Progress 10/01/12 to 12/06/12
Outputs
Target Audience: Nothing to report due to death of PI in 2012 Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Nothing to report due to death of PI in 2012.
Publications
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: The primary goal of this project is to study the mechanisms of action of insecticides, including the modes of development of resistance to insecticides so that the knowledge we acquire will be used to help developing most effective strategies of controlling serious insect pests without increasing the chance of developments of hideous resistances by those pests. The following is the description of the work accomplished in the form of PhD thesis by Mohamed A.I. Ahmed, a graduate student I have been supervising. Background: The current strategy for control of vector mosquitoes of human diseases relies heavily on the use of pyrethroids on bed nets, indoor spraying and larvicidal applications (i.e. throughout all stages of the life cycle of the pest. Such a heavy reliance on only one type of chemicals is likely to lead to future problems of increasing resistance development. A useful approach to avoid rapid development of resistance would be to avoid excessive, uniform, region-wide uses of high dose application of only one type of insecticides with a common action mechanism, and instead minimize the extent of reliance on them and, second introduce insecticides with very different modes of action following god examples from successful IPM programs. Formamidines are unique insecticides and acaricides that elicit multiple effects in controlling insects. The primary objective of this study is to establish that formamidine chemicals are effective synergists for the increasing the effectiveness of most commonly used pyrethroids on Aedes aegypti mosquitoes to reduce the current heavy reliance on pyrethroids. The results will be disseminated in the form of publications of our findings in scientific journals. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
In the current study, two typical formamidines, amitraz and chlordimeform, were assessed for their actions on pyrethroids, and neonicotinoids on the 4th instar larvae of Aedes aegypti, a vector for dengue disease. Amitraz and chlordimeform indeed showed significant reduction effects on behavioral tests and delayed effects on life cycle. Furthermore, the synergistic effects of amitraz on those 2 different classes of larvicides was very significant on deltamethrin and fenvalerate type pyrethroids, but it showed no synergistic action on permethrin. . Furthermore, we could show that formamidines are also effective synergists against newly developed neonicotinoids sa well. For instance, against neonicotinoids amitraz showed significant synergizing effect in terms of causing their mortality. Particularly effective was its action on newer type of neonicotinoids such as dinotefuran and thiamethoxam. These two new class of neonicotinoids are being considered to be the most promising group of insecticides to combat the problems of pyrethroid-resistaqnt mosquitoes. The potential impact of this work is to help minimizing the use of pyrethroids on vector mosquitoes in order to reduce the chance of those pests of the public health importance.
Publications
- Mohamed A. I. Ahmed, PhD thesis, 2011. entitled "Biochemical and Molecular Biological Studies on the Action of Formamidines in Synergizing Insecticidal Action of Pyrethroids and Neonicotinoids in Aedes aegypti Mosquitoes"
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: In this project period I completed the study on the action mechanism of diflubenzuron, a prototype chitin synthesis inhibitor that has been used as a powerful insecticide. The reason why we studied this insecticide is that its action mechanism has remained mystery for many years. Unlike the traditional chitin synthesis inhibitors this class of benzoylurea insecticides does not inhibit chitin synthesis in fungi. Moreover, many attempts to study its possible action site have failed, since any type of disruption of the integrity of insect cuticles always has made this compound inactive in the past.. This observation has led our research team to suspect that the target of this insecticide might be related to the system engaged in intracellular trafficking of chitin building materials. Indeed after many trials, we could obtain intracellular vesicles from newly molted integuments American cockroaches, which respond well to diflubenzuron (DFB) to alter its ion-exchanging activity. Particularly affected were the activity of exchanging Ca2+ and K+ ions, which were also activated by exogenous ATP and GTP. Based on this observation, we hypothesized that those intracellular vesicles might belong to the class of ABC-transporters. Our efforts to characterize the sub-type of this DFB target vesicle resulted in identification of "sulfonylurea receptor" as the most likely sub-type. This information was submitted to Pesticide Biochemistry and Physiology, and subsequently published in that journal. PARTICIPANTS: Gamal Abo-Elgher, a postdoctoral scientist (supported by the Government of Egypt) Phillip Fujiyoshi, a postdoctoral staff research associate. Both worked in my laboratory at Dept of Environmental Toxicology at University of California Davis TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Outcome/Impact. Identification of this target site for this chitin synthesis inhibitor made it possible to understand for the first time that this new sub-type of "sulfonylurea receptor" (SUR) does exist in insect cuticle. This is quite surprising, since SURs in higher animals are best known for their function in pancreas to control insulin secretion upon stimulation by glucose. In fact reduction in the glucose responding function of SUR in pancreas is the initial trigger for Type 2 diabetes in human. Hence there are two major impacts: one is the discovery of SUR in insect integuments and the other is this discovery that benzoylurea type insecticides could also stimulate the ion exchanging SUR functions as in the case of therapeutic agents for type 2 diabetes (such as the most frequently used compound called Glibenclamide) provides a new idea for future development of therapeutic drugs for type 2 diabetes.
