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Annual report : National Institute of Environmental Health Sciences (Volume 1985) online

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Sufficient evidence of a link between the genotoxic and carcinogenic properties
of chemicals exists to justify the use of genotoxicity data in the evaluation of
a chemical's potential carcinogenicity. Determination of a genetic effect in the
same animals and by the same routes of exposure and dose levels may provide the
more relevant information than in vitro studies. This assay system may provide
that type of data from the precTirbmc toxicity studies and contribute valuable
information to the decision of whether or not to pursue a chronic toxicity
study.



863



OAK RIDGE NATIONAL LABORATORY
(Department of Energy)
Oak Ridge, TN 37830
(Y01-ES-40118)

TITLE: Mouse Endogenous Retroviral Long Terminal Repeat Elements for Studying
Gene Transposition in Environmental Carcinogenesis

CONTRACTOR'S PRINCIPAL INVESTIGATOR: Dr. Wen K. Yang

PROJECT OFFICER (NIEHS): Raymond W. Tennant, Chief

Cellular and Genetic Toxicology Branch

DATE CONTRACT INITIATED: January 10, 1984

CURRENT ANNUAL LEVEL: $339,187

PROJECT DESCRIPTION

OBJECTIVES : The project addresses the important question of whether endogenous
retrovirus and related proviral elements in a murine model system are func-
tionally transposable elements which may be potential targets of environmental
genotoxic agents. The conceptual framework for this research effort is based on
the well known role of retroviruses in insertional mutagenesis and car-
cinogenesis and the structural analogy between retroviruses and transposable
elements. The mouse genome contains many related families of endogenous pro-
virus and proviral-1 ike elements that are likely to be involved in transposition
or other events related to genetic damage. This project focuses on charac-
terizing the structure and function of a unique class of proviral-1 ike elements
that are distantly related to murine leukemia virus.

METHODS EMPLOYED : Recombinant DNA cloning of LTR and Insertion Sequence 23
(IS23) containing fragments from Balb/c and RFM/Un mouse DNA has provided a
variety of endogenous proviral-1 ike elements. These clones are being charac-
terized by restriction enzyme mapping and nucleotide sequence analysis. The
inheritance of these elements are being studied by a comparative analysis of the
integration site in various strains and subspecies of the mouse.

MAJOR FINDINGS AND PROPOSED COURSE : The proviral unit designated as IS23 was
found to contain a repeated sequence reiterated approximately 200 times in the
mouse genome. The IS23 provirus is integrated within a region that is repeated
approximately 10 times. The IS23 probe exhibits a mouse strain specific DNA
restriction fragment pattern and unexpectedly recognizes a male specific
fragment. Evidence that this male specific IS23 fragment is lost from certain
chemically transformed NIH-3T3 cell clones and from tumors arising from chemi-
cally transformed cells will be confirmed and extended in the coming year.
Solitary LTR structures have also been isolated from leukemias and their role in
leukemogenesis will be examined by identifying their genomic location with
respect to cellular oncogenes. Finally, an experimental transposition model
system will be constructed by introducing molecular clones of these proviral
elements into human or other species cells that do not have an endogenous
background of related sequences.



864



SIG NIFIC ANCE TO BIOMEDICAL^ RESEARCH AND THE PROGRAM OF THE JNSTTTUTE: Identifi-
cation ot mammaiTah franspoisaVl e~~eTe^ents T ahlPthefr roTe fn "mutagenesis and car-
cinogenesis is of considerable importance in contemporary biomedical research.
The information gained from research on retroviral related elements will benefit
the long-term goal of developing rel i able Jji vitro tests to measure DNA transpo-
sition as a potentially significant genetic endpoint. The aoili-cy to detect
such events in a sensitive and precise manner and the ability to identify
environmental agents with the potential to cause such effects are fundamental
goals of the CGTB/NTP/NIEHS.



865



NATIONAL INSTITUTE OF OCCUPATIONAL SAFETY & HEALTH

Cincinnati, Ohio 45226 «.

