Role of organophosphorous pesticides and acetylcholine in breast carcinogenesis

ABSTRACT

Breast cancer is the leading cause of cancer-related death in women worldwide. Several studies have addressed the association between cancer in humans and agricultural pesticide exposure. Evidence indicates that exposure to organophosphorous pesticides such as parathion and malathion occurs as a result of occupational factors since they are extensively used to control insects. On the other hand, estrogens have been considered beneficial to the organism; however, epidemiological studies have pointed out an increased breast cancer risk in both humans and animals. Experimental female rat mammary gland cancer models were developed after exposure to parathion, malathion, eserine, an acetylcholinesterase inhibitor, and estrogen allowing the analysis of the signs of carci- nogenicity as alteration of cell proliferation, receptor expression, genomic instability, and cell metabolism in vivo and in vitro. Thus, pesticides increased proliferative ducts followed by ductal carcinoma; and 17β-estradiol increased proliferative lobules followed by lobular carcinomas.

The combination of both pesticides and either eserine or estrogen induced tumors with both types of structures followed by mammary gland tumors and metastasis to the lung and kidneys after 240 days of a 5-day treatment. Studies also showed that these pesticides and eserine decreased three to five times the acetylcholinesterase activity in the serum compared to controls whereas terminal end buds increased in number, being inhibited by atropine. Genomic instability was analyzed in such tissues (mp53, CYP1A2, c-myc, c-fos, ERα, M2R) and pesticides increased protein expression that was stimulated by estrogens but inhibited by atropine. Eserine also transformed the epithelium of the rat mammary gland in the presence of estrogen and increased the number of terminal end buds after treatment inducing mammary carcinomas. Then, enzymatic digestion of such structures gave rise to cells with increased DNA synthesis and induced anchorage independence. Thus, there were changes in the epithelium of the mammary gland influencing breast carcinogenesis. Furthermore, these substances and acetylcholine also showed the signs of carcinogenicity in vitro as cell proliferation, receptor expression (ERα, ErbB2, M2R), genomic instability (c- myc, mp53, ERα, M2R), and cell metabolism. A unique cellular model is also presented here based on the use of MCF-10 F, a non-tumorigenic cell line that represents a valuable clinically translatable experimental approach that identifies mechanistic links for pesticides and estrogen as suspect human carcinogenic agents.

1. Introduction

Breast cancer is a major health threat and it is the leading cause of cancer-related death in women worldwide [1]. The Global Cancer Project regarding the incidence and mortality of breast cancer showed that accounted for 24.2 % of all new cancer cases and 13 % of all cancer deaths among women worldwide in 2018 [2]. Its etiology seems to be associated with internal (inherited mutations, hormones) and external (radiation, smoking, and diet) factors [3]. It is a heterogeneous disease not only at the molecular level but also in its pathologic and clinical aspects [4,5]. The mammary epithelium is composed of luminal cells, which express receptors to respond to ovarian hormones, including estrogen receptor alpha (ERα) then it is considered a typical marker of the luminal epithelial phenotype in breast cancer cells, and a good indicator for endocrine therapy of breast cancer [6]. The normal proliferating rat mammary epithelial cells rarely express ERα; whereas estrogen receptor beta (ERß) can be detected in 30–47 % of proliferating epithelial cells [7]. Also, 17β-estradiol/ERα signaling promotes the differentiation of mammary epithelia along a luminal/epithelial lineage, in part through transcriptional activation of luminal/epithelial-related transcription factors [8].

Analyses of cancer deaths indicated that women usually have triple- negative breast carcinoma, a molecular subtype of breast cancer tumor that does not express estrogen, estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER2) [4, 9–11]. Tamoxifen and Herceptin have been the most successful treat- ment of breast cancer patients with positive ER-α and Her-2 [12]. These markers have not only shown the usefulness of therapy in positive cases (60–80 %) in the clinic but they have also indicated the aggressiveness of hormone-receptor negative cases, and such cases call for the use of chemotherapeutic agents [13–15].

The association between cancer in humans and agricultural pesticide exposure has been addressed [16]. Environmental factors are among the major influencing components causing an increase in the incidence of breast cancer, evidence indicates that exposure to pesticides occurs as a result of occupational (e.g. spraying fields) work as well as environ- mental factors such as drinking water and food [3]. Pesticides constitute a diverse class of xenobiotics encountered frequently in the environment that are extensively used for the protection of crops and for increasing the yield of agricultural products. Several organophosphorous pesticides (OPs) are widely used in public health programs in all market sectors (i. e., agriculture, home and garden, industry, commerce, and government) in the USA and abroad and currently comprise approximately 35 % of insecticides [17].