Publications
- Fumio Matsumura (2010). Studies on the action mechanism of benzoylurea insecticide to inhibit the process of chitin synthesis in insects: A review on the status of research activities in the past, the present and future prospects. Pestic. Biochem. Physiol. 97:133-139.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Recent reports indicate the existence of breast cancer cells expressing very high levels of the Arylhydrocarbon receptor (AhR), a ubiquitous intracellular receptor best known for mediating toxic action of dioxin and many other organochlorine pollutants. Positive correlation between the degree of AhR overexpression and states of increasing transformation of mammary epithelial cells appears to exist. These observations have raised many questions such as why and how AhR is overexpressed in breast cancer and whether its overexpression make them more susceptible to the effects of environmental pollutants so as to accelerate their progression to advanced forms of cancer, or not. To address those questions, we hypothesized that AhR overexpression occurs in cells experiencing deficiencies in normally required estrogen receptor (ER) signaling, and the basic role of AhR in such cases is to guide the affected cells to develop orchestrated cellular changes aimed at substituting the normal functions of ER. At the same time, the AhR serves as the mediator of the cell survival program in the absence of ER signaling. METHODS: We subjected two lines of Michigan Cancer Foundation (MCF) mammary epithelial cells to 3 different types ER interacting agents for a number of passages and followed the changes in the expression of AhR mRNA. The resulting sublines were analyzed for phenotypical changes and unique molecular characteristics. RESULTS: MCF10AT1 cells continuously exposed to 17-beta-estradiol (E2) developed sub-lines that show AhR overexpression with the characteristic phenotype of increased proliferation, and distinct resistance to apoptosis. When these chemically selected cell lines were treated with a specific AhR antagonist, 3-methoxy-4-nitroflavone (MNF), both of the above abnormal cellular characteristics disappeared, indicating the pivotal role of AhR in expressing those cellular phenotypes. The most prominent molecular characteristics of these AhR overexpressing MCF cells were found to be overexpression of ErbB2 and COX-2, indicating that those cells are likely to show inflammatory responses and proliferate faster than normal cells. Furthermore, we could demonstrate that the progression of those cells to become malignant cancer cells are clearly promoted by a number of environmental pollutants. CONCLUSION: One of the main causes for AhR overexpression in these MCF breast cancer cells appears to be the loss of ERalpha functions. This phenomenon is likely to be based on the mutually antagonistic relationship between ER and AhR. PARTICIPANTS: Wong PS, Li W, Vogel CF, Matsumura F Wong PS is Assistant Project Scientist, Center for Health and the Environment (role as the senior author). Li W is a postdoctoral trainee in Matsumura's Laboratory (postdoctoral trainee). Vogel CF is Assistant Scientist, Environmental Toxicology (role as the senior author). Matsumura F is Professor, Department of Environmental Toxicology. All of them actively participated in this project. All belong to the University of California Davis. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
It is well known that the development of breast cancer is greatly influenced by environmental factors such as exposure to organochlorine pollutants as shown by a number of epidemiological studies. However, the reason why those environmental pollutants promote breast cancer is not clarified yet. This study has addressed the meaning the recent finding that the Ah receptor (i.e. the dioxin receptor) is over-expressed in some types of breast cancer. First, we determined that Ah receptor over-expression is closely associated with the estrogen receptor-negative breast cancer, and second established that thus overexpressed Ah receptor assists the progression of those pre-cancer cells to become more malignant type of cells, particularly when they are exposed to dioxin and other types of organochlorine pollutants. Perhaps the most important outcome of this work is that the Ah receptor could become an important target of future development of therapeutic approaches, being the critical component of those breast cancer cells to advance into the malignant form.