(Y01-ES-40121)

TITLE: In Vitro Tests for Workplace Co-carcinogens

CONTRACTOR'S PRINCIPAL INVESTIGATOR: Or. J. Bohrman

PROJECT OFFICER (NIEHS): Robert Langenbach, Ph.D., Head

Metabolic Activation Group, CGTB

DATE CONTRACT INITIATED: September 30, 1982 (Y01-ES-20094)

March 30, 1984 (Y01-ES-40121)

CURRENT ANNUAL LEVEL: $171,000

PROJECT DESCRIPTION

OBJECTIVES : The objective of this research is to further develop and validate
an in vitro assay for tumor promoters and then to test (coded) up to 40 chemicals
for which the NTP has carcinogenesis data and in vitro genetic toxicity data.

METHODS EMPLOYED : The system being studied is the V79 cell metabolic coopera-
tivity assay developed by Trosko and colleagues. Chinese hamster V79 cells
which are resistant to 6-thioquanine (6-TG) are co-cultivated with V79 cells
sensitive to 6-TG. In the co-cultivation, when metabolic cooperativity exists,
the 6-TG resistant cells are killed and their cloning efficiency decreases. In
the presence of chemicals with promoting activity, metabolic cooperativity is
decreased and an increased number of 6-TG resistant colonies survive.

MAJOR FINDINGS AND PROPOSED COURSE : Two laboratories under NIOSH and NTP 1 s
supervision are looking at the effect of varying basic parameters in the assay.
These parameters include: pH effects, optimum Ca ++ levels, optimum time between
addition of test chemical and 6-TG, required duration of the assay, and statis-
tical approaches. With the developed protocol, up to 40 coded chemicals with
an NTP data base will be tested. The general ability of the assay to distinguish
between carcinogens and noncarcinogens will be determined. Furthermore, the
assay will be studied for its ability to identify carcinogens which act only by
promoting mechanisms as suggested by their lack of genetic toxicity in vitro .
The data from the metabolic cooperation assay will be analyzed in combination
with carcinogenicity and genetic toxicity results.

SIGNIFICANCE TO BIOMEDICAL RESEARCH AND THE PROGRAM OF THE INSTITUTE : Most
short-term assays for chemical carcinogens are based on the induction of genetic
damage. Therefore, chemicals which cause or promote cancer cells by nongenetic
mechanisms are not readily detected in short-term assays currently used by the
NTP. The development of an assay which detects such chemicals would greatly
enhance the spectrum of carcinogens which short-term tests detect.



866



DUKE UNIVERSITY MEDICAL CENTER

Durham, North Carolina 27710

(N01-ES-55091)

TITLE: Task 1: Development of Human Cell Assay Systems for Genetic Toxicity

CONTRACTOR'S PROJECT DIRECTORS: Dr. S. Strom

Dr. G. Michalopoulos

PROJECT OFFICER (NIEHS): Robert Langenbach, Ph.D., Head

Metabolic Activation Group, CGTB

DATE CONTRACT INITIATED: May 1, 1985

CURRENT ANNUAL LEVEL: $187,626

PROJECT DESCRIPTION

OBJECTIVES : The project is a three-year study to develop and utilize a cell-
med i a te d~Ty st.em where freshly isolated human hepatocytes are used for meTaUolic
activation and co-cultivated human target cells are used to detect genetic
damage. The overall objective is to determine the utility of a human cell-
mediated system in screening for genotoxic agents. Some specific objectives
are: (1) analysis of individual variation of carcinogen activation by human
hepatocytes; (2) measurement ot UUS in the human liver cells and of mutation and
SCE induction in human fibroblast for four uncoded and 12 coded chemicals; (3)
comparison of human hepatocytes and rat hepatocytes for metabolic activation and
human fibroblast and V/9 cells as genetic targets; (4) improve freezing tech -
niques for liver cells; and (5) compare human liver cells and human S9 for
metabol ic activation in genetic toxicity assays.