Increased cancer risk has been associated with several OPs in epidemiological studies, including case-control studies in the USA [18], Canada [19], and Italy [20], nested case-control studies of structural pest control workers in Florida [21] and farmworkers in California [22], and among licensed pesticide applicators in the prospective Agricultural Health Study (AHS) cohort [23]. Evidence from human studies has been scarce but studies on OPs linked to cancer risk through epidemiological studies have been largely conducted in predominantly male populations; however, there are also prospective studies examining the use of indi- vidual OPs and the risk of multiple cancer sites in women; the use of specific OPs and cancer incidence among female spouses of pesticide applicators was evaluated in the prospective AHS cohort where the use of OPs was confirmed in an increased risk of several hormonally-mediated cancers like breast and ovary [23]. Thus, many of the cancer sites examined included breast, ovary, uterus, thyroid, and lung as major public health concern since they are commonly diagnosed and are important contributors to cancer deaths among women [24]. In vivo and in vitro studies have shown that such substances (DDT, poly- chlorinated biphenyls, 4-nonylphenol,4-octylphenol) can promote mammary cancer [25,26].

Following a new approach for analyzing carcinogens developed by the International Agency for Research on Cancer (IARC) and previous studies, the substances reviewed in this study have been classified based on 10 common characteristics that reflect the properties of a cancer- causing agent, such as ‘is genotoxic’, ‘is immunosuppressive’ or ‘mod- ulates receptor-mediated effects,’ and are different from the hallmarks of cancer, which are the properties of tumors [27–29]. Several com- pounds have been established as carcinogens, according to IARC criteria, which means that they are capable of inducing malignant transformation of normal cells in animals and humans, generally, four groups define the carcinogenicity of a chemical: group 1 refers to compounds that are carcinogenic to humans; group 2 refer to com- pounds that are probably (Group 2A) and possibly (Group 2B) carci- nogenic to humans, where the term probably carcinogenic means a higher level of evidence than possibly carcinogenic; group 3 refers to compounds that are not classifiable as to its carcinogenicity to humans, and group 4 includes compounds that are probably not carcinogenic to humans [27–29].

OPs are based on the common chemical structure of phosphoric acid esters, among them parathion (P) [O, O-Diethyl O-(4-nitrophenyl)- phosphorothioate)] [30,31] and malathion (M) (O,O-dimethyl-S- 1,2-bis ethoxy carbonyl ethyl phosphorodithionate) are similar across species, rapidly absorbed, distributed, and metabolized to their bioactive metabolite, paraoxon, and malaoxon, respectively humans [32].

Eserine (Ese), (3,S-cis)-1, 2, 3, 3, 8, 8 -Hexahydro-1, 3, 8-trimethyl- pyrrolo (2,3-b) indol-5-ol methylcarbamate, is an ester obtained from the Calabar Bean, the seeds of the vine Physostigma venenosum [31]. It is an acetylcholinesterase (AChE) inhibitor, as M and P increasing acetylcholine (ACh) availability which in turn could stimulate cholin- ergic receptors producing nicotinic and muscarinic effects as secretions and contractions in the glands [31]. It is incorporated through the epithelium of the skin, mouth, and respiratory tract and responsible for the hydrolysis of choline esters found in the body, including the ACh present at the cholinergic synapse and used as a miotic drug [33–35]. Under normal physiologic conditions, the cholinergic receptors are usually operative through ACh action, and atropine, a para- sympatholytic alkaloid known to occupy the muscarinic cholinergic receptors, interferes with the action of ACh and used as an antidote to ACh inhibitors [30,31,33].

Mammary gland development is associated with its topography and is modulated by age, hormones, and parity history, it is formed by a single main lactiferous duct that divides into secondary ducts at birth called terminal end buds (TEBs) and alveolar buds (ABs) that evolve into lobules [35,36]; these studies characterized the TEBs and demonstrated a high proliferative growth fraction of the cell cycle with high suscep- tibility to neoplastic transformation attributed to the cell kinetic of the epithelium, therefore, considered the site of origin of human breast carcinomas. Thus, a model was developed exposing Sprague-Dawley rats to 7,12-dimethylbenz[a]anthracene (DMBA), this chemical carcinogen induced 100 % of mammary carcinomas with a latency period of 86 days [37–41].

