and between annexin (early apoptosis) and methoxychlor. While a weak negative correlation is noticed regarding heptachlor, methoxychlor, and PIM1 and between Bcl-2 and methoxychlor.

Apoptosis is a type of cell death that plays a role in various pathological conditions as well as in regulation of tumor progression. Induction of apoptosis is actively used by the immune system to eliminate abnormal cells and to prevent the tumor development. Higher incidence of cancers is related to disruption of apoptotic mechanisms.²⁹

Many molecular markers play a crucial role in carcinogenesis. For example, Bcl-2 is involved in regulation of apoptosis. It may have a role in breast cancer by raising the apoptosis threshold.³⁰ In addition, CD95 is a type I cell surface glycoprotein that transduces apoptosis in sensitive cells. Increased CD95 has been reported in various cancers.³¹ On the other hand, annexins are involved in cell growth and proliferation and alteration of their expression is associated with cancer.³²

AlegrÍa-Torres et al.³³ documented that DDT and its metabolites are able to induce apoptosis in many cell lines such as human peripheral blood mononuclear cells. Heptachlor was also reported to inhibit apoptosis and thus triggering proliferation of rat hepatocytes.³⁴ A chronic apoptotic stimulus can lead to a high rate of tissue regeneration with elevation of the risk of mitotic errors that may predispose to cancer development³⁵ which could explain the findings in the current work.

Additionally, it was reported that HCB causes an imbalance between cell death and cell proliferation, resulting in an increased number of apoptotic cells. Consequently, HCB was suggested to be a tumor co-carcinogen in rat mammary gland.³⁶ It was claimed that HCB-induced apoptosis is mediated through increased CD95 and caspase-8.³⁷

Moreover, it was observed that DDT constituents (i.e. o,p′-DDT, o,p′-DDE, p,p′-DDT, and p,p′-DDE) induced cell proliferation.³⁸ In addition, there is an evidence that these chemicals might also cause cancer via other mechanisms, including the induction of enzymes that produce genotoxic intermediates and DNA damage,³⁹ and disruption of the epigenomic landscape in cancers.⁹

Chatterjee et al.⁴⁰ also reported that pesticides induced a significantly higher level of apoptotic receptors expression (annexin V and CD95) in addition to an increased percentage of cells in the G0/G1 phase compared to the control group in the bone marrow progenitor cells. However, there was a significant reduction of the cell fraction in the S/G2/M phase, indicating an enhanced level of stem/progenitor cellular apoptosis and impaired cell proliferation following pesticide exposure. This suggests a higher rate of premature cell senescence and restricted cell cycle pattern. The inhibition of cell replication and cell cycle arrest at the G0/G1 phase also signifies the toxic effect of pesticides.⁴¹

The present findings could partly explain the molecular mechanisms induced by organochlorine pesticides to be involved in the pathogenesis of breast cancer. It was reported that these pesticides even at very low levels can cause cancer through their tumor-promoting properties, immunosuppressive effects, endocrine-disrupting impacts, and potential estrogenic activity leading to disturbance of the normal estrogen–progesterone balance^42
–44 and hence these environmental toxic pollutants could contribute to the risk of breast cancer.

Additionally, it was shown that exposure to OCs may modulate cell-cycle control and other molecular parameters in human mammary cells.^28,45 In other words, certain molecular changes have to occur in epithelial cells to convert normal cells into tumor cells. Thus, OCs may promote oncogenesis and cancer development through enhanced cell proliferation, decreased cell-cycle arrest, and decreased apoptosis⁴⁶ as evidenced in the current work.

Limitations of our study do exist, mainly that there are no adipose tissue samples are available from healthy controls as it seems unethical to take a tissue biopsy from healthy individuals.

In conclusion, the higher levels of organochlorine pesticides in the tissue specimens of breast cancer and the resultant molecular dysfunction highlight a possible association. Future cancer studies should analyze the interaction between those environmental contaminants with endogenous hormones and with cellular dysfunction. No safe limits of exposure to OCs have yet been determined. Hence, continued exposure to these persistent pollutants should be considered especially in developing countries where alarming rates of BC incidence exist.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This is a research project funded by Mansoura University Institution.

References

Andreotti

Hou

Laura

. Body mass index, agricultural pesticide use, and cancer incidence in the Agricultural Health Study cohort. Cancer Causes Control 2010; 21(11): 1759–1775.

Fenik

Tankiewicz

Biziuk

. Properties and determination of pesticides in fruits and vegetables. Trends Anal Chem 2011; 30: 814–826.

