ANTICANCER RESEARCH 33: 231-240 (2013)
Role of Pesticides in the Induction of Tumor Angiogenesis SALIMATH P. BHARATHI1, HARSH M. RAJ1*, SMITA JAIN2*, BASU DEV BANERJEE2, TANZEEL AHMED2 and VINOD KUMAR ARORA3 1Department
of Studies in Biotechnology, University of Mysore, Mysore, Karnataka, India; Biology and Molecular Biology Laboratory, Department of Biochemistry and 3Department of Pathology, University College of Medical Sciences and GTB Hospital, University of Delhi, Dilshad Garden, New Delhi, India
2Environmental
Abstract. Due to their estrogen-mimicking ability, pesticides are considered as prime etiological suspects of increasing tumor incidence, although a direct link is still undefined. The present study aimed to identify the effect of xenoestrogens (lindane, propoxur and endosulfan) at 20 mg/l each on tumorigenesis, by evaluating endothelial cell proliferation, H3 thymidine incorporation, wound healing, ascites formation and secretion, shell less Chorio Allantoic Membrane (CAM) formation using in vitro, as well as in vivo, models. The genotoxic effect of xenoestrogens in terms of DNA damage was also studied. The results showed that the endothelial cell proliferation, H3 thymidine incorporation, wound healing, CAM formation were increased following xenoestrogen exposure, but the intensity of angiogenesis was dependent on the structural homology of these xenoestrogens to endogenous estrogen. Moreover, lindane was the most potent angiogenesis stimulator followed by propoxur and Endosulfan. Further studies were undertaken to examine lindane for its possible carcinogenicity. However, no effect was observed on the integrity of DNA after exposure to these xenoestrogens. Great attention has been attributed on the undesired sideeffects of xenoestrogens, chemical substances that include pesticides, which mimic the activity of the female hormone estrogen in terms of structure and function. Among different classes of pesticides, organochlorines (OCP) and carbamates attract widespread concern due to their high
*These Authors contributed equally to this study. Correspondence to: Professor B.D. Banerjee, Room no. 237, Environmental Biochemistry and Molecular Biology Laboratory, Department of Biochemistry, University College of Medical Sciences (University of Delhi) and GTB Hospital, Dilshad Garden, New Delhi-110095, India. Tel: +91 1122135362, Fax: +91 1122590495, e-mail:
[email protected] Key Words: Pesticides, cancer, angiogenesis, xenoestrogen.
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biological activity, reduced rate of degradation, and thus long-term environmental persistence, and high biomagnification in the food chain. Therefore, it is necessary to carefully monitor their deleterious effects. Several reports have shown that chronic pesticide exposure might be a risk factor for increasing incidence of cancer, suppression of the immune system, sterility among males and females, and endocrine and neurological disorders (13). Exposure to environmental carcinogens, including herbicides and insecticides, has long been implicated in the development of many common types of cancer such as those of the lung, colon, breast, and testis (4, 5). Therefore risk assessment of such environmental contaminants became an important issue of public health policy. Endosulfan and lindane, widely used OCPs, are important insecticides, which act by blocking the function of gamma amino butyric acid (GABA) neurotransmitter at the site of GABAA receptor-chloride channel complex. Both these OCPs are known neurotoxicants and endocrine disruptors. It has been shown that malpractice in formulation and/or application of pesticides has greatly contributed to the incidence of leukemia, brain tumors, lung cancer, breast cancer and non-Hodgkin's lymphoma (4, 6, 7). Recently we found significant endosulfan residues in patients with prostate cancer and suggested that xenoestrogenic activity of the endosulfan in association with a genetic predisposition imparts a great risk for prostate cancer (8, 9). Several in vitro studies have also reported on the carcinogenic action of endosulfan and showed that it facilitated estrogen-dependent cellular proliferation by stimulating estrogen receptor (ER)-α and by simultaneously inhibiting ER-β in MCF-7 human estrogen-sensitive breast cancer cells (10). Similarly, the toxic effects of another OCP, lindane was observed in terms of genital malformations, reduced fertility and induction of some tumors in lindane-exposed rodents (11). However, some studies have refuted the carcinogenic effects of xenoestrogens. Hence, it is of considerable interest to investigate the effect of OCPs in carcinogenesis initiation and promotion.