The present work aims to summarize those experimental rat mam- mary gland models to study the role of P, M, Ese, 17β-estradiol (E2), ACh, and atropine in breast carcinogenesis in vivo and in vitro.

2. The effect of parathion, malathion, and eserine on breast carcinogenesis in vivo

An experimental rat mammary gland cancer model was developed with female Sprague-Dawley rats, 39 days old that received injections of a saline solution, Ese, P, M, At, and a combination of substances twice a day for 5 days to study the stepwise transformation of normal mammary gland epithelial structures into malignant ones [42]. Results showed that there was no significant difference in the density of TEBs of the mammary gland in the P-, M-, Ese-treated rats or the combination in comparison to controls in 21-day-old rats, and ABs were absent. How- ever, in 44-day-old animals, such treatment significantly increased TEB density whereas AB density decreased as seen in whole-mount prepa- rations and histological examination of mammary tissues where the number of the epithelial layers increased followed by mammary carci- nomas. Atropine alone or in combination with the pesticides or Ese caused inhibition of TEB density in comparison with controls and an increase in AB density similar to that of controls. OPs and Ese induced changes in the mammary gland epithelium influencing carcinogenesis and also affected the nervous system by increasing the cholinergic stimulation.

3. The effect of malathion and estrogen on breast carcinogenesis in vivo

Epidemiological studies have pointed to an increased breast cancer risk in both humans and animals [45–49]. Estrogens are known to control the growth of many carcinomas in experimental animals and humans; however, estrogens have been considered beneficial to the organism based on a variety of hormonal effects demonstrated by a strong relationship between endogenous estrogen levels and such risk [50–55]. It seems that estrogen risk increases with continuous doses of E2 and with the length of treatment [56]; however, even slightly elevated levels of circulating estrogens seem to be a risk factor for breast cancer [50,51,57]. This role of endogenous estrogen in human breast carcinogenesis is supported by finding high levels of it in serum or urine [52,53].

It can be hypothesized that a possible mechanism for breast cancer induction is the consequence of excessive estrogenic stimulation pro- moting cell proliferation of normal breast epithelial tissue [58]. In other words, errors in cell division can develop malignant phenotype errors such as DNA copying errors, and translocations [59].

Another experimental mammary gland model was developed in fe- male Sprague-Dawley rats, and they were injected M twice a day for 5 days and sacrificed after 10, 20, 30, 60, 90, 124, 240, and 400 days [43, 44]. When animals were treated with M the TEBs increased in number and as time progressed ducts were formed with numerous epithelial layers per square millimeter in comparison to Ct causing malignant phenotypic alterations. Such structures were referred to as ducts in stage of proliferation (dsp/mm2) until tumors were formed. Pathology of such tumors revealed a similarity to ductal carcinomas as described by the World Health Organization (WHO) [36]. Under the same conditions, the effect of E2 was studied, progressive alterations in lobules were observed and the density of TEBs per square millimeter decreased while lobules became markedly abnormal, with large and dilated numerous congested tubular structures filled with pink deposits that increased in size and referred to as secretory lobules [43,44]. Pathology of such tumors revealed lobular carcinomas similar to those described for patients by WHO [36]. The combination of both substances induced greater cellular changes than either treatment alone indicated by increased proliferative ducts and secretory lobules inducing mammary gland tumor formation. Fig. 2A shows whole-mount preparation of rat mammary gland: a) Ct, b) M-, c) E2-, and d) combination of both substances-treated rats. A cross-section of a) normal ducts and alveoli, b) lobules of rat mammary gland formed by the effect of E2, c) by the combination of M plus E2 with higher magnification, and d) cross-section of lobular rat mammary gland tumor immunoperoxidase stained with M2R can be observed in Fig. 2B. The effect of M, E2, and combination of both on a) bars that show the average number of ducts in stage of proliferation (dsp/mm2) and b) and the average number of lobules per mm2 after 30, 124, 240 days of a 5- day treatment compared with Ct is found in Fig. 2C. Fig. 2D shows the effect of M, E2, and combination of both on mp53, CYP1A2, c-myc, and c-fos protein expression in rat mammary gland tissues after 240 days in comparison to Ct by a) western blot in MCF-10 F cell line and b) its quantification based on the relative unit of densitometry of such expression.

