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DEPARTMENT Ok I-~EALTH& HUMAN SERVICES Public Health Service National Institutes of Health National Institute of Environmental Health Sciences Research Triangle Park, N.C. 27709 U.S. Department of Energy Oak Ridge National Laboratory Post Office Box E Oak Ridge, Tennessee 37830 Energy Programs and Support Division Subject: Request for Proposal Number NIH-ES-86-15 You are requested to submit a proposal for a project entitled, "Development of Image Processing Methodology for Comparative Analysis of Proteins from Description of Work. Your proposal should be submitted for the period September 30, 1986 through September 29, 1987 and should include the following: Pour ( 4 ) copies of a technical proposal must be submitted. The technical proposal should include: a. discussion of the objectives to be met and general approach to be taken toward accomplishment of the work outlined in Attachment A , as well as any significant milestones to be achieved. b. a listing of the names and titles of individuals to be assigned to the project. Resumes should be provided for the key personnel c. a floor plan of dedicated space and a list of the equipment proposed for use under the project. Include evidence to indicate that these facilities will permit performance of the assigned tasks at the proposed capacity within the indicated timeframe. d. a description of the Agency's policy on safety and security. Agency will be expected to comply with the National Toxicology Program's Health and Safety minimum requirements. Three (3) copies of a business proposal must be submitted with at least one (1) copy bearing the original signature of an authorized Officer. The Agency, as a minimum, must submit a cost proposal fully supported by cost and pricing data adequate to establish the
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reasonableness of the proposed costs. The attached Instructions f o r Submission of Cost and Pricing Data (Attachment B) is recommended for use in the preparation of your proposal. Copies of your proposal should be sumbitted to the following address no later than August 5 , 1986: National Institute of Environmental Health Sciences Contracts Management Office Post Office Box 12874 4310 South Miami Boulevard Research Triangle Park, North Carolina 27709 If you have any questions concerning this requirement, please contact Dorothy G. Williams at (919) 541-7893. Contiacting Officer, OAM Attachment A - Description of Work Attachment B - Instructions f o r Submission of Cost and Pricing Data
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NATIONAL TOXICOLOGY PROGRAM w:Development of Image Processing Methodology f o r Comparative Analysis of Proteins from Two-Dimensional Gel Electrophoresis : Monitor Early Changes in Multiple Cellular Enzymes and Proteins after Carcinogen Treatment, with the Ability to Store, Rapidly Retrieve and Analytically Contrast the Effects of Various Chemical Treatments. To determine the critical metabolic parameters that differentiate between chemical carcinogen susceptible and resistant cells. Chemical carcinogens and - mta gens are metabolized by a complex of enzymes comprlsed of the monooxygenases as the activation fraction, and the epoxide hydro las es and cytoplasmic transfer as es as the detoxification fraction. The primary function of the metabolic processing of xenobio tics is to render them biochemically inert and water soluble for more facile excretion from the organism. This enzymatic array has evolved a s the major mode of detoxifying chemicals, and while the liver is the main site of this activity,the monooxygenase system appears t o be ubiquitous i n mammalian tissues. Since many environmentally prevalent toxic substances are chemically stable it is necessary for the tissues to deactivate them by der iv at iz ation into innocuous forms (Coon et al., 1980). However, cellular processing for parent carcinogens and mutagens toward detoxification form the highly reactive intermediates that are now known to be the carcinogenic and mutagenic forms of the molecule (Harris and Cer rut ti, 1982). grown substantially over the last decade, and meta boli te profiles for various chemicals from tissues and cells i n many species have been (Miller and Miller, 1977) and all species form the same family of metabolites for any given chemical carcinogen (Selkirk et al., 1982). Unfortunately, for a given carcinogen, we are then faced with the reality that our current theoretical concepts and experimental metabolic data base cannot explain the reasons for the wide range of susceptibility to malignant transformation by a given chemical among test species. Since all short-term and long-term carcinogen and mutagen as says rely on the same type of metabolic activation system (Douglas, 19841, itis necessary t o probe further into the metabolic reasons for the relative differences in susceptibility for a wide spectrum Of chemical carcinogens. The valid extrapolation of animal carcinogen i city data to human risk requires the most complete understanding of the respective differences and similarities i n the processing rate or specific modulation of the critical ' enzymes for activation and detoxification. The data base on carcinogen metabolism has From this vast compendium of data two generalizations have It appears that all active carcinogens are electro phil es