Publications
- Wong PS, Li W, Vogel CF, Matsumura F. (2009). Characterization of MCF mammary epithelial cells overexpressing the Arylhydrocarbon receptor (AhR). BMC Cancer. 2009 Jul 15;9:234. Department of Environmental Toxicology and the Center for Environmental Health Sciences, University of California, One Shields Ave., Davis, CA 95616, USA. patwong@ucdavis.edu
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The main objective of this project has been to find the reason why the exposures to certain pesticides have consistently been associated with human diseases. In this project period we have made a concerted effort to address the question on the rising incidents of lymphoma among those people who are exposed to a variety of pesticide such as farm workers and farmers. For this purpose we have selected dioxin, which is found among many pesticide products as a contaminant. Using three different lymphoma cell lines we could demonstrate that the exposure to dioxin causes those cells to be come resistant to apoptosis, which is a hallmark sign of cancerous cells, giving them the survival advantages over normal cells. This action of dioxin is accompanied with the increased inflammatory state of lymphoma cells that is caused by the activation of cyclooxygenase-2 (Cox-2). Inhibition of this enzyme by NS-398, a common anti-inflammatory drug, prevented this action of dioxin to cause apoptosis resistance in those lymphoma cells. The importance of this action of dioxin has been demonstrated in mice which developed the advanced level of lymphoma as a result of their exposure to dioxin, particularly in lymph nodes. In view of the importance of this discovery we have reported this finding in American Journal of Pathology, a well respected and a widely circulated scientific journal. PARTICIPANTS: Vogel CF is Assistant Scientist, Environmental Toxicology (role as the senior author). Li W is a postdoctoral trainee in Matsumura's Laboratory (postdoctoral trainee). Sciullo E is a PhD Graduate student, Toxicology-Pharmacology Graduate Group, and Research Assistant in Matsumura's Laboratory (graduate student training). Newman J is a postdoctoral researcher in Hammock's Laboratory (provided the analytical data on prostaglandins). Hammock B is Professor of Entomology (Dr Newman's supervisor). Reader JR is a Staff Pathologist at Comparative Pathology Lab, Veterinary School (conducted the MET, micropositron emission topography imaging experiments) Tuscano J is Professor of Hematology and Oncology, UCDMC (provided his expertise on lymphoma's). Matsumura F is Professor of Environmental Toxicology and Entomology. All participants belong to University of California Davis. TARGET AUDIENCES: The intended target audiences are: a) lymphoma specialists, b) scientists in the field of Agricultural Safety, c) risk assessment specialists in the government agencies (e.g. USEPA, CDC, CalEPA,), d) toxicologists, who are working on pesticides and dioxins, and e) health scientists, who are interested in Environmental Oncology. PROJECT MODIFICATIONS: For this study period, the project has been modified to add dioxin to the list of insecticidal compounds. This expansion of the scope of this project was necessary, since dioxin is found as a contaminant in many pesticide products, and since dioxin is the most potent cancer causing component in those products. Therefore, it would be a mistake, if we do not consider the contribution of dioxin to the toxic action of those pesticides.
Impacts
Approximately 4.5 million people worldwide are living with various forms of lymphoma, and an estimated 300,000 people die each year from lymphoma. In terms of incidence and death, non-Hodgkin's lymphoma (NHL) is the second fastest growing cancer in the United States and the third fastest growing in the rest of the world. NHL has grown in incidence by 80% since the early 1970s in the United States. The cause for the increasing incidence of NHL and leukemia is still primarily unknown; however, research has focused on at least four possible factors that may contribute to the development of lymphoma, including genetic factors, autoimmune disorders, viruses such as human immunodeficiency virus, and exposure to pesticides. Epidemiological studies have linked exposure to environmental contaminants such as polychlorinated biphenyls and polychlorinated dibenzodioxins/furans (PCDD/PCDF) with increased incidence of NHL and myeloid leukemia. A hallmark of leukemic/lymphoma cells is the ability to proliferate in the absence of exogenous growth factors and to escape apoptosis. As a consequence of a suppressed apoptotic response, malignant cells can escape from immune control, accumulate genetic defects, and proliferate to expand their cell population. Therefore, inhibition of DNA damage- or stress-induced apoptosis plays an important role in the development of lymphoma/leukemia and has been suggested as a mechanism of carcinogenic action of AhR-activating agents like TCDD. The most important outcome of this finding is that the development of dioxin-mediated lymphoma can be antagonized by treating mice with an anti-inflammatory drug, NS-398. Such information could provide the idea of developing a preventive and therapeutic approach in the future to reduce the occurrence of this hideous type of disease such as lymphomas and leukemia's based on the principle discovered in this study.
Publications
- Vogel CF, Li W, Sciullo E, Newman J, Hammock B, Reader JR, Tuscano J, Matsumura F. 2007. Pathogenesis of aryl hydrocarbon receptor-mediated development of lymphoma is associated with increased cyclooxygenase-2 expression. Am J Pathol.171(5):1538-48.