METHODS EMPLOYED : Human liver tissue is obtained from organ donors or surgical
specimens and perfused to obtain isolated, intact, viable hepatocytes. The
hepatocytes are then co-cultivated with human fibroblast in the presence of the
chemical and the genetic endpoints UDS, SCE, and mutation measured. Metabolite
analysis of the chemicals is done by HPLC.

MAJOR FINDINGS AND PROPOSED COURSE : Initial experiments will improve techniques
for obtaining viable human hepatocytes and use the hepatocytes in a human cell-
mediated system to determine the genetic effects of aflatoxin B]_, dimethyl nitro-
sarnine, acetyl aminofl uorene, and benzo(a)pyrene. The metabolism and metabolic
activation to genotoxins of these carcinogens in hepatocyte preparations from
different humans will be compared. Early studies will also focus on improving
techniques to freeze hepatocytes for subsequent use in genotoxicity assays.

SIGNIFICANCE TO BIOMEDICAL RESEARCH AND THE PROGRAM OF THE INSTITUTE : The study
addresses the problem of utilizing human cells in determining the genetic
toxicity of chemicals. Because species differences in response to chemical
carcinogens exist, it is useful to determine the effects of chemicals in human
cell systems, in addition to rodent cell system, to better predict human health
hazard. The long-term outcome of the study will be a clearer understanding of
the role of human cells in short-term assays.



867



UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL GENTEST LIMITED PARTNERSHIP
Chapel Hill, North Carolina 27514 Woburn, Massachusetts 01801

(N01-ES-55092) (N01-ES-55093 ) '

TITLE: Task II: Development of Human Cell Assay Systems for Genetic Toxicity

CONTRACTOR'S PROJECT DIRECTORS: Dr. D. Kaufman (N01-ES-55092)

Drs. C. Crespi and B. Pennman (N01-ES-55093)

PROJECT OFFICER (NIEHS): Robert Langenbach, Ph.D., Head

Metabolic Activation Group, CGTB

DATE CONTRACT INITIATED: May 1, 1985

CURRENT ANNUAL LEVEL: (N01-ES-55092 ) - $252,810

(N01-ES-55093) = $488,292

PROJECT DESCRIPTION

OBJECTIVES : The project is a three-year study to develop and utilize a human
eel 1 system for the detection of genotoxic chemicals in which metabolic activa-
tion of the cnemicals and measurement of genetic endpoints can be accomplished
in the same cells. As most cell systems used in genetic toxicity systems have
lost varying degrees of metabolic capability, a primary objective is to improve
the metabolic capacity of the cells so that more useful, reliable, and sensitive
human cell systems are available for short-term testing.

METHODS EMPLOYED : Human lymphobl astoid cells will be used in the studies and /
attempts made to improve metabolic capabilities by fusion with metabolically
active human cells, transfer of specific chromosomes, or selective cloning.
Genetic endpoints measured will be mutations, DNA repair, and chromosome
aberrations.

MAJOR FINDINGS AND PROPOSED COURSE : Initial experiments will determine which
classes of chemical carcinogens the lymphobl astoid cells cannot metabolize.
Metabolic capability will be assessed by induction of genetic endpoints and
by HPLC identification of metabolites produced. Chemicals which are not
metabolized by the parent cell line will be identified and attempts made to
modify metabolic capability. Experiments to fuse the lymphoblastoid cells with
established human cell lines of known metabolic capacity (e.g., Hep G2 or
endometrial cells) and select for hybrids with enhanced metabolic capability.
Alternatively, selective cloning of cells or use of cell sorting in the presence
of the test chemical to identify cells within the mass population with the
ability to metabolize the chemical will be employed. In later studies, 12 (UNC)
and 30 (Gentest) coded chemicals will be tested in the metabolically altered
human cell system for genotoxic effects.

SIGNIFICANCE TO BIOMEDICAL RESEARCH AND THE PROGRAM OF THE INSTITUTE : Most
cells used in vitro to assess the genetic toxicity of a chemical have lost the
metabolic capabil ity they possessed in vivo . The present study addresses the
uses of human cells in genetic toxicity assays and simultaneously searches for
methodologies to enhance in vitro metabolic capability. The development of



868



cells in which both metabolism and measurement of genetic endpoints can be
accomplished should yield more reliable and sensitive cell systems for short-
term tests. Furthermore, the use of human cells could produce systems ^hich
are more predictive of hazard to humans.