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The burgeoning chemical carcinogen data base clearly shows a l l metabolic products are identical for a given chemical inall eukaryotes and have markedly similar patterns of InaCrOfBOleCular binding. With the exception of a single generalization that all chemical carcinogens form reactive electrophiles, there is no unifying concept t o explain the specific carcinogenic mechanism for chemicals of widely divergent structure. large number of metabolic studies over the l a s t decade exhibit a marked phylogenetic conservation for xenobiotic metabolism with similar activation and detoxification pathways. relatives usc ep tl bil it y and resistance t o malignant transformation between species, tissues and cells for carcinogens and mutagens (Langenbach e t al., 1983). Furthermore, there is great variation I n susceptibility between species for a given carcinogen. assays best exemplifl the concept of variable susceptibility. Specialized assay systems have evolved for often unknown biochemical reasons, which are uniquely sensitive t o a given chemical class or mode mammary tumor system a r e excellent for polycyclic carcinogens, while other species are not assen dtl ve to these chemicals. of unique sensitivity is aflatoxin B1 where rat,ferret and trout develop liver tumors a t microgram doses while the mouse is highly resistant t o hepatoma formation by this chemical. Yet there is a wide spectrum of The mouse skin model for initiation / promotion and the rat This metabolic enigma of an apparent identity in overall metaboliam witha concommitant susceptibility Variance for malignant transformation i n different species has eluded explanation. It becomes critical to attempt to understand the biochemical and genetic mechanisms for t h i s paradoxical situation where a given carcinogen w i l l produce the putative reactive intermediate i n two test species and will exhibit a clear dose response i n one species and be completely refractory i n the other, It is becoming increasingly important t o understand i f species variance to chemical carcinogenesis is a function of the genetic regulation of the . vast array of enzymes induced d u r i n g activation and detoxification of the The god of this project is t o determine which portions of the metabolic pathwaysare more or it ical in forming, maintaining, and directing the ultimate cafiinogenlc or mutagenic species of the molecule toward its final biological fate. monitor a feu enzymes i n the metabolic scheme. only a small number of the components and are sufficiently labor Intensive to prohibit large experiments that would Include many time points and various chemical treatments. However, an analytical system that could simultaneously assay dl1 the critical cellular proteins would markedly enhance our understanding of t h e kinetics of each component i n the enzymatic pathway by determining which proteins are altered by chemical treatment, and how susceptible and resistant cells differ In I n the past i t was only possible and practical t o Standard assays exist for The proposed research w i l l establish an analytical system that is capable of qualitatively and quantitatively measuring cellular proteins. Using
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two-dimensinal gel electrophoresis and computer image analysis technology i t will be possible to accurately monitor per tub at ions Ln the array of cellular proteins during chemical carcinogen treatment. Since both susceptible and resistant cells to malignant transformation have been shown t o produce the identical array of metabolites for any given carcinogen,the enzymatic processing of the chemical may be altered in such a way as t o shunt the metabolism of the carcinogen toward or away from formation of the reactive intermediate. By monitoring the enzyme systems responsible for activation and detoxification in the cell and their alterations by both carcinogenic and noncarcinogenic isomers, i t is expected that the pathways critical for activation and detoxification will be discernible when compared to identical protein profiles from However, it is first necessary t o develop the methodology toits maximum level of reproducibility. Therefore this project will compare the prote in profiles of both human and rodent cells to ascertain basic differences inthe cytoplasmic and nuclear protein composition in control and chemically treated cells. of selected rodent and human cellular proteins. digitized, processed inan image analyzer, and stored in the computer for comparison against the protein maps from carcinogen-treated cells. Special image analysis algorithms wLll determine qualitative and quant it at ive per tub at ions in cellular proteins between control and chemically treated cells. The first chemical probes in this research will be the use of carcinogenic and noncarcinogenic isomers of the same polycyclic hydrocarbon molecule. Benzo(a)pyrene and dibenz(a,h)anthracene and their excellent probes since the latter compounds have been predicted by the current bay-region hypothesis to possess significant carcinogenic potential. However, i n vivo and In vitro studies have shown benzo(e)pyrene The plan will be first to prepare master maps These profiles will be -- --’. and di be oz(a,c)anthracene to be inactive as initiators. Background History and Needs Ched cal carcinogens and mutagens are metabolized by a complex of enzymes comprised of the mnooxygenases as the act lv ation fraction, and the epoxide hydro las es and cytoplasmic transfer as es as the detoxification fraction. The primary