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Progress 01/01/07 to 12/31/07
Outputs
Exposure to beta-Hexachlorocyclohexane (beta-HCH), a contaminant of the hexachlorohexane pesticide lindane, has been implicated as a risk factor in the development of breast cancers in epidemiological studies. Previous studies in our laboratory have demonstrated the ability of beta-HCH to elicit its actions via a ligand-independent activation of the estrogen receptor through increased c-Neu (= erbB2 or HER-2) expression and kinase activation in both the BG-1 and MCF-7 cell lines. In addition, long term exposure (33 passages) to beta-HCH was shown to promote the selection of MCF-7 cells which exhibit a more metastatic phenotype. METHODS: In this current study, we decided to investigate the long-term effects of beta-HCH in both the MCF10AT1 cell line which was derived from a normal epithelial cell line by stably transfecting a mutated c-Ha-ras and a MMTV-Neu mouse model for mammary cancer in vivo. MCF10AT1 cells were exposed for 20 passages with beta-HCH, 4-OH-Tamoxifen
(Tam), or 17-beta-estradiol (E2) after which cells were analyzed for proliferation rates and mRNA expression by RT-PCR. In our in vivo studies, MMTV-Neu mice were injected with beta-HCH and observed for tumor formation over a 70 week period. RESULTS: beta-HCH and Tam selected MCF10AT1 cells demonstrated increased mRNA expression of MMP-13 (collagenase-3) a marker of increased invasiveness. Beta-HCH treatment was also seen to increase the expression in a number of proto-oncogenes (c-Neu, Cyclin D1, p27), cell status markers (Met-1, CK19), and the inflammatory marker NFkappaB. Previous studies, have demonstrated the role of these markers as evidence of malignant transformations, and further illustrate the ability of beta-HCH to be carcinogenic. To demonstrate beta-HCH's tumorigenic properties in an in vivo system, we used an MMTV-Neu mouse model.MMTV-Neu is a c-Neu over expressing strain which has been shown to spontaneously develop mammary tumors at later stages of aging. In this
experiment, beta-HCH exposure was shown to both accelerate the appearance (~8 weeks for median tumor-free period) and incidence (~25% increase at the end of the test period) of tumors when compared to control mice receiving only the corn-oil vehicle. CONCLUSION: Based upon these results, it was concluded that beta-HCH does act as a breast cancer promoter which exerts its tumorigenic activity via increased c-Neu expression.
Impacts
The results of this work clearly establish that at least one of the typical chlorinated hydrocarbon insecticides, beta-HCH, which has been frequently found in human breast tissue samples, indeed promotes breast cancer based on both an in vitro and an in vivo model of carcinogenesis. This point is very important, since the results of several epidemiological studies have been ambiguous on this question, as well as being highly controversial. Therefore, providing this experimental evidence serves as one of the ways to help settling this controversial issue. Furthermore the findings of this work also help to indicate that those pesticides showing estrogenic properties are likely the ones promoting human breast cancer and that the type of breast cancer develops from human exposure to this type of environmental factors is likely the estrogen receptor positive and the c-Neu over-expressing type.
Publications
- Wong PS, Matsumura F. 2007. July. Promotion of breast cancer by beta-hexachlorocyclohexane in MCF10AT1 and MMTV-neu mice. BMC Cancer 7:130.
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Progress 01/01/06 to 12/31/06
Outputs
Many pesticides have been found to cause estrogen like actions in wild animals as well as in experimental animals. Yet, assessing their estrogen like actions in models systems in the laboratory has not been easy. The main problem has been that most assay methods rely on detecting the direct action of those pesticides to bind to the estrogen receptor to approximate their ER activation potential. Such methods, however, do have drawbacks, since most of pesticideshave been found to activate the ER via different mechanisms than just binding directly to this receptor. Therefore, there is an acute need to develop a reliable assay method that will detect several types of the estrogen-like actions of a pesticide regardless of its ER binding ability. Using a serum-free BG-1 ovarian cell culture model, we investigated the ability of several oganochlorine pesticides to act like estrogen. This assay method takes the advantage of ovarian cells to increase their activity of cell
proliferation through several different types of estrogen-like stimuli. These effects were abolished using a pure ER antagonist, ICI 164,384, thus implicating the requirement of ER for these events. Using these methods, we could observe concentration-dependent cell proliferation in BG-1 cells as the results of their exposure to extremely low concentrations of a variety of organochlorine pesticides, including DDT, beta-HCH, heptachlor epoxide, toxaphene and endosulfan I. The range of concentrations of those pesticides causing such estrogen-like effects was typically a few parts per billion, the levels often found in a variety of food products particularly in animal products. Development of such a methodology helps to assess the potentials of a variety of pesticides and environmental pollutants to act as potential endocrine disruptors in the future.
