More than 70% of sporadic breast cancers are attributable to long-term exposure to environmental factors, such as chemical carcinogens, etc.; this multiyear, multistep and multipath disease process involves cumulative genetic and epigenetic alterations to induce progressive carcinogenesis of breast cells from non-cancerous to precancerous and cancerous stages (1
). Over 200 chemical mammary carcinogens have been experimentally detected to acutely induce cancerous cells in cultures and tumors in animals at high doses of micro- to millimolar concentrations (1
). A high-dose approach may serve as a proper way to study occupational exposure; however, considering that chronic exposure of human tissues to low doses of carcinogens is responsible for most human cancers, a chronic low-dose approach might be a more proper way to study the environmental exposure most often responsible for human breast cancer development. A new approach is needed to reveal environmental mammary carcinogens, at low and bioachievable levels, capable of inducing human breast cell carcinogenesis.
We have been developing a model to mimic breast cell carcinogenesis occurring with accumulated exposures to low doses of environmental carcinogens (6
). We used the environmental carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (B[a]P) at picomolar concentrations, like those detected in patients (10
), to repeatedly treat immortalized, non-cancerous, human breast epithelial MCF10A cells in culture to progressively induce acquisition of cancer-associated properties (6
). NNK is considered one of the most potent lung carcinogens in tobacco products (14
); although gastric administration of NNK into rats results in DNA–adduct formation in the mammary gland and development of mammary tumors (14
), NNK is not currently recognized as a breast carcinogen. B[a]P, a family member of polycyclic aromatic hydrocarbons, is considered an environmental, dietary and tobacco carcinogen, its metabolites forming strong DNA adducts and causing DNA lesions and it is recognized as a mammary carcinogen in rodents (3
). Studies using human cell lines for genotoxicity tests and studies of adduct formation reveal genotoxic activity of NNK and B[a]P at concentrations as low as 25 mmol/l and 25 μmol/l, respectively (22
). Our cellular model reveals the ability of NNK and B[a]P, at a bioachievable level of 100 pmol/l, to chronically and progressively induce carcinogenesis of MCF10A cells (6
). Hence, our model system takes a new sensitive approach of validating low doses of environmental mammary carcinogens in chronic induction of human breast cell carcinogenesis.
It has been shown that a short-term exposure of MCF10A cells to the B[a]P metabolites B[a]P-quinones at 10 μmol/l for 10 min induces reactive oxygen species (ROS) elevation (23
), and exposure of normal human bronchial epithelial cells to NNK at 1–5 μmol/l for 24 h induces cell proliferation (24
). It has been postulated that ROS elevation and cell proliferation increase cell susceptibility to DNA damage induced by carcinogens, contributing to cellular carcinogenesis (25
). The oxidative DNA damage caused by ROS includes strand breaks and nucleotide modifications, resulting in mutations and contributing to cellular transformation (19
). Activation of the extracellular signal-regulated kinase (ERK) pathway also contributes to cell proliferation and phosphorylation of histone H2AX (27
), the latter of which (on serine 139) is widely used as an indicator for DNA damage (28
). In addition, B[a]P, at high doses ranging from 0.02 to 1 μmol/l, has been shown to induce cell proliferation and DNA damage in breast adenocarcinoma MCF7 cells (29
). However, it is not clear whether picomolar levels of NNK and B[a]P are able to induce ROS elevation and cell proliferation in breast cells with short-term exposure, contributing to induction of carcinogenesis associated with long-term exposure.
Epidemiologic and experimental studies have shown that various dietary polyphenolic compounds, which are widely found in vegetables, fruits and tea, possess anticancer, antiproliferative, antioxidant and apoptotic activities (1
). The use of green tea to increase the body’s antioxidant activity is becoming increasingly popular in the Western world (32
). A typical brewed green tea contains 30–45% green tea catechins (GTCs), including epicatechin (EC), epicatechin-3-gallate (ECG), epigallocatechin (EGC) and epigallocatechin-3-gallate (EGCG) (31
). GTCs have been shown to be more effective antioxidants than vitamins C and E (33
), and their order of effectiveness as radical scavengers is ECG > EGCG > EGC > EC (24
). Animal studies show that GTCs are able to suppress rat mammary carcinogenesis induced by 7,12-dimethylbenz[a]anthracene and N
). Laboratory studies also have shown that GTCs possess inhibitory and apoptotic activity in the growth of human breast cancer cells in cultures (36
). Epidemiological studies have examined the benefits of tea consumption for breast cancer prevention, and some evidence has indicated that green tea consumption may help prevent breast cancer recurrence in early stage cancers; however, the results are controversial (31
). In addition, studies show that EGCG and EGC exhibit higher toxicity than ECG and EC in inducing cellular DNA damage (40
). Therefore, additional studies are needed to clarify the effectivity of individual GTCs, at non-cytotoxic levels, used in protection of breast cells from carcinogenesis in order to safely and effectively reduce the health risk of sporadic breast cancer.
Previously, we used our model system to detect the ability of a dietary GTC extract containing 60% total catechins, at non-cytotoxic concentrations (<40 μg/ml), to suppress chronically B[a]P-induced carcinogenesis of breast epithelial cells (8
). In this study, we used our model system to pursue the mechanisms of NNK and B[a]P in inducing breast cell carcinogenesis and identify targeted end points transiently or constantly induced by short-term and long-term exposure to both carcinogens, respectively. Then, we used NNK- and B[a]P-induced end points as targets to identify preventive agents capable of intervening in the cellular carcinogenesis. We identified the essential role of ROS in modulating the ERK pathway leading to cell proliferation and chromosomal DNA damage in NNK- and B[a]P-induced breast cell carcinogenesis. We also revealed the preventive activity of individual EC, ECG, EGC and EGCG, at non-cytotoxic levels, in suppression of NNK- and B[a]P-induced breast cell carcinogenesis.