Kamel

Moussa

Abonorag

. Occurrence and possible fate of organochlorine pesticide residues at Manzala Lake in Egypt as a model study. Environ Monit Assess 2015; 187: 4161–1463.

Abou Arab

AAK

Abou Donia

Enb

. Chemical composition, metals content and pesticide residues in raw, pasteurized and UHT milk and their dietary intake. J Egypt Soc Toxicol 2008; 39: 111–121.

Sharaf

Elserougy

Hussein

. Organochlorine pesticides in breast milk and other tissues of some Egyptian mothers. American-Eurasian J Agric Environ Sci 2008; 4: 434–442.

Nabi

Ahmad

Ghufran . Link between the occurrence of various forms of cancer and chronic exposure to pesticides. Basic Res J Med Clin Sci 2014; 3(12): 131–135. Available at: http://www.basicresearchjournals.org/medicine/.

Ferlay

Soerjomataram

Ervik

. GLOBOCAN 2012 v1.0, cancer incidence and mortality worldwide: IARC Cancer Base No. 11. Lyon: International Agency for Research on Cancer, http://globocan.iarc.fr/2013 (accessed 10 June 2014).

World Cancer Research Fund International. http://www.wcrf.org/index.php (2014, accessed 16 January 2016).

Knower

Leung

. Endocrine disruption of the epigenome: a breast cancer link. Endocr Relat Cancer 2014; 21: T33–T55.

10.

Petreas

Smith

Hurley

. Distribution of persistent, lipid-soluble chemicals in breast and abdominal adipose tissues: lessons learned from a breast cancer study. Cancer Epidemiol Biomarkers Prev 2004; 13: 416–424.

11.

Spyratos

. DNA content and cell cycle analysis by flow cytometry in clinical samples: application in breast cancer. Biol Cell 1993; 78: 69–72.

12.

Ormerod

Tribukait

Giaretti

. Consensus report of the task force on standardization of DNA flow cytometry in clinical pathology. Anal Cell Pathol 1998; 17: 103–110.

13.

Cifone

De Maria

Roncaioli

. Apoptotic signaling through CD95 (Fas/APO-1) activates an acidic sphingomyelinase. J Exp Med 1994; 180: 1547–1552.

14.

Yoshino

Ami

Terao

. Upgrading of flow cytometric analysis for absolute counts, cytokines and other antigenic molecules of Cynomolgus Monkeys (Macaca fascicularis) by using anti-human cross-reactive antibodies. Exp Anim 2000; 49: 97–110.

15.

Dragowska

Lopes de Menesez

Sartor

. Quantitative fluorescence cytometric analysis of Bcl-2 levels in tumor cells exhibiting a wide range of inherent Bcl-2 protein expression: correlation with western blot analysis. Cytometry 2000; 40: 346–352.

16.

Akl

Ayoub

Mohyeldin

. Olive phenolics as c-Met inhibitors: (−)-oleocanthal attenuates cell proliferation, invasiveness, and tumor growth in breast cancer models. Plos One 2014; 9(5): e97622.

17.

Park

J-H

Cha

. exposure to dichlorodiphenyltrichloroethane and the risk of breast cancer: a systematic review and meta-analysis. Osong Public Health Res Perspect 2014; 5(2): 77e–84e

18.

Del Pup

Mantovani

Luce

. Endocrine disruptors and female cancer: informing the patients (Review). Oncol Rep 2015; 34(1): 3–11.

19.

Uysal

Bozcuk

Karakılınc

. Pesticides and cancer: the first incidence study conducted in turkey. J Environ Pathol Toxicol Oncol 2013; 32(3): 245–249.

20.

Koppen

Covaci

Van Cleuvenbergen

. Persistent organochlorine pollutants in human serum of 50–65 years old women in the Flanders Environmental and Health Study (FLEHS). Part 1: Concentrations and regional differences. Chemosphere 2002; 48: 811–825.

21.

Crinnion

. Chlorinated pesticides: threats to health and importance of detection. Altern Med Rev 2009; 14: 347–359.

22.

Salerno

Carcagnì

Sacco

. An Italian population-based case-control study on the association between farming and cancer: are pesticides a plausible risk factor? Arch Environ Occup Health 2016; 71(3): 147–156.

23.

Arrebola

Belhassen

Artacho-Cordón

. Risk of female breast cancer and serum concentrations of organochlorine pesticides and polychlorinated biphenyls: a case–control study in Tunisia. Sci Total Environ 2015; 520: 106–113.

24.