Impacts
Some of pesticides have been found to cause side effects to animals and humans, which closely mimic the action of estrogen. Such effects found in live experimental animals include increased weight of uterus, early maturation of female offspring and increased incidence of breast cancer. Yet, no reliable detection methods for such estrogen-like actions of pesticides are available. The method developed in our laboratory is based on a line of cultured human ovarian cells (called BG-1) which is known to respond to very low concentrations of estrogen to rapidly proliferate. Using this approach we could detect the estrogen-like effects of a number of pesticides at concentrations at least 1/1000 of previously reported levels. Such levels of some of those pesticides are already found among human tissues, and therefore are of great concern to the society already. The availability of this method makes it possible to start the process of identifying those potentially hormone
mimicking chemicals, and thereby alert the regulatory agencies for their potential hazards to human and the environment.
Publications
- Wong PS, Matsumura F. 2006 April
- Serum free BG-1 cell proliferation assay: a sensitive method for determining organochlorine pesticide estrogen receptor activation at the nanomolar range. Toxicol In Vitro. 20(3):382-94. Epub 2005 Oct 19
- Hansen ME, Pessah IN, Matsumura F. 2006 March 15
- Heptachlor epoxide induces a non-capacitated type of Ca2+ entry and immediate early gene expression in mouse hepatoma cells. Toxicology. 220(2-3):218-31. Epub 2006 Feb 15
- Park S, Matsumura F. 2006 January 16
- Characterization of anti-apoptotic action of TCDD as a defensive cellular stress response reaction against the cell damaging action of ultra-violet irradiation in an immortalized normal human mammary epithelial cell line, MCF10A. Toxicology. 217(2-3):139-46. Epub 2005 Oct 6.
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Progress 01/01/05 to 12/31/05
Outputs
One mechanism of carcinogenic action of TCD (=dioxin) on breast cells is that it causes significant inhibition of proper differentiation of mammary duct epithelial cells and thereby increases the number of terminal end buds, which are susceptible to other carcinogens (Fenton et al., Toxicol Sci 2002;67:63-74; Brown et al., Carcinogenesis 1998;10:1623-1629; Lamartiniere, J Mammary Gland Biol Neoplasia 2002;7:67-76). To address this topic, we selected MCF10A, a line of immortalized normal human breast epithelial cells as an in vitro model. An initial effort was made to optimize the cultural condition of MCF10A cells to promote the cell differentiation effect of insulin. Under this condition, TCDD clearly antagonized the action of insulin only in the presence of Cholera toxin that is known to promote the differentiation of normal human breast epithelial cells. To test the hypothesis that TCDD induced c-Src kinase activation is casually related to this compound's
antagonistic action against insulin, we treated MCF10A cells with two c-Src blocking agents, an anti-Src antisense oligonucleotides blocker and a known specific inhibitor of c-Src kinase, PP-2 and studied the effect of insulin and TCDD on cell proliferation. The results showed that in cells treated with either of these two c-Src blocking agents, the antagonistic effect of TCDD disappeared. It was also found that agents which specifically block the activation of ERK could also abrogate the action of TCDD to suppress insulin signaling. Together, these results indicate that the mechanism of the antagonistic action of TCDD on insulin signaling is mainly mediated through c-Src signaling through activation of ERK.
Impacts
The significance is the same as previous years; Epidemiological evidence indicates there is positive correlation between the incidence of breast cancer and high residue levels of organochlorine compounds such as DDT B-HCH, PCB and dioxin; no explanation for such a correlation has been offered. The finding clearly indicates that dioxin interferes with the normal action of insulin to facilitate the normal growth of mammary epithelial cells into well differentiated lobular duct structures. Pinpointing the critical action site of these pollutants is very important from the viewpoint of designing the therapeutic strategy in the future.
Publications
- Wong, P.S. and Matsumura, F. 2005 Serum free BG-1 cell proliferation assay: A sensitive method for determining organochlorine pesticide estrogen receptor activation at the nanomolar range. Toxicol In Vitro, PMID: 16242299.