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ANTICANCER RESEARCH 33: 231-240 (2013) Propoxur, [2-(1-methylethoxy) phenyl methylcarbamate] is an economically important insecticide widely used in aerosols to control household insects. The mutagenic and genotoxic effects of propoxur are well-established (12-14) but only few studies, till date, have demonstrated the xenoestrogenic activity of propoxur in relation to estrogen- related cancer in vitro. Here we determined the carcinogenic effect of propoxur using in vitro as well as in vitro models. Angiogenesis, formation of new blood vessels from existing ones, is an important mechanism for supplying essential nutrients and oxygen to cells present at distance from established vessels. It usually occurs during embryonic development and under certain physiological circumstances in the adult, including wound healing. In addition to its important role in normal physiological processes, angiogenesis contributes to the pathology of a number of diseases including tumor progression and metastasis. Initiation of angiogenesis involves the activation of certain genes via cell signaling, to produce certain growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and angiopoietins, for stimulating the growth of blood vessels and inhibitors, such as thrombospondin, endostatin and angiostatin. VEGF, a signal protein which belongs to the plateletderived growth factor family, plays an important role in vasculogenesis, i.e. formation of blood vessels de novo, as well as in angiogenesis during embryonic development and other pathological conditions such as injury, cancer and arthritis. Binding of VEGF to its affinity receptors on endothelial cells stimulates a complex series of events, including the secretion of metalloproteases and matrixdegrading enzymes, cellular movement into the newly-created space, endothelial cell division, proliferation, vessel formation and metastasis (15). Lack of concrete evidence regarding the carcinogenic effect of endosulfan, propoxur and lindane and its association with estrogenic-mimicking activity of these pesticides, if any, necessitates an identification of the angiogenic potential of these pesticides with special reference to tumorigenesis. Furthermore, the complexity of the role and mechanism of action of xenoestrogens in several physio-pathological conditions remains undefined, to date, due to limited knowledge on estrogen-responsive genes and downstream signaling. Hence, the present study was designed to identify the angiogenic potential of endosulfan, propoxur and lindane using in vitro as well as in vivo models, and their roles in carcinogenesis, in terms of cellular proliferation and de novo blood vessel formation at the site of tumor. To our knowledge, the present study is the first to determine the role of growth factors such as VEGF in the xenoestrogen-induced cellular abnormalities and provides an insight into the molecular mechanisms involved therein.
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Materials and Methods Chemicals and reagents. Endosulfan (99.9% purity), lindane (99.7% purity) and propoxur (99.4% purity) were obtained from AcccuStandard, New Haven, CT, USA. Stock solutions (1000 mg/l) of each pesticide were prepared using 0.1% dimethylsulfoxide (DMSO). The working solution of each pesticide was 20 mg/l. 3HThymidine was obtained from Baba Atomic Research Centre, Mumbai, India. Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS) and penicillin-streptomycin were purchased from Invitrogen, (California, USA). Poly-2-hydroxyethylmethacrylate, Hank’s balanced salt solution (HBSS) and DMEM Ham’s F-12 were procured from Sigma Aldrich, St. Louis, USA. Fertilized eggs were taken from a Government poultry farm, Bangalore, India. Matrigel was purchased from Chemicon International (Millipore Corporation, Massachusettes, USA). All other reagents used were of the highest analytical grade. Animals, in vivo tumor model and cell lines. Swiss albino mice (6-8 weeks old) were obtained from the animal house, Department of Studies in Zoology, University of Mysore, Karnataka, India. Ehlrich ascites tumor (EAT)/mouse mammary carcinoma cells maintained in our laboratory were used for in vivo transplantation. The animal experiments were approved by the Institutional animal care and use committee, University of Mysore, Karnataka, India. Human umbilical vein endothelial cells (HUVECs), endothelial basal medium (EBM) and endothelial growth medium (EGM) were obtained from Cambrex Biosciences, Walkersville, MD21793, USA. The MCF-7 (breast cancer) cell line was requested from the National Centre for Cell Sciences, Pune, India. Isolation of human peripheral blood mononuclear cells (PBMC). PBMCs were isolated as previously described (16) from peripheral blood, freshly-collected in heparinized vacutainers from healthy human volunteers who attended the blood bank of the University College of Medical Sciences (University of Delhi) and Guru Teg Bahadur Hospital, New Delhi, India, after obtaining informed consent. The study was approved by the Institutional Human Ethics Committee, UCMS. Briefly, 5 ml blood were layered carefully over an equal volume of Histopaque 1077 (Himedia, Mumbai, India) and were subjected to density gradient centrifugation for 30 min at 3000 revolutions per minute (rpm). PBMCs were collected from the buffy layer formed at the plasma–Histopaque 1077 interface and diluted in HBSS. After washing, PBMCs were suspended at 2 × 106 cells/ml in RPMI-1640 media fortified with 2 mM L-glutamine, 50 U/ml of penicillin, 50 μg/ml of streptomycin and 10% fetal bovine serum for further investigations. Assay for proliferation of cells. 3H-Thymidine incorporation assay was carried as described previously (17, 18) in order to study the effect of xenoestrogens on cellular proliferation in vitro. MCF-7 cells were plated onto separate 12 well plates (50,000 cells/well) in DMEM and grown in 5% CO2 at 37˚C for 48h. On third day, 3H-Thymidine (1 μCi/ml medium) was added prior to the addition of xenoestrogens (20 mg/l each of propoxur, lindane and endosulfan) and vehicle (control) into individual wells. After two days, the cells were washed with PBS and high molecular weight DNA was precipitated using 10% trichloroacetic acid at 4˚C for 30 min followed by washing with ice-cold H2O. The precipitate was solubilized in 0.2 N NaOH/0.1% sodium dodecyl sulfate (SDS) and
Bharathi et al: Pesticides in the Induction of Tumor Angiogenesis
3[H] radioactivity was determined by liquid scintillation counter. The experiment was performed in duplicates and repeated twice.