A study on the effect of M, E2, and combination of both on 2-catechol estrogen (2-CE) and 4-catechol estrogen (4-CE) concentrations in blood of rats showed that there was no formation of any characteristic peak in Ct, M-, and E2-treated animals [62]; however, the combination of both induced the two peaks corresponding coincident with mammary gland tumor formation after 240 days suggesting an important biomarker in blood for mammary gland cancer as seen in Fig. 3A, tumor formation analyzed by High-Performance Liquid Chromatography (HPLC) with electrochemical analysis of serum of animals: a) Ct, b) M-, c) E2- d) M + E2-treated animals after 240 days of a 5-day treatment. E2 alone and M + E2-treated rats showed characteristic peaks of 2-CE and 4-CE whereas animals treated with M alone showed characteristic peaks.

An epidemiological study indicated that E2 modulated the signs of carcinogenicity in vivo altering metabolism as shown by CE formation with greater risk for breast cancer than the high 16- α hydroxylation of estrogens [50]. In general, the major CE metabolites of the estrogens are the 2-CE and 4-CE and such conversion occurs through the cytochrome P450 family, activating many pro-carcinogenic chemicals [50,63]. The pathway to 4-CE is the one that leads to the endogenous carcinogenic of CE 3, 4-quinones [64–67]. Studies done to detect 31 estrogen metabo- lites in a biopsy from tissues of breast cancer patients by HPLC with electro-chemical analysis reported that 4-CE was more common than 2-CE in breast cancer [67]. It is known that the oxidation of the catechols gives rise to reactive quinones causing DNA damage and redox cycling to generate reactive oxygen species that can cause oxidative damage [68].

4. The effect of eserine and estrogen on breast carcinogenesis in vivo and in vitro

Another rat mammary gland experimental model was developed with Ese in vivo and in vitro. Female Sprague-Dawley rats, 44 days of age, were treated with Ese for 10 days followed by E2 for 30 days [69]. Ese and E2 play a crucial role in cell proliferation and transformation of rat immunostained with antibodies for c-Myc (1D), mp53 (1E), ERα (1 F) as seen in a) normal ducts, treated with b) M, c) At, and d) combination of both. M significantly greater than Ct, At, and a combination of both after 60, 90, 124, and 240 days. G Effect of M and At on protein expression in histological cross-section of rat mammary gland tissues immunostained with antibodies for M2R as seen in a) normal ducts, treated on mp53, CYP1A2, c-myc, and c-fos protein expression in rat mammary gland tissues after 240 days in comparison to Ct by a) western blot and b) its quantification based on the relative unit of densitometry of such an expression. M, E2, and M plus E2 protein expression significantly greater than Ct (P < 0.05). E-H Effect of M and E2 on protein expression in histological cross-section of rat mammary gland tissues immunostained with antibodies for c-Myc (2E), mp53 (2 F), ERα (2 G), and M2R (2 H) in a) normal ducts, treated with b) M, c) E2, and d) combination of both. Tissues derived from animals were obtained out of a repository of paraffin blocks performed in a previous study [43,44,61].

The total number of cells by the effect of parathion (P), and atropine (At), P plus At; acetylcholine (ACh), ACh + At; E2, and ACh plus E2 in comparison with MCF-10 F (Ct) cell line after 7 days in culture; P, Ach, Ach + E2 significantly greater than Ct, At, P + At, and E2 (P < 0.05), and b) invasive index (%) of Ct, E2, P, P + E2, M, M + E2 scored 20 h after plating onto matrigel basement membranes using modified Boyden’s chambers constructed with multi-well cell culture plates and cell culture inserts (P < 0.05). D Representative images of a) MCF-10 F cell line (Ct) under phase contrast and transmission electron microscopes. b) Colonies obtained by anchorage-independency assay: Ct, E2, P, M, P + E2; M + E2, Ach, and At cell lines. E Effect of a) E2, P, and combination on c-Ha-ras, mp53, and ErbB2 protein expression by western blot in comparison with Ct; b) E2, M, M + E2, P, P + E2 on RAS1: c-Ha-ras gene expression (11p14.1), at codon 12 and 61 and c) P, P + E2, M + E2, E2, M in comparison with Ct on c-myc in MCF-10 F cell line. F a) A graph that shows the effect of P, E2, and a combination of both on ERα, ERß, and ErbB2 protein expression. P, P + E2 significantly greater than Ct and E2 (P < 0.05) and b) Representative images of immunofluorescence staining; Results were obtained by computational quantification of immune-fluorescence-stained images coupled with a confocal microscope. Tissues derived from animals were obtained out of a repository of paraffin blocks performed in a previous study [43,81].