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Progress 01/01/04 to 12/31/04
Outputs
One of the proposed mechanisms of carcinogenic action of TCDD (=dioxin) on breast cells is that it causes significant inhibition of proper differentiation of mammary duct epithelial cells and thereby increases the number of terminal ends buds, which are susceptible to other carcinogens (Fenton et al., Toxicol Sci 2002; 67:63-74; Brown et al, Carcinogenesis 1998;10:1623-1629; Lamartiniere, J Mammary Gland Biol Neoplasia 2002;7:67-76). To address this topic, we selected MCF10A, a line of immortalized normal human breast epithelial cells as an in vitro model. An initial effort was made to optimize the cultural condition of MCF10A cells to promote the cell differentiation effect of insulin. Under this condition, TCDD clearly antagonized the action of insulin only in the presence of Cholera toxin that is know to promote the differentiation of normal human breast epithelial cells. To test the hypothesis that TCDD induced c-Src kinase activation is casually related to this
compound's antagonistic action against insulin, we treated MCF10A cells with two c-Src blocking agents, an anti-Src antisense oligonucleotides blocker and a known specific inhibitor of c-Src kinase, PP-2 and studied the effect of insulin and TCDD on cell proliferation. The results showed that in cells treated with either of these two c-Src blocking agents, the antagonistic effect of TCDD disappeared. It was also found that agents which specifically block the activation of ERK could also abrogate the action of TCDD to suppress insulin signaling. Together, these results indicate that the mechanism of the antagonistic action of TCDD on insulin signaling is mainly mediated through c-Src signaling through activation of ERK. 2005 Wiley Periodicals, Inc. J. Biochem Mol Tox
Impacts
Epidemiological evidence indicates that there is a positive correlation between the rising incidence of breast cancer and high residue levels of organochlorine compounds such as DDT B-HCH, PCB and dioxin. Yet, so far no explanation for such a correlation has been offered. The finding reported in this publication clearly indicates that dioxin interferes with the normal action of insulin to facilitate the normal growth of mammary epithelial cells into well differentiated lobular duct structures. Pinpointing the critical action site of these pollutants is very important from the viewpoint of designing the therapeutic strategy in the future.
Publications
- TCDD causes Suppression of Growth and Differentiation of MCF10A, Human Mammary Epithelial Cells by Interfering with Their Insulin Receptor signaling Trhough c-Src Kinase and ERK Activation Sujin Park, Olga Mazina, Akira Kitagawa, Patrick Wong, and Fumio Matsumura, Biochem Molecular Toxicology received 24 July 2004; revised 10 October 2004; accepted 15 october 2004, Vol 18, Number 6, 2005 p1-11
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Progress 01/01/03 to 12/31/03
Outputs
Due to its lipophilicity and persistence, an organochlorine compound, B (Beta)-hexachlorocyclohexane (B-HCH), is known to frequently accumulate in human adipose and breast tissues. An epidemiological study has indicated that exposure to B-HCH could be one of the significant environmental risk factors for the development of human breast cancers. Additionally, B-HCH has recently been identified as an environmental estrogen capable of activating estrogen receptor (ER) through a ligand-independent pathway. In the present investigation, we examined the impact of long-term in vitro exposure to B-HCH on cell transformation and the metastatic potentials of MCF-7 cells. We found that continuous exposure of MCF-7 cells to B-HCH at 100 nM and 1 uM to 17B-estradiol (E2) at 1 nM for up to 13 months (33 passages) not only enhanced their transformation tendencies but also promoted their invasiveness. Western blot analysis revealed that B-HCH induced transformation-related
biochemical changes in MCF-7 cells, such as a decline in the levels of ERalpha and p44/42 MAP kinase and a significant increase in expression of c-ErbB2 and MMP-9 levels. In contrast, long-term E2 treatment resulted in the downregulation of ERalpha and p44/42 MAP kinase and upregulation of MMP-9 only, but no changes in c-ErbB2. Together, these results indicate that these biochemical changes induced by B-HCH are consistent with the events taking place in these cells to promote the phenotypical expression of transformed cells. Our results provide the in vitro mechanistic basis supporting the hypothesis that B-HCH is one of the epigenetic risk factors assisting the progression of breast cancer cells to an advanced state of malignancy.
Impacts
For the first time, we have clearly established that a pesticide, B-HCH causes malignant transformation of human breast mammary gland epithelical cells when they are exposed to this pesticide or a long time (over one year). Breast cancer is a serious disease, affecting 180,00 women every year in the United States. Not only that, breast cancer is a type of cancer affected greatly by environmental factors. The type of malignancy observed in these cells resembles that of tamoxifen resistant phenotype, meaning that they have become drug resistant and estrogen dependent.
Publications
- Zou, E. and Matsumura, F. 2003. Long-term exposure to B-hexachlorocyclohexane (B-HCH) promotes transformation and invasiveness of MCF-7 human breast cancer cells. Biochemical Pharmacology, 66, 831-840.