Cohn

Merrill

Krigbaum

. DDT exposure in utero and breast cancer. J Clin Endocrinol Metab 2015; 100(8): 2865–2872.

25.

Boada

Zumbado

Henriquez-Hernandez

. Complex organochlorine pesticide mixtures as determinant factor for breast cancer risk: a population-based case control study in the Canary Islands (Spain). Environ Health 2012; 11: 28.

26.

Farooq

Joshi

Nookala

. Self-reported exposure to pesticides in residential settings and risk of breast cancer: a case–control study. Environ Health 2010; 9: 30. DOI: 10.1186/1476-069X-9-30. PMCID: PMC2909990 (accessed 15 March 2015).

27.

Recio-Vega

Velazco-Rodriguez

Ocampo-Gomez

. Serum levels of polychlorinated biphenyls in Mexican women and breast cancer risk. J Appl Toxicol 2011; 31: 270–278.

28.

Fucic

Gamulin

Ferencic

. Environmental exposure to xenoestrogens and oestrogen related cancers: reproductive system, breast, lung, kidney, pancreas, and brain. Environ Health 2012; 11: S8.

29.

Olimón-Andalón

Aguilar-Lemarroy

Ratkovich-González

. Proapoptotic CD95 L levels in normal human serum and sera of breast cancer patients. Tumor Biol 2015; 36: 3669–3678.

30.

Guiro

Patel

Greco

. Investigating breast cancer cell behavior using tissue engineering scaffolds. Plos One 2015; 10(3): e0118724.

31.

Peter

Budd

Desbarats

. The CD95 receptor: apoptosis revisited. Cell 2007; 3: 447–450.

32.

Fatimathas

Moss

. Annexins as disease modifiers. Histol Histopathol 2010; 25: 527–532.

33.

Alegría-Torres

Díaz-Barriga

Gandolfi

. Mechanisms of p, p′-DDE-induced apoptosis in human peripheral blood mononuclear cells. Toxicol In Vitro 2009; 23:1000–1006.

34.

Okoumassoun

Averill-Bates

Gagne

. Assessing the estrogenic potential of organochlorine pesticides in primary cultures of male rainbow trout (Oncorhynchus mykiss) hepatocytes using vitellogenin as a biomarker. Toxicology 2002; 178: 193–207.

35.

Pikarsky

Porat

Stein

. NF-κB functions as a tumor promoter in inflammation-associated cancer. Nature 2004; 431: 461–466.

36.

Randi

Cocca

Carbone

. Hexachlorobenzene is a tumor co-carcinogen and induces alterations in insulin-growth factors signaling pathway in the rat mammary gland. Toxicol Sci 2006; 89: 83–92.

37.

Ogasawara

Watanabe-Fukunaga

Adachi

. Lethal effect of the anti-Fas antibody in mice. Nature 1993; 364: 806–809.

38.

Andersen

Andersson

Arnold

. Comparison of short-term estrogenicity tests for identification of hormone disrupting chemicals. Environ Health Perspect 1999; 107(1): 89–108.

39.

Yanez

Borja-Aburto

Rojas

. DDT induces DNA damage in blood cells. Studies in vitro and in women chronically exposed to this insecticide. Environ Res 2004; 94: 18–24.

40.

Chatterjee

Basak

Chaklader

. Pesticide induced alterations in marrow physiology and depletion of stem and stromal progenitor population: an experimental model to study the toxic effects of pesticide. Environ Toxicol 2011; 29(1): 84–97.

41.

Iglesias

Cairney

Ferrier

. Annexin A8 identifies a subpopulation of transiently quiescent c-kit positive luminal progenitor cells of the ductal mammary epithelium. PLOS One 2015; 10(3): e0119718.

42.

Tabrez

Priyadarshini

Priyamvada

. Gene–environment interactions in heavy metal and pesticide carcinogenesis. Mut Res 2014; 760: 1–9.

43.

Pestana

Teixeira

Faria

. Effects of environmental organochlorine pesticides on human breast cancer: putative involvement on invasive cell ability. Environ Toxicol 2015; 30(2): 168–176.

44.

Shanle

. Endocrine disrupting chemicals targeting estrogen receptor signaling: identification and mechanisms of action. Chem Res Toxicol 2011; 24(1): 6–19.

45.

Rivero

Luzardo

Henríquez-Hernández

. In vitro evaluation of estrogenic/androgenic activity of the serum organochlorine pesticide mixtures previously described in a breast cancer case–control study. Sci Total Environ 2015; 537: 197–202.

46.