the number and size of TEBs after 10 day-treatment in vivo in comparison to Ct. There was an increase in the number of cells per duct by Ese demonstrated by histomorphometric analysis of mammary glands. Furthermore, there was an increase in the number of cells per ducts in the Ese plus E2 treatment in comparison to Ct. However, there was a decrease in the number of lobules per duct and alveoli per lobules after 40-day Ese-treatment. Then E2 induced adenocarcinomas in 2 out of 12 rats with a latency of 180 and 245 days. Cells were obtained from such tissues after enzymatic digestion of mammary glands. Such enzymatic digestion of TEBs and ABs indicated that cells of ABs did not grow in 14-day cultures. However, cells derived from TEBs from Ese-treated-rats significantly increased DNA synthesis and induced anchorage indepen- dence in comparison to Ct. Ese induced changes in the DNA synthesis, this was demonstrated in the rat submandibular gland after a 5-day in vivo treatment [70]. Such results also confirmed that TEBs are major targets related to rat mammary carcinogenesis and estrogen can be initiator and promoter in this process.

Studies confirmed that E2 plays an important role in regulating the growth of normal, premalignant, and malignant cell breast epithelial cells [71] and it increased the growth fraction of human breast epithelial cells, that is the total compartments of the cells engaged in the cell cycle, DNA-LI, the length of the S phase, and the total length of the cell cycle after 5 days in culture in comparison to Ct [72], E2 also increased DNA synthesis shown by DNA-LI and [3H]-thymidine incorporation into DNA in organ culture of fibrocystic disease lesions as well as carcinomas in situ of premenopausal women in culture [73]. Furthermore, E2 promotes proliferation, migration, and invasion in breast cancer cell lines even at low concentration through ERα [74], whereas high concentration induced apoptosis independent of the presence of such receptor [75] and long-term exposure increased the risk of breast cancer in several animal species, as well as in women [76].

5. The effect of OPs, E2, and ACh on breast carcinogenesis in vitro

A previous study has demonstrated that the MCF-10 F human breast epithelial cell line retains the characteristics of a normal breast mam- mary epithelium [77] and it has one of the signs of carcinogenicity such as immortalization as defined by IARC [78]. Even though such cell line is ER-negative, very sensitive to E2, and it increases cell proliferation after 10 days [79].

Increased proliferation is a sign of carcinogenicity and it was demonstrated by the effect of P and when is combined with atropine or alone decreased such effect [80,81]. ACh alone and combined with E2 also increased cell proliferation, which was corroborated by anchorage-independent growth whereas atropine decreased such effect. Fig. 3C a) shows the total number of cells by the effect of P, At, P + At; ACh, ACh + At; E2, and ACh plus E2 in comparison to MCF-10 F (Ct) cell lines after 7 days in culture, and b) invasive index (%) of Ct, E2, P, P + E2, M, M + E2 scored 20 h after plating onto matrigel basement mem- branes using modified Boyden’s chambers constructed with multi-well cell culture plates and cell culture inserts. Fig. 3D shows representative images of a) MCF-10 F cell line (Ct) under phase contrast and transmission electron microscopes, b) colonies obtained by anchorage-independency assay by the effect of E2, P, M, P + E2; M + E2, ACh, and atropine (At) in comparison to Ct. Fig. 3E shows the effect of a) E2, P, and combination on c-Ha-ras, mp53, and ErbB2 protein expression by western blot in comparison to Ct, b) E2, M, M + E2, P, P + E2 on RAS1: c-Ha-ras gene expression (11p14.1) at codon 12 and 61, and c) on c-myc in P, P + E2, M + E2, E2, and M groups in comparison to Ct.

The activation of membrane receptors is the main molecular mech- anism underlying the action of chemicals involved in breast cancer development. It seems that P, M, and E2 modulate receptor expression since ERα, ERß, and ErbB2 protein expression increase in the MCF-10 F cell line when compared to controls as seen in Fig. 3F a) and b) representative images of immunofluorescence staining, results were obtained by computational quantification of immune-fluorescence stained images coupled with a confocal microscope.

The term non-neuronal cholinergic system was introduced to point out the idea that ACh is present in cells independent of neurons, and that it can act on cells themselves or in neighboring cells [99]. Many epithelial cells express a cholinergic autocrine loop in which ACh acts as a growth factor to stimulate cell proliferation [100], using M3R to activate AKT/MAPK signaling through the epidermal growth factor re- ceptor (EGFR) in human colon cancer cells, which were inhibited by At, a cholinergic antagonist [90,93]. However, not only ACh has been found in non-neuronal tissues, but also the enzyme AChE that has been asso- ciated with processes such as cell adhesion, differentiation, prolifera- tion, and apoptosis [101].