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Progress 01/01/02 to 12/31/02
Outputs
Previously we reported a novel phenomenon that some organochlorine compounds mainly act through activation of c-Neu tyrosine kinase without being strong agonists for the estrogen receptor. In this study we tested the possibility of developing an assay system to identify estrogenic compounds acting through this c-Neu-mediated mechanism. We describe herein an assay that utilized foci formation of MCF7 cells as an endpoint, antibody 9G6 to neutralize the c-Neu-mediated pathway and 4-hydroxytamoxifen to block the ER. Aroclors 1242 and 1248, 2,2',3,5',6-pentachlorobiphenyl (PCB 95), 2,2'dichlorobiphenyl (PCB), cis- and trans- permethrins, and chlorothalonil were found to render estrogenic effects through this c-Neu-mediated mechanism, while alpha and beta endosulfans appeared to act through a pathway independent of the c-Neu-mediated one. Pentachloronitrobenzene was found to be capable of antagonizing the 17Beta-estradiol effect, which has never been reported previously.
Impacts
The above finding indicates that those pesticides promote carcinogenic transformation of human breast cells, and thereby support the hypothesis generated by epidemiological evidence that the presence of those long lasting residues in human bodies may be linked to rising incidence of breast cancer.
Publications
- Zou E., Hatakeyama, M. and Fumio Matsumura (2002). Foci formation of MCF7 cells as an in vitro screening method for estrogenic chemicals, Environmental Toxicology and Pharmacology 11, 71-77.
- Hansen, M. and Matsumura, D. (2001) Down-regulation of particulate protein kinase ce and up-regulation of nuclear activator protein-1 DNA binding in liver following in vivo exposure of B6C3F1 male mice to heptachlor epoxide, J Biochem Molecular Toxicology, Vol 15, Number 1.
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Progress 01/01/01 to 12/31/01
Outputs
The effects of the organochlorine (OC) liver tumor promoter heptachlor epoxide (HE; 0, 0.1, 1, 10, and 50 microM) on several cellular tumor promoter-sensitive parameters were studied in mouse 1c1c7 hepatoma cells in an effort to identify the most sensitive biomarker for OC promoter exposure and the critical pathway and target of OC promoters. The levels of Ca2+ in the endoplasmic reticulum (ER) store, connexin43 (Cx43), PLCgamma(1), nPKCvarepsilon, and AP-1 DNA binding in nucleus were studied to screen for effects induced by submicromolar HE levels. While all the parameters tested elicited effects, particulate PLCgamma(1) and AP-1 DNA binding were found to be the most sensitive parameters affected by HE on both dose and temporal bases. Their levels were increased with 10- to 100-fold lower HE concentrations than were required to affect nPKCvarepsilon or Cx43. Further, with the lower HE dosages, particulate PLCgamma(1) and nuclear AP-1 were positively modulated by HE
after 1 h versus 3 or 72 h for nPKCvarepsilon and Cx43. Ca2+ store depletion was probably the third most sensitive parameter, after AP-1 and PLCgamma (1). These results suggest the tyrosine kinase growth factor receptor pathway is the probable critical pathway for HE-induce tumor promotion with the critical target most likely being upstream of PLCgamma(1) and AP-1. This work also demonstrates that upon exposure to a tumor promoter such as HE, many hepatocellular effects or changes result, suggesting that a cellular-program shift occurs similar to that described by the resistant hepatocyte model after exposure to a carcinogen or enzyme inducer.
Impacts
Our finding indicates that heptachlor, an insecticide commonly used in the past, shows cancer promoting activities. Our work also provides the mechanistic information why heptachlor causes cancer promotion, and, therefore helps others to formulate the preventative and/or therapeutic strategies to reduce the incidence of this type of cancer.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
Progress Report: Prostate cancer is currently the second leading cause of cancer mortality, after lung cancer, in American men. Each year in the United States approximately 185,000 men are diagnosed with prostate cancer, and approximately 40,000 die from the disease. While the number of diagnosed cases has been rising for the past 30 years, our understanding of the causes and development of prostate cancer lags behind that of other common cancers, such as breast and colon cancer. Several epidemiological studies have identified an association between agrochemical exposure and elevated rates of prostate cancer in farm workers and pesticide applicators. The oncogene erbB-2 codes for an EGF receptor-family tyrosine kinase (erbB-2/HER-2/neu) that is implicated in several types of cancer, including prostate and breast cancers. In the hormone-dependant prostate cancer cell line LNCaP, erB-2 is overexpressed and functions in hormonally regulated mitotic signaling pathways. In
this cell line we found that erB-2 kinase was activated by agrochemicals of different chemical classes: the organochlorine insecticides B-hexachlorocyclohexane (B-HCH), o,p'-dichlorodiphenyltrichloroethane (o,p'-DDT), and heptachlor epoxide; the pyrethroid insecticide trans-permethrin; and the fungicide chlorothalonil (O,p'-DDT) also cause phosphorylation of mitogen activated protein kinase (MAPK) and cellular proliferation of the androgen-dependent LNCaP line, whereas no proliferative effect was observed in the androgen-independent PC-3 line.