Cell migration assay. Cell migration assay was carried out as described previously (19). MCF-7 cells (1×105) were seeded into a 6-well plate and cultured to a confluent monolayer in DMEM complete media. The wells were washed gently with PBS and cells were then serum-starved overnight. The next day, the cells were treated with 100 μl of mitomycin (5 ng/ml) for 2 h. The monolayer was wounded by vertically scratching the surface with a sterile 10-μl micropipette tip in presence of PBS. The wells were again washed gently and the detached cells were aspirated along with the PBS. Two milliliters of basal media was added followed by in vitro treatment with propoxur, lindane and endosulfan (20 mg/l each)/vehicle/VEGF and incubation at 37˚C in 5% CO2. The initial wound healing and the movement of cells in the scratched area was photographically monitored at 0, 6, 12 and 24 h after the treatment. The selected pesticide dose exhibited non-lethal effects in vitro as described in our previously published studies (16). Matrigel tube formation assay. Tube formation of HUVECs was performed as described previously (20). A 96-well pre-chilled tissue culture plate was coated with 50 μl of Matrigel/well (Becton Dickinson Labware, Bedford, MA, USA) and the latter was allowed to solidify at 37˚C for 1 h, followed by the addition of 150 μl of EBM. HUVECs (5×103 cells), which were serum-starved overnight, were seeded onto the polymerized Matrigel and incubated with propoxur, lindane and endosulfan (20 mg/l each) for 2 h at 37˚C. Ten nanograms of human recombinant VEGF (rVEGF) were added to each well and the plate was further incubated for 24 h at 37˚C in 5% CO2 followed by the addition of EGM. Enclosed networks of complete tubes, thus formed, were counted and photographed at ×40 magnification, in five randomly chosen fields, using an Olympus inverted microscope (CKX40; Olympus, NY, USA) connected to a digital camera. Shell less chorio allantoic membrane (CAM) assay. CAM assay is a well-established assay used to assess angiogenesis ex vivo (21). Fertilized eggs were surface sterilized with 70% alcohol and incubated at 37˚C for 48 h. Eggs were gently rolled periodically. On day 3, eggs were removed from the incubator and swabbed with 70% alcohol, then 2 ml of albumin were removed by making a small hole in the pointed end of the egg using 2-ml syringe with 21-gauge needle; the hole was closed with the parafilm and the eggs were returned to the incubator. On day 4, eggs were cracked open and placed on the hammocks. The egg preparation was covered with a sterile petri dish and transferred to a humid incubator at 37˚C for 48 h. On day 6, propoxur, lindane and endosulfan (20 mg/l each) was administered individually by placing a 2-mm sterile filter disc, saturated with the respective pesticide, directly over a blood vessel, preferably at a major bilateral site on the yolk sac membrane and the eggs were further incubated for 48 h. On day 8, the holes of the eggs were re-opened and inspected for development of neovascularization in the area below the coverslip and photographed. Effect of xenoestrogens on body weight, ascites volume, cell number and peritoneal angiogenesis in EAT-bearing mice. To study the procarcinogenic or antitumor effects of xenoestrogens, 5×106 EAT cells, maintained in our laboratory, were injected intraperitoneally (i.p.) into four groups of mice (n=6 per group), as described previously (22). These cells exhibited an exponential growth period from the
Figure 1. Effect of xenoestrogens (20 mg/l each) on H3 thymidine incorporation in MCF-7 cells. The values are expressed as the mean±SEM. Lindane and propoxur induced a higher rate of H3 thymidine incorporation as compared to vehicle-treated cells (control group). The rate of thymidine incorporation in endosulfan-treated cells was less than lindane- and propoxur-treated cells.