869



BROWN UNIVERSITY UNIVERSITY OF KENTUCKY

Providence, Rhode Island 02912 „ Lexington, Kentucky 40506-0225
(N01-LS-5-5095) (N01-ES-5-5106) W

TITLE: Validation and Testing of Chemicals in a Drosouhila Aneuploidy Detection
System

CONTRACTOR'S PROJECT DIRECTOR: Stanley Zimmering, Ph.D. (rJ01-ES-5-5095)

Christopher Osgood, Ph.D. (M01-ES-5-5106)

PROJECT OFFICER (NIEHS): James M. Mason, Ph.D., Geneticist

Environmental Mutagenesis Group, CGTb

DATE CONTRACT INTIATED: N01-ES-5-5095: March 1, 1985

N01-ES-5-5106: June 1, 1985

CURRENT ANNUAL LEVEL: N01-ES-5-5O95 = 5184,330

N01-ES-5-5106 = $144,382

PROJECT DESCRIPTION

OBJECTIVES: The purpose of this contract is to develop a test system in
TJrosoplnTa" for screening environmental chemical s for their ability to induce
TheiTpToTdy, i.e., changes in chromosome number. The use of a test for aneuploidy
will allow the identification of chemicals which induce certain types of cnrpjrio-
somal aberrations but may not be identified as mutagenic in the standard "short
Te~n^mTJtl^e7Te"sTs~te s t systems now in use.

METHO DS EMPLOYE D: Standard genetic manipulations are employed utilizing well
clfa'rac ten zed" YH inked mutants and chromosomal aberrations i n Pro soph n J a mel_a_no-
gaster. The proposed assays for aneuploidy involve feeding test chemical s to
Te"maTe~ larvae, mating these females to tester males and scoring for the occurrence
of progeny resulting from changes in chromosome number. Each chemical will be
tested at 2-3 doses.

MAJOR FINDINGS AND PROPOSED COURSE: Three separate strains have been established
ThaTwill allow aneuploid progeny "to be recognized in a one-generation screen in
which half of the regular progeny die. These three strains are being compared for
sensitivity and reliability by treating each in parallel with a number of positive
control compounds. The compounds have been chosen to represent different classes
of chemicals known to interact with the mitotic/meiotic apparatus. The results
from these tests will be used to determine the strain that will be used in future
assays for chemically-induced aneuploidy in Drosophila. As soon as the appropri-
ate strain is chosen, testing of known positive and negative compounds will begin
in a dual laboratory study to evaluate the test system.

SIGNIFICANCE T O BIO MEDICAL RES EARCH AND THE PRO GRAM OF THE _ INSTITUTE: A large
Tractufn of spontaneous abortion in h uma n s 'arid certain serious genetic diseases
(e.g., Down's syndrome) are caused by aneuploidy. A few chemicals are known to
induce aneuploidy; however, there is no fast, reliable, well-defined developed
test to detect such chemicals on a large scale. This project is designed to de-
velop a test system to be used as part of the Cellular and Genetic Toxicology
Branch mutagen screening program. The use of a test for aneuploidy will allow the
Program to identify chemicals which induce certain types of chromosomal aberra- c
tions that would not be identified as mutagenic in other mutagenesis test systems.

870



CHEMICAL PATHOLOGY BRANCH



871



CHEMICAL PATHOLOGY BRANCH
Summary Statement

Mission : During FY 85 the Chemical Pathology Branch continued its three major
functions: (1) support of the National Toxicology Program (45%); (2) support of
intramural research (30%); and (3) independent research (25%).

Section Work Areas : Histology and Electron Microscopy, Tumor Pathology,
Toxicologic Pathology, Experimental Pathology, and Laboratory Animal Management.