Impacts
Our finding indicates that some of these pesticides exhibit the property to activate in human prostate cancer cells this important proto-oncogen, c-Neu, which is known to play a pivotal role in pathogenesis of prostate cancer. Such a finding raises the possibility that their residues in human tissues are acting as androgen mimics in promoting the development of prostate cancer.
Publications
- Select Agrochemicals Increase ErbB-2 Tyrosine Kinase Activity and Cell Proliferation in the Prostate Cancer Cell Line LNCaP. Tessier, D.M. and Matsumura, F. (2000) Toxicol. Sciences, In Press.
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Progress 01/01/99 to 12/31/99
Outputs
Several studies have shown that some organochlorine compounds act like estrogen in certain animals and vitro cell culture systems, and therefore, there is a possibility that they could promote the process of tumorigenesis in breast cancer cells. In our previous study, two representative organochlorines, 1,1,1-trichloro, 2-o-chlorophenyl-2'-p-chlorophenyl ethane (o,p'-DDT) and B-1,2,3,4,5,6-hexachlorocyclohexane were found to directly activate the protein tyrosine kinase of Neu (c-erbB-2 proto-oncogene product) immunoprecipitates isolated from MCF-7 breast cancer cells. In the current study, we also found that 4,4,5-trichlorophenoxy-acetic acid (2,4,5-T) and 1nM and 1-HCH isomers at 100 nM could also significantly activate protein tyrosine kinase of Neu immunoprecipitates in a cell-free system. We also found that organochlorines result in an increase of Neu protein tyrosine kinase after intact cell treatment in estrogen-depleted medium. This Neu kinase activation by
B-HCH (100 mM) was blocked when the cells were pretreated with Neu mRNA antisense oligonucleotide. Endogously added a-, b-, and y-HCH, o,p'-DDT, 2,2'-dichlorobiphenyl (2,2'-PCB), and 2,4,5-T at 100 nM were found to promote foci formation in postconfluent cultures of this cell line. This stimulatory effect caused by 17B-estradiol, o,p'-DDT, and b-HCH on foci formation was inhibited by coincubation with Neu monoclonal antibody (p< 0.05). Those two events induced by organochlorines (i.e., Neu kinase activation and foci formation) seemed causally correlated.
Impacts
(N/A)
Publications
- MATSUMURA, F. , 1999. Correlation between the activation of neu tyrosine kinase and promotion of foci formation induced by selected organochlorine compounds in the MCF-7 model system. J Biochem Molecular Toxicology, V13, number 6, 296-302.
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Progress 01/01/98 to 12/01/98
Outputs
The kdr-type resistance is characterized as the specific resistance mechanism of many pest insects effective against DDT, pryethroids and prethrins. During this reporting period we have studied the molecular biological basis of kdr resistance in Blatella germanica. Complementary DNA sequences encoding calmodulin, partial structures of calmodulin dependent protein kinase II (CaM-kinase II) and an L-type-like calcium channel al subunit (IVS5-IVS6-Ef hand region) were identified and compared between susceptible and kdr strains of German cockroach, Blatella germanica. For this purpose, polymerase chain reactions (PCR) were used to obtain their sequences using cDNA from poly(A) + RNA isolated from their heads and thoraces. No mutation differences were found in all three sequences of calcium-regulating proteins between susceptible and strain. Northern blot analysis, however, showed reduced expressions of CaM-kinase II mRNA in two kdr strains. Western blot analysis with an
antibody preparation against CaM-kinase II on protein levels confirmed the above strain difference in the tuter of this enzyme. In contrast, the levels of calmodulin as well as that of an L-type-like calcium channel gene expression were not different between susceptible and kdr strains.
Impacts
(N/A)
Publications
- INAGAKI, S., KAKU, K., DUNLAP, D.Y. and MATSUMURA, F. Sequences of cDNAs encoding calmodulin, and partical structures of calmodulin kinase, and a calcium channel of kdr-resistant and -susceptible German cockroaches, Blattella germanica.
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