sixth or seventh day after tumor injection and the animal succumb to death on twelveth to fourteenth day, due to tumor burden. To identify whether xenoestrogens inhibited tumor growth and angiogenesis in vivo, propoxur, lindane and endosulfan (100 μg/kg body weight each) were dissolved in 0.1% DMSO (vehicle) and injected i.p. daily for seven days into three groups of EAT-bearing mice from the sixth day of EAT transplantation. The remaining group of EAT-bearing mice was treated with PBS dissolved in the vehicle (0.1% DMSO) for seven days. Growth was recorded daily for all animals from the day of transplantation. The body weight of mice of all the four groups was monitored from day 1 to day 13. On day 13, the animals were sacrificed, 2 ml of saline was injected i.p., and a small incision was made in the abdominal region to collect the EAT cells along with ascitic fluid in a sterile polypropylene tube containing 2 ml of saline which was then centrifuged at 3000 rpm for 10 min at 4˚C. The volume of ascites was calculated by subtracting the volume of the saline injected from the volume of the supernatant collected. The cell number was determined by the trypan blue exclusion method using a hemocytometer. After collection of cells along with the fluid, the incision in the abdominal wall was extended and the exposed peritoneum was examined for vascularization and photographed. Immunohistological analysis (Hematoxylin & Eosin staining) for microvessel density (MVD) scoring. The effect of the xenoestrogens on the angiogenic response induced by VEGF was further verified by analysis of microvessel density in EAT-bearing mice which were treated regularly for seven days with the pesticides after the sixth day of EAT cell transplantation. The peritoneum of the mice treated with or without xenoestrogens was fixed in formalin, de-hydrated with alcohol and embedded in paraffin. Five micrometer sections were taken using a microtome and stained with routine hematoxylin and eosin stain. The MVD was determined by the ‘hotspot’ method (23) using a Nikon binocular microscope. In brief, 10 fields with highly vascularized areas were screened at low magnification (×10), and further magnification was changed to high-power field (HPF) (×40) and the microvessels were counted. Effect of xenoestrogens on DNA fragmentation in vitro. Cell viability of freshly-isolated PBMCs was determined by the trypan blue exclusion
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ANTICANCER RESEARCH 33: 231-240 (2013)
Figure 2. Effect of xenoestrogens on MCF-7 wound healing. Photographs taken at 0, 6, 12 and 24 h at ×40 magnification. Cell migration was high in lindane- and propoxur-treated cells, but less than the one in vascular endothelial growth factor (VEGF)-treated cells. Endosulfan treatment weakly-promoted wound healing compared to lindane, propoxur and VEGF.
assay (14). Suspension (1 ml) containing 2×106 cells was seeded into 24-well plate with propoxur, lindane and endosulfan (20 mg/l each). Cells were incubated for 24 h at 37˚C in a humidified atmosphere of 95% air and 5% CO2 in an incubator (MCO-15AC; Sanyo, Japan). DNA fragmentation assay was then carried out by using the method previously described (24). Briefly, the cultured PBMCs were collected by centrifugation at 3000 rpm for 5 min at 4˚C. The precipitate was washed twice with PBS followed by immediate lysis with 400 μl lysis buffer [1% TritonX, 50 mM Tris–HCl, (pH 7.5), 20 mM EDTA]. The lysate was centrifuged at 4500 rpm for 5 min at 4˚C in a microcentrifuge (Eppendorf) and the supernatant containing DNA was removed. The supernatant was then incubated at 50˚C for 3 h after mixing well with 20 μl of 10% SDS and 5 μl RNase A (10 mg/ml). Subsequently, 5 μl proteinase K (15.6 mg/ml) was added and the mixture was further incubated at 37˚C for 3h. DNA was precipitated by ethanol, separated by centrifugation and dissolved in Tris-EDTA buffer. Aliquots of DNA from the different treatment groups along with a 100-bp marker were electrophoresed using 1% agarose gel at 80 V/18 A for 3 h. The bands were visualized using a trans-illuminator and photographed using the geldoc 2000 gel documentation system (Bio-rad Laboratories Ltd, UK). Statistical analysis. Data are presented as the mean±standard error of the mean (SEM). p-Values