ACh has been associated with pathways involving mitogen-activated protein kinase (MAPK), phosphoinositide-3-kinase (PI3K), and Ras ho- molog gene family [102,103], modulating different processes such as cell proliferation and tumorigenesis in the breast, lung, colon, among other organs [104]. Thus, it has been hypothesized that ACh production can play a key role in regulating tumor cells or tissues that are not innervated by cholinergic neurons (e.g., breast and bronchial epithelial cells) [84].

Besides ACh [104–107] numerous neurotransmitters have been associated with cancer as signaling molecules in tumor cells, among them, norepinephrine [108], dopamine [109], gamma-aminobutyric acid [110].

The M3R antagonists inhibit growth in melanoma, lung, gastric, colon, pancreatic, ovarian, prostate, brain, and breast cancer in vivo as well as in vitro [84,90,92,118,122–124]. Cancers derived from these tissues similarly express a cholinergic autocrine loop and ACh secreted by cancer or neighboring cells interact with M3R expressed on the cancer cells to stimulate tumor growth. However, ACh does not only promote proliferation through M3R but also cell migration of small-cell lung cancer [122,125,126].

Novel observations indicate that the activation of muscarinic acetylcholine receptor expression in cancer plays an essential role in many pathological processes [84,112] since they are present in human tumors as colon, prostate, pancreas, lung, brain, stomach, blood (lym- phoma and leukemia), ovaries, and breast and specifically in cell lines derived from cancer of the breast [123], brain [127], colon [93], lung [92,100,122], ovary [128], pancreas [129], prostate [130,131], skin [132,133], stomach [134], and uterus [135]. Furthermore, ACh can induce cell proliferation, through modulation of EGFR probably through the M3R to activate the EGFR signaling [83,94,98,104,136] as well as differentiation, apoptosis, and cell to cell interaction [137].

A summary of the role of exposure to OPs, E2, and ACh is presented in Fig. 4D. OPs, E2, Ese, and ACh act as autocrine growth factors stim- ulating cell proliferation [80,138,139] and inducing tumor formation through muscarinic receptors as demonstrated by in vivo and in vitro studies. Exposure to OPs and E2 does not only has a significant effect on the normal breast but also the Ese since it induced changes in the epithelium of the mammary gland influencing breast carcinogenesis, which affected the nervous system by increasing the cholinergic stim- ulation [69]. It was first demonstrated that an increase in ACh levels induced by OPs was associated with mammary gland tumor formation in vivo. The mechanisms included AChE inhibition among other characteristics.

This study presents several cellular models that show the signs of carcinogenicity by the effect of M, P, Ese, E2, and ACh demonstrating features of cell transformation in vivo as histological changes and tumor formation from tissues derived from rats besides increased cell prolif- eration, anchorage independence, invasive capabilities, modulation of receptor expression, genomic instability as epithelial-to-mesenchymal transition (EMT) [44,80,81,138,140–142], oxidative stress [141], and cell metabolism [43,139,141]. This review also presents a cellular model based on the use of MCF-10 F, a human tissue-derived, immortalized, non-tumorigenic and ER-negative breast cell line that represents a valuable clinically experimental approach that minimizes extrapolation and thereby, uniquely facilitates the clinical translation of the data. This allowed us to analyze several signs of carcinogenicity by the effect of M, P, Ese, E2, and ACh demonstrating features of cell transformation in vitro such as increased cell proliferation, anchorage independence, invasive capabilities, modulation of receptor expression, genomic instability [44, 80,81,138,140–142], oxidative stress (SO2) [141], and cell metabolism (CYPs) [43,139,141], also revised by Calaf et al. [142].

6. Conclusions and future perspectives

This review presents a comprehensive summary of the impact of several substances in breast carcinogenesis. MYCi975 Unique in vivo and in vitro experimental rat mammary gland cancer models were developed by exposure to OPs as M, P, and Ese in combination with E2 and ACh allowing the study of the stepwise transformation of epithelial structures into malignant ones. The cellular and molecular endpoints altered in response to such treatment represent relevant issues in carcinogenesis. These are unique experimental approaches that identify mechanisms to link the role of OPs with E2 and ACh as possible and probable agents of risk in breast carcinogenesis.