function of the metabolic processing of zeaobiotic si & t o render them biochemically inert and water soluble for more facile excretion from the organism. This enzymatic array has evolved as the major mode of detoxifying chemicals, and while the liver is the main site of this activity,the monooxygenase system appears t o be ubiquitous i n mammalian tissues. Since nany environmentally prevalent toxic substances are chemically stable it is necessary for the tissues to deact iv ate them by der iv at iz ation into innocuous forms (Coon etal., 1980). However, cellular processing for parent carcinogens and mutagens toward detoxification form the highly reactive intermediates that are now known t o be the carcinogenic and mutagenic forms of the molecule (Harris and Cer rut ti, 1982). The database on carcinogen metabolism has grown substantially over the last decade, and meta boli te profiles for various
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chemicals from tissues and cells in many species have been elucidated. From this vast compendium of data two generalizations have been derived. I t appears that all active carcinogens are electro phil es(Miller and Miller, 1977) and all species form the same family of metabolites for any given chemical carcinogen(Selkirk et al., 1982). Unfortunately,fora given carcinogen, ve are then faced with the reality that our current theoretical concepts and experimental metabolic database cannot explain the reasons for the wide range of susceptibility to malignant trans for - mation by a given chemical among test species. long-term carcinogen and mutagen as says rely on the same type of metabolic activation system (Douglas, 1984) it is necessary t o probe further into the metabolic reasons for the relative differences in susceptibility for a wide spectrum of chemical carcinogens. mal carcinogen i city data to human risk requires the most complete understanding of the respective differences and similarities in the proces sing rate or specific modulation of the critical enzymes for activation and detoxification. Since all short-term and The valid extrapolation of aniData, Data Rights,Patents, and Copyrights All data and materials shall be available to the National Toxicology Program project officer and/or his designee for inspection or use a t any time during the IAA. Division of the Oak Ridge National Laboratory through the term of the processed in the Biology Divisi o a,all relevant data stored in the computer * protocols and standard operating procedures developed by the laboratory,data generated and collected, and reports and manuscripts prepared by the Biology Division on this project. The data and samples shall be stored in the Biology The data set includes proteins extracted from cells and tissues And At the conclusion of the project the data shall be shipped tothe NTP unless the project officer and principal investigator agree that it is i n the best interest of the project to leave some or all of the data, or copies of the data, in the Biology Division at ORNL. Data refers t o records on paper and/or other collection media, but as such not limited t o computer disks, tapes, or microfiche. The agency shall acknowledge the support of the Department of Health and Human Services,National Institutes of Health, National Institute of Environ?nen&al Health Sciences, whenever publicizing the work under this agreement i n any media. Support shall be acknowledged as follows: " This work was conducted for the National Toxicology Program under the National Institute of Environment Health Sciences, Interagency Agrsement
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Replication, Dissemination, or Use of the Results These results should be presented for use of the general chemical carcino genesis community to help understand the mechanism of action of chemical carcinogea processing i n cells susceptible to malignant transformation. The data should be published as collaborative efforts between the principal investigator and the project officer. No information may be released however without prior written authorization by the NTP project Post-Award Administration and Monitoring 2. Gel equipment will be ut liz ed and the system fine tuned for reproduci bly electro ph ore sing 20 two-dimensional gels simultaneously. Algorithms will be prepared for developing master control gel images, and the ability to search multiple gels for specific proteins will be The principal investigator will submit a semiannual report .3 . 4. Final report reviewing the results of all studies (by the end of the interagency agreement term). Statement of Uork The Natiottel Toxicology Program utilizes various short-term and chronic assays for tibxicity, carcinogen es is, and genotoxic effects. For the vast majority of environmentally prevalent chemicals, some form of metabolic activation is required by the cell ' s microsomal, monooxygenase complex t o form reactive inter a edi at es for manifestation of the toxic principle. This research program is designed t o broaden the empirical approach t o assessing the role of metabolism of chemicals i n malignant transformation susceptible and resistant cells. The overall plan is to utilize bath cell types as probes t o understand why certain species, tissues, and
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cells are predisposed toward malignant transformation by chemicals and compare the metabolic competence, and …
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