Staffing : The Chemical Pathology Branch consists of 11 comparative
pathologists, 1 laboratory animal veterinarian, 12 technicians, 3 secretaries,
and 1 stay-in-school student. Or. Roger Alison, formerly of Huntingdon,
England, joined the Branch in February 1985. Dr. Linda Uraih, formerly of
International Research and Development Corporation (IRDC), joined the 3ranch in
March 1985. Dr. Steven Stefanski , having recently completed a pathology
residency and masters program at Michigan State University, joined the Branch in
April 1985. Dr. Kunitoshi Mitsumori, formerly of the National Institute of
Environmental Toxicology, Tokyo, Japan, joined the Branch in May 1985.
Dr. Michael Elwell, formerly of Walter Reed Army Institute of Research
(transferred from the Army to the Public Health Service), joined the Branch in
July 1985. Dr. Kenneth Pierce of Texas A & M University, and Dr. Jeffrey Wilson
of Sandoz, Basal, Switzerland, are working in the Branch as National Institutes
of Health Guest Workers.

Accompl ishments :

1. Management of Quality Assurance Program for the National Toxicology
Program - During FY 85 the Chemical Pathology Branch continued
responsibility for evaluating the quality of pathology conducted in
bioassays performed by the NTP. This included the review of 14 sub-
chronic, 19 interim sacrifices, and 21 chronic bioassays during the
first nine months of FY 85 (Tables 1, 2, and 3).

2. Establishment of the NTP Archives in Research Triangle Park adjacent to
the NTP - The move from Rockville, Maryland, was accomplished in
November 1984 and the "downtime" for the Archive was less than one
month. Over 300 studies and 5.5 million slides are now available to
NTP and approved persons on an easily accessible computerized system.

3. Implementation of TDMS - During FY 85 the Toxicology Data Management
System (TDMS) was implemented in 3 additional contractor laboratories
(total of 11) and 4 interagency agreement sites. There are presently
162 chemicals on TDMS and all new starts will be put on the system. A
one-day meeting of advisors reviewed the pathology nomenclature code
table and added approximately 40 new terms to the computer system.

4. Research Programs - During FY 85 the responsibility for laboratory
animal management was transferred to the Chemical Pathology Branch.
The laboratory animal veterinarian has a strong interest in pathology
and some Branch pathologists have dual boards (Lab Animal Medicine and
Pathology) which will allow a natural collaboration between these two
closely related disciplines.

873



During FY 85 th
pathology data
and the patholo
were instituted
pathology data
pre-QA data rev
the laboratory
found, the path
data is accepta
Archives. The
check, and a ch
able, the data
acceptable, it
include the ori
review. These
of problems and



e Branch placed increasing emphasis on the quality
and meeting GLPs. A total of 30 studies were audi
gy portion reviewed by the Branch. Three major ch

to improve and check the Pathology data. First,
at the end of a 2-year study is now subjected to a
iew while slides, blocks, and wet tissues are stil
If discrepancies, duplications or unexplained da
ology tables are returned to the laboratory. Once
ble, the pathology material and data is submitted
second new step is a slide/block match-up, animal
eck for untrimmed lesions by the Archives. If una
is returned to the laboratory for corrections. If
proceeds to the pathology QA. The third new step
ginal pathologist in the Pathology Working Group (
three new steps have resulted in more prompt resol

fewer post-PWG unresolved pathology issues.



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Research Programs - Studies in support of the National Toxicology
Program included:

a. international meeting and working group on proliferative lesions
of the Brain in Man and Animals co-sponsored jointly by NTP and
Duke University Medical School.

b. modification of the reduced pathology protocol to include slide
preparation but "read downs" for only required tissues. This
preserves most of the savings in cost but is accomplished more
quickly.

c. organizing an international symposium on ovarian tumors in man and
animals .

d. development of a classification scheme and study set for ovarian
tumors in rats and mice.

e. instituting cooperative agreements between Dartmouth Medical
School, Northwestern Medical School, and the University of
Missouri to evaluate the significance of dietary oils and
proliferative exocrine pancreatic lesions found in male rats.

f. evaluate the effect of sampling or the incidence of pancreatic
lesions in vehicle and untreated male and female F344 rats.



l .



completed and submitted for publications the evaluation of the
strain A mouse lung adenoma assay in evaluating the carcinogenic
potential of chemicals.

evaluate the use of immunoperoxidase techniques for identifying
cell types in mouse lymphomas.

transplantation of induced rodent tumors in several NTP studies.
This has allowed a better definition of the biological behavior of
cholangiof ibrosis/cholangiocarcinoma lesions.



874



j. ultrastructural evaluation of the nasal turbinate in control and
methyl isocyanate studies.

k. evaluation of forestomach lesions in methyl bromide studies
utilizing stop studies.

1. evaluation of the salivary sarcomas in the chlorendic acid NTP
study.

m. review and classification of the proliferative gall bladder
lesions found in mice.

n. review of naturally occurring chordomas in F344 rats.

o. institution of a modified classification scheme for proliferative
hepatocellular lesions in the rat. This is based upon classic
pathology terminology utilizing hyperplasia, adenoma, and carci-
noma and will be used by NTP pathologists for two years at which
time the results will be re-evaluated.

p. use of nuclear magnetic resonance (NMR) to assess liver function
in intact animals.

q. preparation of a hematology atlas for peripheral blood and bone
marrow of the F344 rat.

r. use of nuclear magnetic resonance in cooperation with Duke

University to detect naturally occurring and induced tumors in
rats.

s. development of whole body 5-8 micron histology sections for rats
for use in NMR studies.

t. revision of the NTP statement of work to include a more thorough
histopathologic evaluation of acute and 14-day repeated dose
toxicologic studies.

Research Program - Independent studies and collaborative efforts with
other laboratories in TRTP/NTP.

a. mechanism of the dioxin induced myelotoxicity and immunotoxicity
in mice.

b. myelotoxicity of the class of propylene glycols.

c. validation, development, and quality review of clinical pathology
and chemistry in NTP studies.

d. investigation into the two-hit hypothesis of carcinogenesis
utilizing the rat liver model.

e. tissue collection from NTP carcinogenicity studies as well as from
in-house studies for identification on oncogenes.



875



f. publication of a book chapter on pathology QA procedures for
toxicity and carcinogenicity studies.

g. publication of a book chapter on history, naturally occurring
lesions in B6C3F1 mouse and F344 rat and comparison with other
strains used in toxicology.

h. evaluation of immunoperoxidase techniques for murine hematopoietic
tumors and thyroid hormones

i. evaluation of soft-plastic techniques for microscopic examination
of muscle

j. development of histochemical methods for fiber typing of striated
muscle

k. evaluation of subchronic, interim sacrifice, and chronic
toxicologic studies of furan induced liver disease.

1. successful transplantation of furan induced cholangiocarcinomas to
recipient F344 rats

m. special study of 3,3'-dimethylbenzidine induced basal cell
hyperplasia of the skin

n. ultrastructural evaluation of 3,3'-dimethylbenzidine induced
hepatocellular hypertrophy in the rat

o. studies on interstitial and pyelonephritis of the mouse and renal
infarction in the mouse and rat

p. evaluation of the prechronic toxicity of furfural and furfuryl
alcohol

q. study of the subchronic dermal toxicity of vinylcyclohexene
diepoxide in rats and mice

r. urinary bladder and kidney lesions produced in Fischer 344/N rats
and B6C3F1 mice after 13 weeks of 2,2-bis(bromomethyl )-
1 ,3-propanediol administration

s. subchronic toxicity of propantheline bromide in Fischer 344/N rats
and B6C3F1 mice

t. study of the hepatic lesions induced by chlorowax 40 in F344/N
rats

u. study of the neuropathology of 13-week toxicity of orally
administered sodium azide to F344/N rats.



'



876



8. Support of the Intramural Research Program - A great deal of support



Online LibraryNational Institute of Environmental Health ScienceAnnual report : National Institute of Environmental Health Sciences (Volume 1985) → online text (page 85 of 114)